MASARYKOVA UNIVERZITA LÉKAŘSKÁ FAKULTA KONCEPTY LÉKAŘSKÉ FARMAKOLOGIE V ÉŘE PERSONALIZOVANÉ MEDICÍNY A JEJICH IMPLIKACE PŘI VÝUCE FARMAKOLOGIE V 21. STOLETÍ HABILITAČNÍ PRÁCE komentovaný soubor prací navazující na habilitační práci s názvem „Koncepty klinické farmakologie v éře personalizované medicíny” (LF UK Bratislava, 2015) Brno 2017 doc. MUDr. Regina Demlová, Ph.D. Poděkování Děkuji všem svým kolegům a spolupracovníkům za vynikající týmovou spolupráci a podporu při výzkumné činnosti. Za cenné rady, spolupráci a podporu bych chtěla konkrétně poděkovat doc. MUDr. Daliboru Valíkovi, Ph.D., prof. MUDr. Jaroslavu Štěrbovi, Ph.D. a doc. RNDr. Lence Zdražilové Dubské, Ph.D. Za dlouhodobou podporu a oporu děkuji i své rodině. Za vytvoření příznivých pracovních podmínek děkuji rovněž prof. MUDr. Jiřímu Mayerovi, CSc. a vedení Lékařské fakulty MU v Brně a vedení Masarykova onkologického ústavu v čele s prof. MUDr. Janem Žaloudíkem, CSc. OBSAH ABSTRAKT (ČESKY) ............................................................................................1 ABSTRACT (ENGLISH) ........................................................................................2 2. PŘEDMLUVA .................................................................................................3 3. ZÁKLADNÍ FARMAKOLOGIE JAKO TEORETICKÁ NAUKA A KLINICKÁ FARMAKOLOGIE JAKO INTEGRATIVNÍ DISCIPLÍNA S PŘESAHEM DO VŠECH KLINICKÝCH OBORŮ.........................................................................................55 3.1 PŘEHLEDOVÁ ČÁST K PERSONALIZOVANÝM CÍLŮM VE FARMAKOTERAPII NÁDORŮ 9 3.1.1 PERSONALIZOVANÉ CÍLE V ONKOLOGII Z POHLEDU KLINICKÉ FARMAKOLOGIE A PREKLINICKÉM EXPERIMENTU ...........................................1010 3.1.1.1. VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE KLINICKÉ ČÁSTI ..................1111 3.1.1.2. VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE V PREKLINICKÉM EXPERIMENTU .............................................................................................................1414 3.1.2 IMUNOLOGICKÉ ASPEKTY NÁDORŮ VE VZTAHU K PROTINÁDOROVÉ FARMAKOTERAPII.......................................................................................1515 3.1.2.1. VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE V KLINICKÉ ČÁSTI ...............1616 3.1.2.2 VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE V PREKLINICKÉM EXPERIMENTU .............................................................................................................1818 3.1.3 PERSONALIZOVANÁ MEDICÍNA V KONTEXTU HODNOCENÍ ODPOVĚDI DLE „BIOMARKERŮ“ – ÚVOD K VÝZNAMU miRNA ................................................... 211 3.1.3.1 VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE miRNA V PREKLINICKÉM EXPERIMENTU............................................................................................ 22 3.1.3.2. VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE Z POHLEDU KLINICKÉ FARMAKOLOGIE ....................................................................................... 244 3.2 PŘEHLEDOVÁ ČÁST – ÚVOD K METABOLICKÉMU SYNDROMU A ENDOKRINOLOGICKÉMU STATUTU...................................................................2525 3.2.1 METABOLIKCÝ SYNDROM, ZÁNĚT A NÁDOROVÁ ONEMOCNĚNÍ Z POHLEDU KLINICKÉ FARMAKOLOGIE...........................................................................2626 3.2.2 METABOLICKÉ ZMĚNY A ROLE ADIPOKINŮ V PREKLINICKÉM EXPERIMENTU NA ZVÍŘETI ...................................................................................................2626 3.2.2.1 VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE V PREKLINICKÉM EXPERIMENTU .............................................................................................................2828 4. IMPLIKACE PREKLINICKÝCH A KLINICKÝCH AKTIVIT PŘI VÝUCE LÉKAŘSKÉ FARMAKOLOGIE................................................................................................30 4.1 PREGRADUÁLNÍ VÝUKA LÉKAŘSKÉ FARMAKOLOGIE .................................... 30 4.2 POSTRGRADUÁLNÍ VÝUKA LÉKAŘSKÉ FARMAKOLOGIE ................................ 34 5. ZÁVĚR....................................................................................................... 3636 6. REFERENCE................................................................................................... 37 7. SEZNAM KOMENTOVANÝCH PUBLIKACÍ........................................................ 42 8. PŘÍLOHY....................................................................................................... 44 1 ABSTRAKT (česky) Habilitační práce doc. MUDr. Reginy Demlové, Ph.D. na téma „Koncepty lékařské farmakologie v éře personalizované medicíny a jejich implikace při výuce farmakologie ve 21. století” je zpracována formou komentovaného souboru publikovaných vědeckých prací autorky z let 2015-2017 a habilitační práce v oboru Klinická farmakologie obhájené dne 18. 10. 2015 před VR LF UK v Bratislavě, která komentovala klinicko-výzkumné práce před rokem 2015 (práce je přílohou I). Na počátku každé kapitoly je vždy uveden stručný přehled úvodu do problematiky a poté vlastní příspěvek autorky k dané problematice, ať již z hlediska klinické zkušenosti nebo preklinického výzkumu. Úvod habilitační práce nastoluje základní pohled na komplexnost oboru základní a aplikované klinické farmakologie a principy personalizované farmakoterapie v dnešní době. Hlavní část práce přináší informace o principech individualizované medicíny a uplatňování individualizovaného přístupu k pacientovi, definuje farmakologické cíle a vybrané biomarkery protinádorové farmakoterapie, věnuje se jejich genezi od objevu přes validaci až ke klinickým aplikacím. Dále komentuje přístup personalizované farmakoterapie ve vztahu k hostiteli, zejména imunologickým aspektům a roli zánětlivých procesů, které mohou doprovázet metabolický syndrom. Závěr práce se věnuje možné implementaci preklinického i klinického farmakologického výzkumu autorky do pregraduální i postgraduální výuky lékařské farmakologie. 2 ABSTRACT (English) The habilitation (associate professorship) thesis of Regina Demlova on the topic "Pharmacological aspects of personalized medicine and their implications in pharmacology teaching" is comprised of a compendium of author’s selected scientific papers published in the years 2015 – 2017 amended with interpretive comments and the habilitation thesis in the field of Clinical Pharmacology defended on October 18th 2015 at Faculty of Medicine of the Comenius university in Bratislava, which commented on clinical research and scientific papers published before 2015 (attached as an appendix I). There is always a brief overview of the introduction to the issue at the beginning of each chapter and then the author's own contribution to the issue, whether in terms of clinical experience or preclinical research. The overall perspective of the complexity of the basic and applied clinical pharmacology and principles of personalized pharmacotherapy in everyday practice are outlined in the introductory part of the thesis. Major part of the thesis provides information on the principles of individualized medicine and use of the individualized approach to the patient, defines the pharmacological targets and selected biomarkers of anticancer pharmacotherapy, and pays attention to their genesis from discovery to validation and clinical applications. Host-approach of personalized pharmacotherapy, particularly immunological aspects and the role of inflammatory processes that may accompany metabolic syndrome, are discussed in the subsequent text. Last part of the thesis deals with the possible implementation of the preclinical and clinical pharmacological research of the author in the pregraduate and postgraduate teaching of medical pharmacology. 3 2. PŘEDMLUVA Předkládanou habilitační práci v oboru „Lékařská farmakologie” jsem vypracovala jako komentovaný soubor 13 prací se závěry a výstupy, které jsem pojala jako implementaci vlastních výzkumných preklinických poznatků a klinických zkušeností do pregraduální výuky oboru Lékařská farmakologie. Jedná se o práce, které byly publikovány v období od ukončení mého předchozího habilitačního řízení a komentovaný soubor je tvořen 9 full-texty, 3 Meeting Abstracts a 1 akademicky iniciovanou klinickou studií, přičemž 10 z předkládaných prací bylo publikováno v impaktovaných časopisech. Předkládaná habilitační práce navazuje a rozšiřuje již obhájenou habilitační práci z roku 2015 na LF UK v Bratislavě. Ta se soustředila cíleně na komplexní rozpracování konceptu personalizované farmakoterapie v onkologii pohledem klinického farmakologa, a to ve třech základních oblastech zahrnujících personalizovaný výzkum léčiv a jeho zejména farmakogenetické aspekty, s tím související problematiku optimálního designu klinických hodnocení a farmakoekonomiku včetně dopadu na úhradové mechanismy léčiv. Práce byla předložena k obhajobě a obhájena v říjnu 2015 na LF UK v Bratislavě v rámci akreditovaného oboru „Klinická farmakologie”, tedy oboru, který nebyl a není v současné chvíli akreditován pro habilitační a profesorská řízení na žádné z lékařských fakult v České republice. Spis, předkládaný k habilitačnímu řízení na LF MU v Brně, se již věnuje specifickým a rozšiřujícím výzkumným tématům z oblasti preklinické farmakologie v onkologii a psychofarmakologii a dále pak personalizaci protinádorové farmakoterapie ve vztahu k hostitelskému organizmu. Tento spis je potřeba vnímat jako celek, syntézu habilitační práce z roku 2015 (příloha I) a komentovaného souboru navazujících prací s cílovou snahou zasadit vlastní výzkumné aktivity v oblasti farmakologie do kontextu výuky předmětu Lékařské farmakologie na LF MU a představit tak inovativní komplexní pohled klinického farmakologa se zkušeností s preklinickým výzkumem i s dlouhodobou klinickou zkušeností na výuku farmakologie v době personalizované farmakoterapie, na kterou je nutno připravovat studenty již během studia medicíny. 4 Předmět Lékařská farmakologie je v současné době na LF MU vyučován v rámci pregraduálního studia všeobecného i zubního lékařství v 6. a 7. semestru studia. V souladu se stávající akreditací je věnován zejména pochopení základních principů farmakokinetiky a farmakodynamiky v rámci obecné farmakologie a dále pak mechanismům účinků jednotlivých lékových skupin v rámci studia speciální farmakologie. Byť jsou tyto znalosti zásadní a nepodkročitelné zejména v preklinické části studia, tak si během svého působení ve vedení Farmakologického ústavu od roku 2011 stále významněji uvědomuji, že nám chybí systematičtější výuka věnovaná aplikované klinické farmakologii a problémově orientované výuce farmakologie na příkladech personalizované farmakoterapie vybraných onemocnění. S farmakoterapií reálných pacientů se naši studenti potkávají samozřejmě v klinické části studia medicíny, kdy se jim v tomto směru dlouhodobě věnují kolegové, kliničtí lékaři, v jednotlivých oborech medicíny. Tato část patří a vždy bude patřit do curricula klinických oborů, nicméně i tak zůstává stále prostor pro modernější pojetí výuky oboru klinické farmakologie. S mírnou nadsázkou lze v obecné rovině říci, že nám ve výuce chybí „simulace farmakoterapie”. Tento model výuky, uplatňovaný v posledních letech zejména v anglosaských a skandinávských zemích a v zemích Beneluxu, jednoznačně přispívá k vyšší úrovni znalostí farmakologie a jejich aplikaci do reálné klinické farmakoterapie již v době studia medicíny. Závěrečný výstup v předkládané habilitační práci ve smyslu implikací těchto poznatků při výuce Lékařské farmakologie zohledňuje práce publikované v obou spisech tak, aby byly obsaženy prvky jak preklinických, tak klinických výstupů ve výuce pregraduální, ale i postgraduální Lékařské farmakologie. 5 3. ZÁKLADNÍ FARMAKOLOGIE JAKO TEORETICKÁ NAUKA A KLINICKÁ FARMAKOLOGIE JAKO INTEGRATIVNÍ DISCIPLÍNA S PŘESAHEM DO VŠECH KLINICKÝCH OBORŮ Historie farmakologie jako vědního oboru Počátky farmakologie jako exaktní samostatné vědy, zabývající se pochopením účinků látek na lidský organismus, byly položeny přibližně v polovině 19. století. Za zakladatele experimentální farmakologie je považován profesor Rudolf Buchheim (1820 - 1879), který se stal zakladatelem a prvním profesorem Ústavu farmakologie na univerzitě v Dorpatu v Estonsku. Jeho žák, profesor Oswald Schmiedeberg (1838 - 1921), je pak považován za zakladatele moderní farmakologie. V roce 1872 se stal profesorem farmakologie na univerzitě ve Strassburgu, kde ve svých výzkumech mimo jiné prokázal, že „muskarinove účinky” jsou srovnatelné s elektrickou stimulaci n. vagus a svůj objev publikoval v Outline of Pharmacology v roce 1878. Mimo Evropu byla první katedra farmakologie založena ve Spojených státech na Michiganské univerzitě v roce 1890 pod vedením Johna Jacoba Abeleho, rovněž žáka prof. Schmiedeberga. Od těch dob se farmakologie stala živým, otevřeným oborem, který zaznamenal obrovský 6 rozmach, a to zejména ve 20. století s ohledem na rozvoj dalších vědních oborů, zejména organické chemie a biologie (1). Jedním z příkladů té doby, který dokumentuje spolupráci na úrovni chemie, biochemie a farmakologie, je objev aspirinu, léku, který je stále jedním ze základních léčiv používaných v široké terapeutické praxi. Izolací salicinu byla připravena kyselina salicylová a z ní roku 1853 salicylan sodný. Ten však nebyl dobře snášen, působil zejména gastrointestinální obtíže a z tohoto důvodu jej nebylo možno pacientům dlouhodoběji podávat. V roce 1897 německý chemik Felix Hoffmann připravil esterifikovaný derivát kyseliny salicylové, následně jej podal několika pacientům s bolestmi kloubů a zaznamenal výborné terapeutické účinky, současně bez závažnějších nežádoucích účinků (2). Kyselina acetylsalicylová byla roku 1899 patentována pod obchodním jménem Aspirin. Dalším zajímavým objevem, založeným na poznatcích základního farmakologického výzkumu, byl objev β-sympatolytik. Teoretickým východiskem k objevu beta-blokátorů byl článek „A study of the adrenotropic receptors” Američana Raymonda P. Ahlquista, který byl publikován v roce 1948 v časopise American Journal of Physiology. Ahlquist v něm popsal existenci dvou druhů adrenergních receptorů – α a β. Práce byla řadu let ignorována, než byla existence dvou druhů adrenergních receptorů potvrzena experimentálními studiemi C. E. Powela a I. H. Slatera ve výzkumných laboratořích farmaceutické firmy Eli Lilly při pokusech s bronchodilatancii, konkrétně s isoproterenolem. Definitivní potvrzení však přinesl až vývoj prvního beta-blokátoru v polovině 60. let minulého století. První beta-blokátor propranolol (Inderal) vyvinul v roce 1964 geniální britský klinický farmakolog James W. Black, který se snažil anulovat škodlivé účinky adrenalinu a noradrenalinu na srdce (3). Spolu s chemikem J. S. Stephansonem testovali v laboratořích dnes již neexistující farmaceutické firmy ICI řadu látek, než se jim podařilo vyvinout propranolol. Brzy poté bylo doloženo, že tato látka významně snižuje mortalitu a morbiditu nemocných s anginou pectoris. Nemocní léčení propranololem měli po třech letech čtyřikrát nižší mortalitu na infarkt myokardu než nemocní bez léčby (3). Později byly také prokázány prospěšné účinky v léčbě arytmií a hypertrofické kardiomyopatie. Tyto úspěchy stimulovaly další farmakologický výzkum β-adrenergních receptorů, vedly k objevu β2receptorů v bronších a posléze k objevu selektivních β2-mimetik, která se stala důležitými bronchodilatancii. 7 Podstatné je také zmínit se o tom, že ani tehdejší Československo nezaostávalo ve výzkumu nových léčiv, přičemž nejvýznamnější a nejvyspělejší institucí v oblasti objevů a testování nových farmak byl u nás v letech 1951–1990 Výzkumný ústav pro farmacii a biochemii (VÚFB) v Praze, kde byla vyvinuta celá řada léčiv, některých dodnes využívaných v klinické praxi (příkladem Kinedryl, Mesocain, Prothiaden, Valetol nebo jedním z tehdy nejvýznamnějších pak Trimepranol). V sedmdesátých letech byl také zahájen výzkumný program protinádorových léčiv v brněnské firmě LACHEMA. V lékopisné kvalitě byla zvládnuta purifikace platinového komplexu s následnou výrobou lyofilizovaných injekcí PLATIDIAM obsahujících cisplatinu. V roce 1981 byla úspěšně dokončena syntéza methotrexátu, obdobně tomu bylo i v případě antidota leukovorinu, které pak umožnilo zavedení vysokodávkové chemoterapie methotrexátem. Výzkumný tým v krátké době zvládl i syntézu dacarbazinu a tamoxifenu, jejichž lékové formy úspěšně zavedl do výroby a registroval pro použití v klinické praxi (4). Dá se asi obecně říci, že ke konci 20. století, v návaznosti na obrovský pokrok v oblasti genetiky a rozvoji molekulárně-diagnostických metod, se výzkum a vývoj nových léčiv přesouvá směrem k biotechnologicky vyvíjeným léčivům, které mají mnohem komplexnější strukturu, vyšší molekulovou hmotnost, odlišnou farmakokinetiku i farmakodynamické cíle. Současný trend vývoje nových léčiv je charakterizován zejména posunem od „jednodušších, chemicky definovaných molekul” až k dnešní cílené léčbě a léčivým přípravkům pro moderní terapie, jako jsou somatobuněčná a genová terapie nebo produkty tkáňového inženýrství. Tento trend ve vývoji nových cílených léčiv sebou dnes ovšem přináší další výzvy, zejména vymezení jejich mnohem personalizovanějších indikací včetně specifik dávkování a dalších farmakologických principů. V tomto ohledu již farmakologie není pouze teoretickou exaktní vědou jako na svém začátku, ale aplikovanou lékařskou disciplínou, která vede k úspěšné farmakoterapii našich pacientů. 8 Klinická farmakologie a farmakoterapie v reálné klinické praxi Farmakoterapie a preskripce léčivých přípravků je zásadní dovedností, s níž se ve své klinické praxi potkávají v podstatě všichni kliničtí lékaři. Farmakoterapie a léky jsou zdaleka nejčastější formou léčebné intervence, ale také nejčastější příčinou iatrogenního poškození pacientů. Obecně lze říci, že léčivých přípravků preskribovaných lékaři v každodenní reálné klinické praxi přibývá a orientovat se v portfoliu léčiv pro dané onemocnění, znát principy mechanismů účinků, jejich nežádoucí účinky a potenciál klinických interakcí je stále náročnější. Nesmírně se navýšil počet registrovaných léčiv, dnes jich máme už přes jedenáct tisíc, a ani farmakolog nemůže zvládnout znát všechny detailně. Navíc se rychle mění názvy léků, na trh se uvádí velký počet „generických” léčiv a roli hraje i ekonomický tlak plátců péče. V tomto smyslu se v případě farmakologie již nejedná o vědu v užším slova smyslu, ale o translaci farmakologických poznatků do reálné klinické praxe. Je potřeba oprášit termín „účelné farmakoterapie”, což vnímám jako jedno z nejdůležitějších témat současné medicíny ve vztahu k farmakoterapii. Účelná farmakoterapie nikoliv tedy jako restriktivní a regulační prvek, nýbrž jako snaha o dosažení optimálního farmakoterapeutického zásahu přizpůsobeného konkrétnímu pacientovi nejen na základě diagnózy, ale i z hlediska jeho dalších chorob, souběžné medikace, věku nebo farmakogenetických variabilit. Zde vidím také opětovně se objevivší prostor pro renesanci oboru klinické farmakologie, aplikované to lékařské disciplíny, která musí dbát na to, aby se v době personalizované medicíny a stále cílenějšího zásahu farmak nevytratily z farmakoterapie prvky komplexnosti a kontextu reálného pacienta v každodenní lékařské praxi. Každý klinický lékař se s farmakoterapií potkává, a musíme počítat s tím, že do budoucnosti budou kladeny stále větší nároky na znalosti lékařů a větší důraz jejich potřebu se dále vzdělávat i v postgraduálním studiu. Tomu všemu musí předcházet výuka na pregraduálním stupni vzdělávání na lékařských fakultách. Tento aspekt si také dovolím sumarizovat ve 4. kapitole této habilitační práce s implikací vlastních preklinických i klinických farmakologických poznatků, které jsou detailněji diskutovány v kapitolách 3.1. a 3.2, do zejména pregraduální, ale i postgraduální výuky Lékařské farmakologie. 9 3.1 PŘEHLEDOVÁ ČÁST K PERSONALIZOVANÝM CÍLŮM VE FARMAKOTERAPII NÁDORŮ V této části si dovolím navázat na část A 2.2. (Personalizovaná medicína jako „targeted therapy”) v habilitačním spisu z roku 2015, který se věnoval výhradně klinicko-farmakologickému pohledu na personalizaci farmakoterapie (samotný spis je přílohou č. I této habilitační práce). Zmínila jsem zde, že je možno definovat několik atributů ve vztahu k cílovým strukturám a charakteristickým znakům vlastního nádoru, které se spolupodílí na výsledném terapeutickém efektu. Jako zásadní však stále vnímám nutnost holistického pohledu na farmakoterapii, neb stále platí základní farmakologický postulát, že „personalizovaně” musíme vnímat samozřejmě strukturu, na kterou lék působí – „target”, současně však nesmíme opomenout klíčový faktor hostitele – tedy jedince, kterému je léčivo podáváno se všemi jeho možnými specifiky včetně farmakogenetických a imunologických aspektů. V předloženém textu se budu dále věnovat farmakologickému pohledu na možné terapeutické cíle, ať již na úrovni receptoru nebo signální dráhy/drah, s komentářem vybraných publikací, které vznikly na základě vlastních preklinických experimentů nebo klinického výzkumu. V souladu s široce citovanou práci Weinberga a Hanahana (5,6) lze identifikovat deset základních charakteristik nádorové buňky („hallmarks of cancer”), na něž lze v dnešní době farmakologicky cílit, jsou tedy farmakologicky ovlivnitelné. Jsou jimi autokrinní produkce růstových faktorů, necitlivost k regulaci růstu, únik imunitnímu dohledu organismu, neomezený replikační potenciál, zánětlivá reakce, prolongovaná angiogeneze, invazivita do tkání, metastazování, potlačená apoptóza a deregulace buněčného metabolismu. Na tomto místě si dovolím zopakovat, že personalizace farmakoterapie se nemůže omezit pouze na cílení „hallmarks of cancer”, kromě identifikace terapeutického cíle na úrovni receptoru nebo signální dráhy sehrávají ve farmakoterapii nádorových onemocnění současně roli i proměnné ve vztahu k nádorovému mikroprostředí a zejména k nositeli onemocnění, tedy pacientovi. Neléčíme nádor, nýbrž pacienta s nádorem, je tedy nutno vnímat i komplexnost systémových faktorů ze strany pacienta, jimiž jsou jeho věk, pohlaví, komorbidity a komedikace, lékové interakce, nutriční stav, mikrobiom a životní styl, „farmakogenom”, tj. individuální biotransformační kapacita a konečně imunitní výbava pacienta. Koncept personalizované medicíny je tedy potřeba vidět souvztažně „all in one” – definování konceptu 10 personalizované medicíny buď jako „cílené terapie” nebo „cíleného dávkování” je bohužel obvyklou, ale nemedicínskou simplifikací problému (obr. 1, viz také habilitační spis 2015, 12-32). Obr. 1: Koncept personalizované terapie v kontextu konkrétního pacienta (systémové faktory pacienta a základní znaky maligní buňky, upraveno a adaptováno dle Hanahan and Weinberg, 2011) 3.1.1 PERSONALIZOVANÉ CÍLE V ONKOLOGII Z POHLEDU KLINICKÉ FARMAKOLOGIE A PREKLINICKÉM EXPERIMENTU V rámci pokračujících výzkumných aktivit, a to jak klinických v rámci svého působení v Masarykově onkologickém ústavu, tak nověji i preklinických v rámci výzkumné skupiny Farmakologického ústavu LF MU, se snažíme postihnout některé vybrané aspekty personalizované farmakoterapie, zejména směrem k možnostem farmakologického ovlivnění receptorů/signálních drah pro epidermální růstový faktor (EGFR, Her-2 neu), výzkumné aktivity v oblasti imunobiologie nádorů a problematiku miRNA jako možného prediktivního biomarkeru personalizované farmakoterapie (obr. 2). Základní znaky maligní buňky: autokrinní produkce růstových faktorů, necitlivost k regulaci růstu, únik imunitnímu dohledu organismu, neomezený replikační potenciál, zánětlivá reakce, prolongovaná angiogeneze, invazivita do tkání, metastazování, potlačená apoptóza a deregulace buněčného metabolismu Systémové aspekty hostitele: věk, pohlaví, genom, komorbidity, komedikace, životní styl, nutriční stav, mikrobiom, infekce, imunitní výbava 11 Obr. 2: možnosti farmakologického ovlivnění vybraných znaků maligní buňky (upraveno a adaptováno dle Hanahan and Weinberg, 2011) 3.1.1.1 VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE KLINICKÉ ČÁSTI Turjap M, Juřica J, Demlová R. Potential clinical benefit of therapeutic drug monitoring of imatinib in oncology (published in Klin Onkol. 2015; 28(2):105-111) Duchnowska R, Sperinde J, Czartoryska-Arlukowicz B, Mysliwiec P, Winslow J, Radecka B, Petropoulos C, Demlova R. et al. Predictive value of quantitative HER2 and HER3 levels combined with downstream signaling markers in HER2-positive advanced breast cancer patients treated with lapatinib (Meeting Abstract SABSC San Antonio, US. Published in Cancer Research. 2017;77(Suppl.4): 2-05-21. IF 9,122) Farmakologická léčiva s potenciálem ovlivňovat HER-2 receptor, ať již extracelulárně antagonizací vazebného receptorového místa nebo intracelulární inhibicí jeho tyrozinkinázové domény, jsou dnes stěžejní v léčbě Her-2 pozitivního karcinomu prsu. Jednou z látek, která prokázala v reálné klinické praxi zásadní klinický benefit ve smyslu prodloužení přežití, je trastuzumab (7). Trastuzumab antagonizuje receptor pro lidský epidermální faktor 2, který je součástí rodiny čtyř epidermálních receptorů ErbB. Po navázání ligandu na HER2 dochází k dimerizaci 12 a následné aktivaci kináz a spuštění rozdílných signálních cest, které mají za následek proliferaci, motilitu i indukci angiogeneze v nádorové tkáni. Aktivace HER2 vede cestou Ras-MAPK k buněčné proliferaci a inhibici apoptózy cestou PI3KAKT-mTOR (savčí rapamycinový receptor)(8). Další látkou, která ovlivňuje signální dráhu spojenou s epidermálním receptorem, je lapatinib. Lapatinib je potentní a selektivní reverzibilní duální inhibitor receptorů ErbB-1 (EGFR, receptor pro epidermální růstový faktor) a ErbB-2 (HER2). Jedná se o perorálně účinné léčivo s malou molekulovou hmotností, které zasahuje na úrovni intracelulárně umístěné tyrozinkinázové domény obou receptorů, a to kompetitivní vazbou na vazebné místo pro makroergní fosfát ATP. Zabraňuje tak autofosforylaci tyrozinových zbytků intracelulární části receptoru a následné aktivaci signálních drah, jež hrají zásadní roli v buněčném růstu (9). Trastuzumab, společně s imatinibem níže, si dovolím zařadit mezi klinicky nejúspěšnější cílená léčiva. Máme možnost je v klinické praxi používat již více než 15 let, postupem času se stala zlatým standardem v léčbě indikovaných pacientů. Současně si dovolím přiřadit mezi tyto látky i selektivní modulátor estrogenového receptoru tamoxifen, byť mezi cílenými léčivy nebývá zmiňován. Z farmakologického hlediska se ovšem rovněž jedná o cílené léčivo s primárně antiestrogenní aktivitou, modulující cílové místo receptoru, exprimovaného pouze u hormonálně pozitivních nádorů prsu. Další klinicky zásadně úspěšnou látkou je imatinib. Imatinib je perorální selektivní inhibitor tyrosinkinázy BCR-ABL, PDGFR-a i PDGFR-b a KIT. Jeho mechanismus účinku spočívá v kompetitivní inhibici aktivity výše zmíněných tyrosinkináz vazbou na místo určené pro adenosintrifosfát. S patologickou aktivitou kinázy ABL (spojenou s tvorbou fúzního genu BCR/ABL) je spojena chronická myeloidní leukemie (CML) a Ph+ akutní lymfoblastická leukemie (Ph+ ALL), v jejichž indikacích je úspěšně klinicky využíván (10). Imatinib je biotransformován cestou cytochromu P450, přičemž je známo množství významných lékových interakcí. Onkologičtí pacienti navíc často užívají současně množství dalších léčiv, zvyšujících pravděpodobnost takové interakce a svou roli může sehrávat i adherence k léčbě při dlouhodobém podávání. Režimy vycházející z fixního dávkování imatinibu nerespektují interindividuální rozdíly ve farmakokinetice léčiva a je možné, že někteří nemocní tak nedosahují dostatečných plazmatických koncentrací. Na základě evidence z klinických studií lze usuzovat, že existuje vztah mezi plazmatickými koncentracemi imatinibu a klinickou odpovědí (11). Imatinib se 13 proto jeví být vhodným kandidátem pro terapeutické monitorování jeho plazmatických koncentrací. Přehledový článek, který jsme publikovali v Klinické onkologii v roce 2015 (12), předkládá přehled o farmakokinetice, klinicky relevantních lékových interakcích, sumarizuje aktuální stav problematiky stanovení plazmatických koncentrací pro účely optimalizace terapie a dále popisuje možnosti, limity a návrhy pro terapeutické monitorování imatinibu, které chceme zavést v rámci činností klinicko-farmakologické jednotky Farmakologického ústavu LF MU. Další klinicko-výzkumné studie v oblasti personalizované farmakoterapie se věnují problematice klinicky závažných korelací mezi expresí příslušných receptorů/aktivace signálních drah a celkového přežití při léčbě lapatinibem. Předchozí publikované studie studovaly úlohu genů regulovaných estrogenním receptorem (ER) a receptory EGFR (13). Ve spolupráci s klinickými farmakology a onkology v rámci CEEOG (Central Eastern Europe Oncology Group) jsme publikovali výsledky studie zkoumající expresi receptorů ve vztahu k účinnosti lapatinibu (14). Hladiny exprese HER2 a HER3 byly kvantitativně měřeny za použití fluorescenčních metod, pro kvantifikaci hladin exprese HER2 proteinu byl použit test HERmark® (Monogram Biosciences, South San Francisco), exprese HER3 proteinu byla kvantifikována použitím technologie VeraTag® (Monogram Biosciences). Exprese „downstream” signalizačních proteinů (ER, PTEN, Cyklin E, HIF-2alfa, p-p70S6K, p-AMPK a p-MAPK) byla stanovována imunohistochemicky. Všechny hodnocené biomarkery byly korelovány s celkovým přežitím (OS) pacientů léčených lapatinibem a kapecitabinem po progresi na trastuzumabu (n=191). Analýzy celkového přežití byly následně korelovány s expresí HER2 a HER3 za dodržení stratifikačních parametrů (klinické stadium, přítomnosti mozkových metastáz). Parametr celkového přežití byl signifikantně kratší u pacientů pod mezní hodnotou pozitivity testem HERmark® (HR=1,8, p=0,029), a u pacientů s vyššími mediánovými hladinami HER2 (HR=1,7, p=0,009). Zvýšený HER3 vykazoval trend ke korelaci s delším celkovým přežitím (HR=0,66/log; p=0,16), významněji u hormonálně negativních pacientek. Lze vyvozovat, že u těchto pacientek může sehrávat inhibice HER2 a aktivace HER3 významnější roli. Na základě výsledků lze usuzovat, že pacienti s mírně zvýšenou hladinou HER2 mohou mít lepší výsledky a klinicky lépe profitují z podávání lapatinibu po progresi trastuzumabu než ti, kteří mají vysokou expresi HER2. Tento názor podporuje také 14 nedávné publikované výsledky o menším přínosu lapatinibu u pacientů s vysokou expresí HER2 (15). Zdá se, že hladiny HER3 prognózu pacientů významně neovlivňují. Výsledky byly publikovány formou konferenčního abstraktu v prosinci 2016 v rámci San Antonio Breast Cancer Symposium a publikovány v Cancer Research 2017. 3.1.1.2 VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE V PREKLINICKÉM EXPERIMENTU Duchnowska R, Wysocki PJ, Korski K, Czartoryska-Arłukowicz B, Niwińska A, Orlikowska M, Radecka B, Studziński M, Demlova R at al. (CEEOG). Immunohistochemical prediction of lapatinib efficacy in advanced HER2positive breast cancer patients (published in Oncotarget. 2016; 7(1):550-564. IF 5,168) Jak již bylo zmíněno výše, lapatinib inhibuje tyrozinkinázové domény receptorů ErbB-1 (EGFR, receptor pro epidermální růstový faktor) a ErbB-2 (HER2) na intracelulární úrovni (9). V rámci spolupráce s výzkumnou skupinou CEEOG (Central Eastern Europe Oncology Group) na preklinickém výzkumu možných mechanismů vzniku rezistence při podávání lapatinibu, včetně možných interakcí signalizačních drah, jsme zkoumali expresi vybraných proteinů zapojených do signalizačních cest rodiny ErbB. Byla analyzována imunohistochemická exprese fosforylovaného proteinu aktivovaného adenosinmonofosfátem (p-AMPK), proteinkinázy aktivované mitogenem (p-MAPK), fosfo (p) -p70S6K, cyklin E, fosfatázy a tensinového homologu na 270 nádorových vzorcích. Následně byla získaná data korelována s klinickými údaji vývoje onemocnění (16). Exprese p-p70S6K byla nezávisle spojena s delším (HR 0,45, 95% CI:0,25-0,81, p=0,009) a cyklinem E s kratším přežitím bez známek onemocnění (HR 1,83, 95% CI:1,06-3,14, p=0,029). Exprese p-MAPK (HR 1,61, 95% CI:1,13-2,29, p=0,009) a cyklin E (HR 2,99, 95% CI:1,29-6,94, p=0,011) koreluje s kratším a exprese receptoru estrogenu HR 0,65, 95% CI:0,43-0,98, p=0,041) s delším celkovým přežitím. Exprese p-AMPK negativně ovlivnila odpověď na léčbu (HR 3,31; 95% CI:1,48-7,44; p=0,004) a kontrolu onemocnění (HR 3,07; 95% CI: 1,25-7,58; p=0,015). Z výsledků lze usuzovat na skutečnost, že účinnost lapatinibu souvisí rovněž s aktivitou dalších signálních drah - AMPK/mTOR a Ras/Raf /MAPK. 15 Pokračování v dalších výzkumných aktivitách zaměřených tímto směrem by mohl přispět k posouzení klinické užitečnosti kombinace lapatinibu s inhibitory MAPK, spolupráce v rámci CEEOG skupiny s naší účastí i nadále pokračuje. 3.1.2 IMUNOLOGICKÉ ASPEKTY NÁDORŮ VE VZTAHU K PROTINÁDOROVÉ FARMAKOTERAPII Primárním úkolem imunitního systému je ochrana před cizorodými mikroorganizmy a látkami z prostředí. Nádorové buňky vznikají z tělu vlastních buněk, obecně jsou tak pouze slabě imunogenní a je zřejmé, že imunitní dozor organizmu není schopen nádorovému bujení vždy zabránit. Je dokonce známo, že v prvních fázích vzniku nádoru nebo při jeho metastazování může vlastní produkce růstových faktorů nádorovým buňkám napomáhat v jejich růstu. Stimulace imunitního systému může být výraznější v případě nádorů spojených s infekcí onkogenními viry, případně tehdy, pokud nádor začne na svém povrchu produkovat změněné nebo nezvyklé molekuly, které imunitní buňky rozpoznávají jako cizí. Obecně pak hovoříme o imunogenních nádorech, mezi něž patří například maligní melanom, vznikající na kůži nebo sliznicích ze zhoubně přeměněných melanocytů buněk, které jsou v kůži nebo ve sliznici v jistém smyslu „cizorodé”. Nádorové antigeny jsou často buňkou prezentovány v komplexu s MHC I, což umožňuje rozeznání nádorové buňky cytotoxickým T-lymfocytem. Procesem fagocytózy je odumřelá nádorová buňka odstraněna, nádorové antigeny jsou vystaveny MHC II a může dojit ke stimulaci pomocných T-lymfocytů a protilátkové odpovědi. V rámci diskuse o existenci imunitního boje proti nádorovým buňkám je třeba zmínit teorii protinádorového dohledu, která rozlišuje tři odlišné úrovně v procesu boje imunitního systému proti nádorovým buňkám -eliminaci transformované buňky, ustanovení rovnováhy mezi transformovanou buňkou a organismem a únik transformované buňky před kontrolou imunitního systému. Ve většině případů je nádorová buňka v časných stadiích transformace rozpoznána a imunitním systémem zničena. Celý proces zde může skončit nebo přejít do dalších fází. Ve fázi ustanovení rovnováhy je hostitelský imunitní systém a přežívající nádorové buňky ve stadiu dynamické rovnováhy. Tato fáze rovnováhy je z oněch tří popsaných procesů nejdelší a klinicky se nejvíce shoduje s pre-neoplastickým stadiem, které rovněž zůstává nejčastěji nediagnostikované. Poslední fáze je fáze úniku transformované buňky před kontrolou imunitním systémem, vzniká 16 imunoprotektivní nádorové mikroprostředí, dochází tak k růstu nádoru a jeho klinické manifestaci. Je nutno poznamenat, že svou roli sehrává i stav imunity nositele nádoru – pacienta, kdy celková imunosuprese pacienta přispívá ke vzniku imunosupresivního mikroprostředí protektivního pro nádorové buňky. Poznání, že nádory mohou aktivovat regulační mechanismy, které potlačují imunitní odpověď, vedla k novým nadějným imunoterapeutickým přístupům. Principem této strategie může být blokáda inhibičních signálů pro lymfocyty. Monoklonální protilátka proti CTLA-4 a protilátky proti receptoru/ligandu programované buněčné smrti (PD(L)1) jsou již schváleny pro léčbu melanomu, v klinických studiích je celá řada dalších protilátek s obdobným farmakologickým mechanismem účinku (17). Do této kategorie nakonec můžeme zařadit i rituximab, jehož farmakologickým mechanismem účinku je indukce ADCC a CDC vedoucí k léčebné odpovědi. Dalšími ze způsobů, jak stimulovat protinádorovou imunitu, je například tzv. „adoptivní buněčná imunoterapie” pomocí autologních dendritických buněk vystavených nádorovému antigenu a následné použití těchto dendritických buněk jako nádorově pulsní vakcíny (18). I tento přístup je v současnosti předmětem klinických studií včetně vlastní klinické studie LF MU fáze I/II (viz 3.1.2), data z interim-analýzy potvrzují bezpečnost protinádorové vakcíny, data o účinnosti jejich terapeutický přínos budou publikována až po závěrečné analýze klinické studie. 3.1.2.1 VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE V KLINICKÉ ČÁSTI Demlova R., Valik D., Obermannova R., Zdrazilova-Dubska L. The safety of therapeutic monoclonal antibodies: implications for cancer therapy including immuno-checkpoint inhibitors. (published in Physiol Res. 2016; 65(Suppl.4):455-462. IF 1,461) Bilek O, Bohovicova L, Demlova R, Poprach A, Lakomy R, Zdrazilova-Dubska L. Non-Small Cell Lung Cancer-from Immunobiology to Immunotherapy. (published in Klin Onkol. 2016; 29(Suppl.4):78-87) Léčba založená na monoklonálních protilátkách proti kontrolním bodům imunitní reakce je dnes považována za průlomovou metodu v onkologii. Výsledky s anti- 17 CTLA-4 (cytotoxický T-lymfocytární antigen-4) protilátkou ipilimumabem u pokročilého maligního melanom znamenaly doslova revoluci v protinádorové imunoterapii a byly impulzem pro vývoj nových protilátek zaměřených na další kontrolní molekuly (tzv. „checkpoints”) (19,20). Z nich je nutné zmínit protilátky proti receptoru programované buněčné smrti (PD-1) – nivolumab, pembrolizumab, pidilizumab a proti jeho ligandu (PD-L1) – BMS-936559 a MPDL3280A (atezolizumab) (21-25). Výhodou těchto léčiv je kromě jejich účinnosti také jejich potenciální „imunoterapeutická” univerzálnost, což je zásadní pro široké využití u řady malignit. Vývoj a použití imunoterapie dosáhly zatím největších pokroků u pokročilého melanomu a nemalobuněčného karcinomu plic (26), v současnosti pak probíhá řada dalších klinických studií například u nádorů hlavy a krku, nádorů ledvin, ureteliálních karcinomů a řady dalších malignit. Princip účinku „checkpoint” inhibitorů (anti-CTLA-4, anti-PD-1 a anti-PD-L1 protilátek) spočívá v blokádě inhibičních receptorů na buňkách imunitního systému nebo nádoru, a tím prolomení tolerance imunitního systému vůči nádoru. Ipilimumab působí spíše na „centrální úrovni” v orgánech lymfatického systému. Zde, po prezentaci antigenů dendritickými buňkami, blokádou inhibičního CTLA-4 receptoru, udržuje aktivaci T lymfocytů, ty pak putují do nádorové tkáně, kde plní svoji funkci. Protilátky bránící inhibiční interakci mezi PD-1 a PD-L1/L2 naopak sekundárně potencují efektorovou složku imunity „periferně” přímo v nádorové tkáni. Prolomení tolerance vůči nádoru však může být doprovázeno i nežádoucím prolomením tolerance vůči „normálním tkáním”, což vede k vedlejším účinkům, které se svým charakterem blíží autoimunitním onemocněním – tzv. imunitně podmíněné vedlejší účinky (immune related adverse events, ir-AEs). Přehled nejčastějších ir-AEs, k nimž patří kožní toxicita (exantém, pruritus), GIT toxicita (průjem, kolitida), endokrinní toxicita (hypopituitarizmus, hypofyzitida, hypotyreóza, insuficience nadledvin), jaterní toxicita (elevace transamináz, hepatitida), u anti-PD-1 protilátek pneumonitida, včetně managementu toxicit, jsme publikovali v přehledovém článku ve Physiological Research (27). Incidence ir-AEs je vyšší u ipilimumabu (v závislosti na dávce), ve srovnání s anti-PD- 1/ PDL1 protilátkami. Ještě vyšší je u kombinace ipilimumabu a nivolumabu. Vzhledem k vysoké četnosti ir-AEs s rizikem rozvoje život ohrožujících komplikací je nezbytná dostatečná edukace pacienta i odborných lékařů. Byla vypracována řada doporučení, jak při podezření na ir-AEs postupovat. Časné zahájení 18 imunosupresivní léčby kortikosteroidy je zásadním krokem ke zvládnutí příhody, snížení morbidity a případně i mortality. Pokud nejsou kortikoidy dostatečně účinné, přidávají se další imunosupresiva jako infliximab nebo mykofenolát mofetil. Úspěch ipilimumabu u pokročilého melanomu ukázal nové možnosti v léčbě nádorových onemocnění. Imunitně podmíněné vedlejší účinky a jejich vážné komplikace zpočátku vedly k velkým obavám a k rezervovanému postoji u řady onkologů. S postupným získáváním zkušeností však lze toxicitu řešit a při dodržování doporučených postupů je dnes léčba těmito léčiva bezpečná, respektive provázena nežádoucími účinky, které jsou terapeuticky zvládnutelné. 3.1.2.2 VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE V PREKLINICKÉM EXPERIMENTU Klinické hodnocení fáze I/II „Kombinovaná protinádorová terapie s ex vivo manipulovanými dendritickými buňkami produkujícími IL-12 u dětských, adolescentních a mladých dospělých pacientů s progredujícími, relabujícím nebo primárně metastatickými malignitami vysokého rizika“ EudraCT Number:2014-003388-39, zadavatel: LF MU, Farmakologický ústav, odpovědný řešitel LF MU doc. Demlová, Klinika dětské onkologie FN Brno, řešitel prof. Štěrba Zdrazilova-Dubska L, Fedorova L, Pilatova K, Mudry P, Hlavackova E, Matoulkova E, Flajsarova L, Demlova R, Valik D, Sterba J. TKI pazopanib impaires immunostimulatory properties of monocytes: Implication for monocyte-derived DC-based anticancer vaccine preparation (Meeting Abstract, ESMO Immunooncology 2016. Published in Ann Oncol. 2016;27(Suppl.8):18P. IF 11,855) V rámci výzkumné činnosti ACIU (Advanced Cell Immunotherapy Unit) Farmakologického ústavu LF MU se se věnujeme výzkumu a vývoji vakcíny z dendritických buněk (DC vakcína). Takto vyvíjené léčivo somatobuněčné terapie patří mezi tzv. léčivé přípravky pro moderní terapie (ATMP – Advanced Therapy Medicinal Product), kategorie léčiv s významnou regulací na úrovni Evropské 19 lékové agentury (EMA – European Medicines Agency), a to včetně požadavků na provádění klinických studií a výrobu hodnoceného léčivého přípravku. Výroba DC vakcín se řídí správnou výrobní praxí dle GMP (The Rules Governing Medicinal Products in the European Union, Volume 4, Good Manufacturing Practice) a dozor nad výrobní činností provádí SÚKL (Státní ústav pro kontrolu léčiv). Lékařská fakulta Masarykovy univerzity v Brně je držitelem oprávnění k výrobě protinádorových vakcín z autologních dendritických buněk, což je v akademických podmínkách nejen v ČR výjimečný počin. V tomto bodě si dovolím poděkovat vedení Lékařské fakulty MU v Brně za významnou podporu naší práce na poli somatobuněčných terapií. Při vývoji a výrobě DC vakcíny se monocyty periferní krve pacientů vybraných pro imunoterapii získávají v průběhu leukaferézy. Z monocytů se pak připraví velké množství nezralých dendritických buněk v přítomnosti cytokinů GM-CSF a IL-4. Po pěti dnech dojde k diferenciaci monocytů v nezralé dendritické buňky. Další postup závisí na typu nádorového antigenu, který je pro imunoterapii zvažován. Kritickou podmínkou k zařazení do naší klinické studie (viz dále) je odběr vitální nádorové tkáně pacienta (při plánované operaci či reoperaci), která je zpracovávána spolu s autologně leukafereticky získanými leukocyty v čistých prostorách ACIU. Po pohlcení lyzátu nádorových buněk jsou následně dendritické buňky aktivovány a zralé dendritické buňky jsou poté jako protinádorová vakcína podány zpět pacientovi. Výše uvedené imunoterapeutické postupy jistě předpokládají adekvátní pre-existující imunitu a/nebo adekvátní funkci imunitního systému, kdy manipulace imunitního systému spočívá pouze v prezentaci nádorových antigenů v kontextu nespecifické stimulace imunitního systému. Léčivý přípravek je dále specifický tím, že obsahuje živé buňky, nemůže na svém konci projít procesem sterilizace a musí být vyráběn v tzv. čistých prostorách (ČP v kategorii nejvyšší čistoty A/B). Výstupní kontrola kvality DC vakcíny je zaměřena na imunobiologické vlastnosti léčivého přípravku (diferenciace do DC, maturace, schopnost produkovat aktivační cytokiny, schopnost stimulovat imunitní systém v MLR) a na ověření neinfekčnosti (sterilita, nepřítomnost Mycoplasma spp.). Díky tomu, že se v rámci ACIU Farmakologického ústavu LF MU podařilo provést nutné preklinické experimenty a dokončení procesu validace, bylo možno přistoupit k zahájení akademicky iniciované klinické studie. Protokol klinického hodnocení byl schválen Státním ústavem pro kontrolu léčiv, studie je 20 spolufinancována ze zdrojů velké výzkumné infrastruktury CZECRIN (LM2015090), hodnoceným léčivým přípravkem (IMP) je pak vlastní autologní vakcína jako léčivý přípravek moderní somatobuněčné terapie. Studie s názvem „Kombinovaná protinádorová terapie s ex vivo manipulovanými dendritickými buňkami produkujícími interleukin-12 u dětských, adolescentních a mladých dospělých pacientů s progredujícími, relabujícími nebo primárně metastatickými malignitami vysokého rizika” (EudraCT 2014-003388-39, KDO_DC1311) probíhá ve spolupráci s Fakultní nemocnicí Brno, konkrétně na pracovišti Kliniky dětské onkologie pod vedením prof. J. Štěrby a hlavního řešitele prim. P. Múdrého, kde jsou i pacienti v rámci klinického hodnocení léčeni. Primárním cílem studie je prokázání bezpečnosti, sekundárním cílem pak získání pilotních dat o předběžné účinnosti. Součástí sledování pacientů v klinickém hodnoceni KDO_DC1311 je i podrobný imunomonitoring cirkulujících elementů periferní krve. Imunomonitoring, který byl navržen doc. L. Zdražilovou-Dubskou (viz také její habilitační práce Imunobiologické aspekty nádorových onemocnění, Brno 2016), z našeho výzkumného týmu, zahrnuje kvantitativní stanovení MDSC (myeloid-derived stem cells); T-reg; subpopulaci monocytů; maturační a aktivační subsety T-lymfocytů a subsety T-lymfocytů dle exprese ICOS, PD-1, Tim-3; NK-buňky a NKT-like buňky včetně aktivačních znaků; γδ T-lymfocyty včetně subsetů a exprese NKG2D. V rámci interim-analýz (první proběhla po léčbě 5. a druhá po léčbě 10. pacienta léčených dle protokolu klinické studie) vyvstává postupně řada dalších výzkumných otázek ve vztahu DC vakcíny, stavu pacienta ve smyslu jeho preexistující imunity, souběžné onkologické terapie včetně analýzy konkrétních léčiv, které mohou s imunoterapií interferovat. Jedním z těchto léčiv je u dětí „offlabel” podávaný pazopanib. Jedná se o malou molekulu, tyrozinkinázový inhibitor receptorů EGF, PDGF a c-kit, což umožňuje jeho protinádorovou aktivitu zásahem signální trasy pro neoangiogenezi. V klinických studiích u dospělých pacientů prokázal účinnost u metastazujícího světlobuněčného karcinomu ledviny a je registrován k léčbě 1. linie u nepředléčených pacientů a ve 2. linii po selhání cytokinů. Jak jsem zmínila výše, je podáván cíleně v režimu „off-label” i u dětských onkologických pacientů. K posouzení vlivu pazopanibu na biologické funkce monocytů jsme analyzovali periferní krev dětských pacientů léčených pazopanibem a současně zařazených do klinické studie s DC vakcínou. Byla 21 kvantifikována exprese povrchových a intracelulárních molekul, přičemž dvě z osmi vakcín na bázi DC nesplnily imunobiologická „quality-control” kritéria, jako je exprese kostimulační molekuly a produkce IL-12 a nevykazovaly žádnou stimulační schopnost vůči autologním T-lymfocytům. Monocyty vystavené pazopanibu měly zvýšenou expresi CD64 a inhibiční molekulové ILT3, vykazovaly zhoršené imunobiologické vlastnosti relevantní pro DC stimulační funkce. Tyto výsledky jsme zveřejnili formou konferenčního abstraktu v rámci ESMO Immuno-Oncology Symposia v Lausanne s publikací v Annals of Oncology 2016 (28). 3.1.3 PERSONALIZOVANÁ MEDICÍNA V KONTEXTU HODNOCENÍ ODPOVĚDI DLE „BIOMARKERŮ“ – ÚVOD K VÝZNAMU miRNA Studium farmakogenetiky a designování farmakogenetických studií přináší problémy při zkoumání takových jevů, jako jsou normální či abnormální reakce osoby na daný podnět. Důležitým přístupem, jak možno řešit obdobné problémy, je stanovování cílových parametrů biochemické a/nebo genetické povahy, které mohou být měřeny v krátkých časových intervalech. Při návrhu vhodného biomarkeru je potřeba, aby byl dostatečně selektivní vůči sledovanému jevu, měřitelný v dobře dostupném biologickém materiálu a měla by u něj existovat minimální pravděpodobnost spontánní změny. Používaný biomarker by tak mohl být využit jako minimálně invazivní a nenákladný diagnostický test k určení fenotypu jedince a určení, zda je daný fenotypový znak determinován geneticky nebo vyvolán působením exogenních anebo endogenních vlivů. V onkologii jsme schopni využít biomarkerů k uplatnění možností individualizované medicíny ve všech oblastech zdravotní péče: v prevenci (identifikace osob a populací se zvýšeným rizikem vzniku nádorových onemocnění), v diagnostice (diagnostika a staging nádorů, diagnostika toxicity léčby) i v léčbě (individualizace protinádorové léčby na základě rozsahu a fenotypu nádorového onemocnění a prognózy pacienta). V tomto kontextu vhodných diagnostických, prediktivních a prognostických biomarkerů je v posledním desetiletí živě diskutována role miRNA. MikroRNA (miRNA) tvoří velkou skupinu krátkých nekódujících RNA posttranskripčně regulujících genovou expresi. Schopnost miRNA inhibovat translaci onkogenů a nádorových supresorů dává předpoklad jejich zapojení do procesů kancerogeneze, související se samotným vznikem nádorové buňky. Intenzivní výzkum v oblasti biogeneze a biologických funkcích miRNA přináší také poznatky, 22 že miRNA sehrává svou úlohu nejen v oblasti nádorové biologie, ale také v oblasti diagnostické a prediktivní onkologie. Analýza expresních profilů miRNA je proto stále častěji využívána pro účely molekulární diagnostiky nádorových onemocnění, analogicky, jako je tomu u studií založených na DNA čipech a profilování kódujících transkriptů. V kontextu publikovaných studií lze také usuzovat na schopnost vybraných miRNA sloužit jako tkáňové prognostické a prediktivní markery a potenciální terapeutické cíle, zejména u kolorektálního karcinomu, renálního karcinomu a glioblastomu. 3.1.3.1 VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE miRNA V PREKLINICKÉM EXPERIMENTU Merhautová J, Vychytilová-Faltejsková P, Demlová R, Slabý O. Systemic administration of miRNA mimics by liposomal delivery system in animal model of colorectal carcinoma (published in Physiol Res. 2016; 65(Suppl.4):481-488. IF 1,461) Merhautova J, Demlova R, Slaby O. MicroRNA-Based Therapy in Animal Models of Selected Gastrointestinal Cancers (published in Front Pharmacol. 2016;7(329):1-21. IF 4,4) V rámci preklinické výzkumné skupiny Farmakologického ústavu LF MU a ve spolupráci s výzkumnou skupinou Molekulární onkologie II – solidní nádory CEITEC vedenou doc. RNDr. O. Slabým, Ph.D., jsme se věnovali translaci in vitro testů studujících vybrané miRNA v in vivo podmínkách experimentu na zvířeti. MiR-215 je rozpoznaným nádorovým supresorem u kolorektálního karcinomu (CRC). Její biologické účinky a molekulární cíle byly studovány v široké škále in vitro testů na buněčných liniích odvozených od CRC a v rámci těchto experimentů byly skupinou doc. Slabého potvrzeny její tumorově supresorové účinky (29). Vzhledem k hypotetickému terapeutickému potenciálu bylo přikročeno k pokračování výzkumu in vivo. Cílem našich experimentů s pomocí animálních modelů solidních nádorů bylo zejména ověření hypotézy, zda má podávání inhibitorů/prekurzorů terapeutický potenciál včetně jejich farmakologických charakteristik. Pracovní hypotéza pracovala s úvahou navýšení hladin miR-215 v nádorových buňkách a hodnocení tohoto vlivu na změnu jejich fenotypu především ve smyslu snížení 23 proliferace in vivo. V testu tumorigenicity se prokázalo, že stabilně transfekovaná linie HCT-116+/+ s navýšenou expresí miR-215 proliferuje výrazně pomaleji, což se odrazilo na rozdílu objemu tumorů ve srovnání s kontrolní linií. Díky pozitivním výsledkům této studie jsme přistoupili k uspořádání navazujícícho experimentu s modelací eventuálního terapeutického podání u člověka. Pro systémové podání miRNA mimics byl zvolen intravenózní přístup a léková forma liposomů, která byla již několikrát pro dopravení miRNA mimics do tumoru použita (30,31). Z hlediska orgánové distribuce jsme zaznamenali signifikantně zvýšenou expresi v plicích zvířat. Výsledky této studie tak podporují hypotézu plicní akumulace liposomů vytvořených z emulze neutrálních lipidů. Ačkoli se část dávky do tumoru dostala, nebyla dostatečná k tomu, aby se projevil významný tumorově supresorový účinek. Vzhledem k tomu nedošlo ani ke změnám v růstu tumorů, což je velmi pravděpodobně způsobeno zvolenou lékovou formou, kdy liposomy v dostatečném množství neprostupují do nádorové tkáně, ale akumulují se jinde v organismu. Všechny inovativní nosiče léčiv se musí vypořádat s interakcemi s plazmatickými proteiny, s vychytáváním RES a dalšími procesy, které vedou k jejich eliminaci. S přihlédnutím k našim výsledkům lze tedy usuzovat, že NLE liposomy se kumulují v plicní tkáni a byly by tak vhodným nosičem léčiv pro plicní onemocnění. Výsledky byly publikovány v roce 2016 ve Physiological Research (32). Využití NLE liposomů pro dopravu miRNA mimics do nádorů střeva tak bude nutné buď podstatným způsobem optimalizovat (dávka, způsob podání, typ animálního modelu), nebo opustit jako slepou uličku a pokusit se vybrat vhodnější nosič, kdy je v plánu in vitro a in vivo testování nanočástice oxidu železnato-železitého (SPIONs) potažené chitosanem. O této problematice jsme rovněž publikovali přehledový článek, zaměřený na mapování dostupných informací o farmakokinetice a toxicitě terapie založené na miRNA, včetně informací o použitém zvířecím modelu, farmakologických aspektech (chemie oligonukleotidů, systém dodávání, dávkování, cesta podání) a toxikologickém hodnocení (33). 24 3.1.3.2 VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE Z POHLEDU KLINICKÉ FARMAKOLOGIE Merhautova J, Hezova R, PoprachA, Kovarikova A, Radova A, Svoboda M, Vyzula R, Demlova R, Slaby O. miR-155 and miR- 484 Are Associated with Time to Progression in Metastatic Renal Cell Carcinoma Treated with Sunitinib (published in Biomed Res Int. 2015; ID941980. IF 2,476) Merhautova J, Hezova R, Poprach A, Svoboda M, Demlova R, Slaby O. MiRNA associated with time to progression in metastatic renal cell carcinoma patients treated with sunitinib (Meeting Abstract, European Cancer Congress 2015. Published in Eur J Cancer. Sep 2015;51(Suppl.3): S505. IF 6,029) Klinicky orientovaná studie si stanovila jako primární cíl korelaci miRNAbiomarkerů predikujících odpověď na léčbu sunitinibem u renálního karcinomu. Studie byla navržena jako retrospektivní biomarkerová klinická studie a její protokol včetně Informovaného souhlasu schválila Etická komise Masarykova onkologického ústavu (MOÚ). Jednalo se o pacienty Masarykova onkologického ústavu, kde v rámci působení výzkumné skupiny Farmakologického ústavu LF MU na Oddělení klinických hodnocení analyzujeme klinická data pacientů. Sunitinib patří mezi tzv. „small drugs”, látky s malou molekulovou hmotností, jehož farmakologickým mechanismem účinku je inhibice tyrozinkináz receptorů pro růstový faktor z destiček (PDGFR-a a PDGFR-b), receptorů pro vaskulární endoteliální růstový faktor (VEGFR-1, VEGFR-2 a VEGFR-3) a receptorů faktoru kmenových buněk (cKit). Primární indikací je metastazující karcinom ledviny, přičemž klinickým problémem je vývoj rezistence na léčivo s následným selháním léčby a progresí onemocnění. Vývoj rezistence je jistě multifaktoriální, roli mohou sehrávat jak farmakokinetické (hladina léčiva v krvi, aktivní metabolit SU012662), tak farmakodynamické faktory související s cílovým místem v nádorové buňce. Jedním z uvažovaných faktorů může být také role miRNA, kterou jsme v naší studii hodnotili ve vztahu k léčebné odpovědi na sunitinib. Retrospektivně jsme analyzovali nádorovou tkáň pacientů léčených sunitinibem a provedli korelaci ve vztahu k vybraným miRNA (miR-155, miR-374-5p, miR324-3p, miR-484, miR- 25 302c a miR-888) ve vzorcích nádorových tkání. Kaplanova-Meierova analýza prokázala, že dvě z vybraného souboru miRNA signifikantně korelují s časem do progrese onemocnění, a to miR-155 (medián TTP 5,8 vs. 12,8 měsíců, p < 0,01) a miR-484 (5,8 vs. 8,9 měsíců, p < 0,05). U ostatních miRNA nebylo dosaženo statisticky významného rozdílu. Stratifikace pacientů na základě analýzy miRNA by umožnila personalizovanější přístup při léčbě metastatického karcinomu ledvinových buněk. Výsledky byly publikovány v roce 2015 v Biomedical Research a v rámci konferenčního abstraktu v European Journal of Cancer (34,35). 3.2 PŘEHLEDOVÁ ČÁST – ÚVOD K METABOLICKÉMU SYNDROMU A ENDOKRINOLOGICKÉMU STATUTU Metabolický syndrom je dnes široce diskutován jako jeden z velkých medicínských i ekonomických problémů současné doby svázaný s životním stylem ve vyspělých zemích. Je považován za jeden z nejvýznamnějších rizikových faktorů vzniku kardiovaskulárních onemocnění a diabetu mellitu 2. typu, stále častěji je také zmiňována jeho možná role ve vztahu k nádorovým onemocněním. Kombinace dyslipidemie, obezity, arteriální hypertenze, inzulinové rezistence a dalších složek jednoznačně zvyšuje riziko následných zdravotních komplikací. Předpokládá se, že etiopatogenetický mechanismus řady složek metabolického syndromu je společný a souvisí s tukovou tkání. Je známo, že tuková tkáň není složena pouze z diferencovaných adipocytů, na její stavbě se podílí mnoho dalších druhů buněk, jako jsou endotelie, fibroblasty, myocyty, nediferencované adipocyty (preadipocyty) a imunokompetentní buňky, zejména monocyty a lymfocyty (36). Je aktivním sekrečním orgánem produkujícím řadu látek s autokrinním, parakrinním nebo endokrinním účinkem. Za patofyziologický podklad metabolického syndromu je obecně považována inzulínová rezistence, důležitou roli sehrává také dysfunkce tukové tkáně a dysregulace imunitního systému vedoucí k akumulaci makrofágů stimulujících chronický zánět tukové tkáně. Adipocyty, makrofágy a další součásti tukové tkáně produkují ve zvýšené míře prozánětlivé působky (např. TNF-alfa, IL-6) a některé hormony tukové tkáně, nazývané souhrnně adipokiny (37,38). Mezi nejznámější popsané adipokiny patří leptin, adiponektin nebo visfatin. V periferních tkáních pak dochází ke změnám na subcelulární úrovni, poruchám signalizační kaskády inzulínového receptoru, což může přispívat k rozvoji inzulínové rezistence. 26 3.2.1 METABOLIKCÝ SYNDROM, ZÁNĚT A NÁDOROVÁ ONEMOCNĚNÍ Z POHLEDU KLINICKÉ FARMAKOLOGIE V souvislosti s inzulínovou rezistencí, obezitou nebo aterosklerózou se nyní mluví o chronickém zánětlivém stavu nebo metabolicky indukovaném zánětu (39). Chronický zánět je, jak bylo diskutováno již v rámci habilitační práce 2015 (příloha č. 1), jedním z faktorů, které se mohou spolupodílet na vzniku a rozvoji nádorových onemocnění. Působení TNF-alfa je spojeno s chronickým zánětem a podporuje nádorovou angiogenezi a metastazování (40). K metabolizaci a proliferaci maligní buňky přispívá také dostupnost lipidů a dalších makromolekul a inzulinová signalizace. Inzulin, IGF-1, IGF-2 jsou ligandy povrchových receptorů (INSR, IGF1R, IGF2R), jejich aktivace vede k posílení signální transdukce, buněčné proliferaci a inhibici apoptózy (41). Kromě dysfunkce tukové tkáně a dysregulace imunitního systému ve vztahu k zánětu hraje u nádorových onemocnění roli celkový endokrinologický statut pacienta (42). Všechny tyto aspekty je nutno vidět opět souvztažně, s čímž se pojí i systémový pohled na možné ovlivnění metabolického syndromu a jeho komponent včetně možné prevence a ovlivnění průběhu nádorových onemocnění. 3.2.2 METABOLICKÉ ZMĚNY A ROLE ADIPOKINŮ V PREKLINICKÉM EXPERIMENTU NA ZVÍŘETI Kromě všech výše uvedených možných aspektů uvedených v kapitole 3.2.1 je v současné době diskutována i možná souvislost metabolického syndromu s některými psychiatrickými poruchami, zejména u schizofrenie a deprese. Intenzivně se studuje hypotéza, že samotná patofyziologie schizofrenie je spojena s rozvojem metabolického syndromu, preklinický výzkum naznačuje společný základ subchronického zánětu a dysbalanci sekrece adipokinů. Tyto patofyziologické predispozice pro rozvoj složek metabolického syndromu mohou být dále zhoršovány souběžným podáváním farmakoterapie schizofrenickým pacientům. I samotná psychofarmaka, jako například modernější antipsychotika 2. a 3. generace, přináší vedle vyšší účinnosti a lepší snášenlivosti i významné nežádoucí metabolické účinky. 27 Antipsychotika 2. generace můžeme rozdělit do 3 skupin podle mechanismu jejich účinku: 1) selektivní antagonisté dopaminových (D2/D3) receptorů: amisulprid, 2) SDA-serotoninoví, dopaminoví a alfa-1 antagonisté: risperidon, ziprasidon, sertindol, 3) MARTA - multireceptoroví antagonisté: klozapin, olanzapin, quetiapin, zotepin. Selektivní antagonisté dopaminových D2/D3 receptorů neovlivňují jiné neurotransmiterové systémy. Nežádoucí účinky jsou v této skupině antipsychotik vázány výhradně na dopaminový systém, není přítomna nadměrná sedace, somnolence, zvyšování hmotnosti nebo anticholinergní příznaky. SDA (Serotonin and Dopamine Antagonists) antipsychotika blokují významně více serotoninové S2 receptory než dopaminová D2 zakončení a v závislosti na léčivu různě intenzivně i adrenalinová alfa-1 zakončení. Ze skupiny SDA je prvou volbou risperidon, druhou volbou ziprasidon, zejména vzhledem k určitému riziku prodloužení QT intervalu, byť není jednoznačná klinická relevance. Ziprasidon by proto neměl být podáván kardiakům, je naopak vhodný pro obézní nemocné, protože nezvyšuje hmotnost, odobně jako antipsychotikum třetí generace, aripiprazol. MARTA (Multi-Acting Recepor Targeted Agents) antipsychotika ovlivňují jak dopaminový, tak serotoninový, adrenalinový, histaminový a muskarinový systém. Jejich vazba na D2 zakončení je extrastriatálně selektivní s výjimkou zotepinu, s nízkou (klozapin, quetiapin) až středně vysokou (olanzapin, zotepin) obsazeností D2 receptorů. Blokáda serotoninových S2 receptorů je vysoká a podstatně vyšší než D2 zakončení se všemi příznivými důsledky, popsanými u SDA antipsychotik. MARTA antipsychotika jsou smíšenými antagonisty/agonisty muskarinových receptorů s důsledkem zvýšeného uvolňování acetylcholinu, který příznivě ovlivňuje paměťovou složku kognitivní dysfunkce. Nevýhodou těchto léčiv je zvyšování hmotnosti pacienta, určitá sedace a nutnost monitorovat glukozový a lipidový metabolizmus s možným (byť nízkým) rizikem provokace či zhoršení diabetu typu 2. Mezi antipsychotika 3. generace patří látky, které nejsou plnými dopaminergními agonisty, nýbrž dualisty, popř. parciálními antagonisty. Zatím jediným v klinické praxi zavedeným reprezentantem této „třetí” generace antipsychotik je aripiprazol, který vykazuje charakteristiky atypických antipsychotik a má mimořádně dobrou snášenlivost. Klinická praxe zatím u aripiprazolu těží z jeho minimálního působení útlumu a zanedbatelného rizika obezity a diabetu II. typu coby nežádoucího účinku antipsychotické léčby (43). 28 3.2.2.1 VLASTNÍ PŘÍSPĚVEK K PROBLEMATICE V PREKLINICKÉM EXPERIMENTU Horska K, Ruda-Kucerova J, Babinska Z, Karpisek M, Demlova R, Opatrilova R, Suchy P, Kotolova H. Olanzapine-depot administration induces time-dependent changes in adipose tissue endocrine function in rats (published in Psychoneuroendocrinology. 2016; 73:177-185. IF 4,788) Horska K, Ruda-Kucerova J, Drazanova E, Karpisek M, Demlova R, Kasparek T, Kotolova H. Aripiprazole-induced adverse metabolic alteration in polyI:C neurodevelopmental model of schzophrenia in rats (published in Neuropharmacology. 2017; 123:148-158. IF 5,012) V rámci výzkumné skupiny Farmakologického ústavu LF MU, jsme se zaměřili na metabolické nežádoucí účinky vybraných atypických antipsychotik, které významně přispívají ke zvýšení rizika kardiovaskulární morbidity a mortality u pacientů trpících schizofrenií. Přestože se touto otázkou zabývá i řada jiných experimentálních výzkumných skupin, mechanismy vzniku těchto nežádoucích účinků stále nejsou plně pochopeny. V poslední době je věnována pozornost právě také úloze metabolismu tukové tkáně a neurohormonální regulaci a stejnou otázkou jsme se zabývali i v naší práci. Účelem preklinické studie bylo zhodnotit časově závislé účinky podávání olanzapinu v klinicky relevantním dávkování při regulaci energetické homeostázy, metabolismu glukózy a lipidů, hormonů pocházejících z gastrointestinálního traktu a tukového tkáně, které se podílely na regulaci energetické bilance v experimentu na krysích samicích Sprague-Dawley. Léčba olanzapinem nevedla ke změnám sérových hladin ghrelinů, FGF-21 a prozánětlivých markerů IL-1a, IL-6 a TNF-α v jakémkoli časovém bodě studie. Výsledky rovněž naznačují, že došlo k časné změně endokrinní funkce tukové tkáně jako faktoru, který se podílí na mechanismech, jež mohou být základem metabolických nežádoucích účinků antipsychotik (44). 29 Další experimentální práce cílila na podávání atypického antipsychotika aripiprazolu, který jak již bylo zmíněno dříve, představuje antipsychotikum s nízkým rizikem vývoje metabolického syndromu. Cílem této studie bylo vyhodnotit metabolický fenotyp modelu neurodevelopmentu polyI:C a posoudit metabolické účinky chronického podávání aripiprazolu s ohledem na komplexní neuroendokrinní homeostázy. Aripiprazol neovlivnil tělesnou hmotnost, ale způsobil neurohumorální změny. Sérové hladiny leptinu a GLP-1 byly výrazně sníženy, zatímco hladina ghrelinu byla zvýšena. Dále aripiprazol snížil sérové hladiny prozánětlivých cytokinů. Naše údaje naznačují dysregulaci adipokinů a gastrointestinálních hormonů přítomných při chronickém podávání aripiprazolu, které je považována za metabolicky neutrální v polyI:C modelu schizofrenie (45). 30 4. IMPLIKACE PREKLINICKÝCH A KLINICKÝCH AKTIVIT PŘI VÝUCE LÉKAŘSKÉ FARMAKOLOGIE 4.1 PREGRADUÁLNÍ VÝUKA LÉKAŘSKÉ FARMAKOLOGIE Závěrečnou kapitolu předložené habilitační práce, a to v návaznosti na již obhájenou práci z roku 2015 (Příloha č. I), pojímám jako snahu zasadit vlastní výzkumné práce v oblasti farmakologie do kontextu výuky předmětu Lékařské farmakologie na LF MU a dovolím si tak představit komplexní pohled klinického farmakologa se zkušeností s preklinickými výzkumy i dlouhodobou klinickou zkušeností na výuku farmakologie v době personalizované farmakoterapie, na kterou je nutno připravovat naše studenty již během preklinické a klinické části studia medicíny. Předmět Lékařská farmakologie je vyučován na LF MU v rámci pregraduálního studia všeobecného i zubního lékařství v 6. a 7. semestru studia. V souladu se stávající akreditací je věnován zejména pochopení základních principů farmakokinetiky a farmakodynamiky v rámci obecné farmakologie a dále pak mechanismům účinků jednotlivých lékových skupin v rámci studia speciální farmakologie. Z didaktického hlediska farmakologie tvoří jednotu dvou celků: obecné a speciální farmakologie. Obecná farmakologie na základě experimentálních poznatků z oblasti farmakodynamiky a farmakokinetiky léčiv definuje obecně platné zákonitosti biofyzikální a biochemické povahy, projevující se vzájemnou interakcí organizmu a farmaka. Stává se tím zároveň metodologickým návodem pro farmakologii speciální, jež se věnuje konkrétnímu roztřídění farmak z hlediska farmakodynamiky, studuje vlastnosti léčiv v jejich zvláštní specifické podobě, stanovuje jejich farmakokinetiku. Během svého působení ve vedení Farmakologického ústavu od roku 2011 si stále významněji uvědomuji, že byť jsou všechny výše uvedené znalosti zcela zásadní a nepodkročitelné, zejména v preklinické části studia, chybí nám systematičtější výuka věnovaná aplikované klinické farmakologii a problémově orientované výuce farmakologie na příkladech personalizované farmakoterapie vybraných onemocnění. Klinická farmakologie je interdisciplinární obor, který integruje experimentální farmakologii s klinickými a paraklinickými obory s cílem studovat a objektivními 31 metodami hodnotit účinek léku u zdravého i nemocného člověka. Zahrnuje rovněž doporučení a zdůvodnění terapeutického užití určitých skupin léčiv u určitých onemocnění a do její náplně patří i terapeutické monitorování léčiv. V rámci klinické farmakologie se vyčleňují ještě další podobory, např. farmakogenetika jako obor, který se zabývá vlivem jednotlivých farmakogenetických polymorfismů na individuální odpověď na podané léčivo u konkrétního jedince, případně farmakogenomika, obor, který studuje tyto vlivy na úrovni celého genomu. Současný stav výuky klinické farmakologie Klinická farmakologie je v současném akreditovaném programu všeobecného lékařství zavedena jako volitelný předmět „Vybrané kapitoly z klinické farmakologie” pro studenty 5. ročníku, kteří úspěšně absolvovali předmět farmakologie ve 4. ročníku. Sylabus je tvořen 9 přednáškami, předmět je zakončen testem. Témata přednášek se týkají farmakologických principů nejčastěji se vyskytujících nozologických jednotek jako jsou hypertenze, farmakoterapie ischemické choroby srdeční a akutního koronárního syndromu, srdečního selhávání, trombembolických příhod, diabetu mellitu, CHOPN a asthmatu, farmakoterapii nádorových onemocnění, antibiotika a klinicky relevantní lékové interakce. Stávající pojetí výuky klinické farmakologie však neodpovídá plně potřebám vyplývajícím z předpokládaných a vytyčených výstupů z učení, které jsme pro tento předmět optimalizovali v rámci projektu OPTIMED a kde došlo k významnému posunu vzhledem k potřebám posunu úrovně znalostí našich studentů směrem k propojení a kombinaci znalostí základní a aplikované farmakologie. Tato optimalizace výuky je v souladu s vyjádřením pracovní skupiny pro klinickou farmakologii v Evropě při Světové zdravotnické organizaci (46) a jejím primárním cílem je zlepšení péče o pacienta ať už přímo nebo nepřímo vyvíjením lepších, účinnějších a bezpečnějších léčiv a podporou racionálního užívání léčiv (47). Navrhovaná úprava výuky klinické farmakologie Smyslem pregraduální výuky klinické farmakologie musí být efektivní předání znalostí, dovedností a postojů, které budou studenti potřebovat jako lékaři v nadcházejících letech své praxe. Obecným konceptem výuky klinické farmakologie a racionální farmakoterapie v klinické medicíně je příprava studentů k jejich 32 klinické praxi po ukončení studia. Budoucí lékaři musí být obeznámeni s rozhodovacím procesem – jaký lék bude zvolen pro konkrétního pacienta, faktory, které rozhodnutí lékaře ovlivňují a zhodnocení jejich významnosti. Důležitou součástí curricula klinické farmakologie musí být výuka problematiky klinického vývoje nových léků, jež vyžaduje pokročilé porozumění preklinickému výzkumu, stejně jako studium etických, právních a regulačních požadavků na specializované klinické výzkumy v oblasti léčiv. Nedílnou součástí dobře navrženého programu výuky klinické farmakologie je předání znalosti správného a racionálního předepisování, nejen ambulantním, ale i hospitalizovaným pacientům. Výchova kvalifikovaných klinických lékařů je zcela stěžejní a rozhodující pro racionální farmakoterapii a podávání účinných a bezpečných léků v jejich budoucí klinické praxi. Součástí procesu výuky klinické farmakologie musí být i zaměření na problematiku lékových interakcí, ovlivnění funkce parenchymových orgánů, v nichž biotransformace probíhají, vlivů na intervenční terapeutické a diagnostické metody používané v současné medicíně (typicky vztah sérové glukózy a vyšetření PET, obecně vztahy klinické farmakologie a klinické biochemie/hematologie/imunologie, apod.). Navrhovaný integrovaný program seminářů a přednášek sleduje doporučení IUPHAR, Mezinárodní unie základní a klinické farmakologie (48) a harmonizaci mezinárodního curricula výuky klinické farmakologie na lékařských fakultách v Evropě (DELPHI study). V souladu se zavedenými vzdělávacími cíli je koncept výuky klinické farmakologie orientovaný na pacienty, farmakoterapii a dané onemocnění. Klíčovými prvky, které je potřeba zavést povinně do výuky jsou zejména: Základní modul: 1) Obecná praktická klinická farmakologie (zaměřená na individualizaci farmakoterapie), personalizovaná medicína, cílená léčba, inter/intra individuální variabilita 2) Aplikovaná klinická farmakologie - příklady kazuistik specifických pro farmakoterapii nejčastějších onemocnění a zdravotních stavů v rutinní lékařské praxi 33 3) Interaktivní „problem-oriented” výuka zaměřená na specifickou část praktická farmakokinetika, TDM, toxokinetika, specifické populace pacientů (dialyzovaní, imunosuprimovaní atd.) 4) Léčivé přípravky pro moderní technologie, biologická léčba, genová, vektorová léčba, VILP, orphan drugs 5) Racionální principy předepisování (obecné zásady výběru léků, recepturní/žádankový předpis, preskribce pro hospitalizovaného pacienta), generická substituce, úloha pojišťovny, revizního lékaře/SÚKL při použití léčiv v režimu „off-label”, lékové chyby, lékové interakce, polypragmazie (včetně simulace reálných situací) Rozšířený modul: 6) Výzkum a vývoj léčiv (preklinické a klinické hodnocení, translace výstupů z prekliniky do klinického hodnocení, PASS, PAESS) – farmakologie využitá v rámci klinických hodnocení 7) Regulace léčiv (ve vývoji, během registračního procesu, po uvedení na trh, stažení přípravku,…), srovnání s potravinovým doplňkem – rizika jejich použití 8) Farmakovigilance (ve vývoji, během registračního procesu, po uvedení na trh), rozpoznání a hlášení NÚ, kritéria CTCAE a etické aspekty používaní léčiv („off-label” use, léky na mimořádný dovoz, repurposing drugs, compassionate use…), monitoring nových léčiv v klinické praxi se zaměřením na farmakovigilanci a HTA, etické problémy neschválených léčebných postupů 9) Problematika zdravotnických prostředků 10)Evidence - based medicine – kritické zhodnocení literatury, interpretace výsledků klinických studií a case reportů, lékařské úvahy a situace, které se nejčastěji vyskytují v rutinní lékařské praxi Ve výuce budou uplatněny různé formy učení: didaktické a interaktivní přednášky, jednodenní výukové semináře založené na řešení problémů, diskuze v malých skupinách, učení formou rolí (lékař/pacient), samostudium a společné řešení seminárních prací, kvízy a výuka prostřednictvím IT – TDM, vyhledávání informací. Cílem výuky je rovněž podpořit kritické myšlení založené na znalostech tak, aby absolvent dokázal samostatně vyhodnocovat nově přicházející informace vždy ku 34 prospěchu pacientů. Podstatné je rovněž kontinuální zařazování nejnovějších poznatků a trendů do výuky tak, aby absolvent předmětu vždy odcházel se soudobými informacemi. K tomu bude využito moderních technologií a jejich možností, včetně využití simulačních nástrojů umožňující reálné modelování situací a jejich analýzu. Aktivní studenti dostanou příležitost podílet se na realizaci pokročilých výzkumných projektů klinických hodnocení a budou mít možnost spoluautorství v souvisejících publikacích. 4.2 POSTGRADUÁLNÍ VÝUKA LÉKAŘSKÉ FARMAKOLOGIE Studium klinické farmakologie zůstává procesem celoživotního učení, jelikož je integrální, esenciální a celoživotní kompetencí každého klinického lékaře. Současně je celoživotní výzvou pro klinické farmakology a další lékařské pedagogy vyučující tento obor. V rámci Farmakologického ústavu LF MU je tento koncept podporován spoluprací lékařů, klinických farmakologů a klinických farmaceutů, v současné době je také diskutováno založení konzultačního centra pro farmakologickou interpretaci hladin léků (TDM) a lékových interakcí ve spolupráci s klinickými laboratořemi a lékárnami fakultních nemocnic. Dalšími oblastmi, které patří do náplně oboru klinické farmakologie a ve kterých se díky vlastním zkušenostem podílíme i na postgraduálním vzdělávání, jsou také: Translační a klinický výzkum: v rámci Farmakologického ústavu LF MU jsme v roce 2011 založili Centrum pro akademické klinické studie, podílíme se na provádění vlastních klinických hodnocení s významným inovativním potenciálem (KDO_DC1311), včetně samotného designu počátečních fází klinických hodnocení léčiv (fáze I, I/II). To vše se daří realizovat i díky výzkumné infrastruktuře CZECRIN (Czech Clinical Research Infrastructure Network), jejíž jsem hlavní řešitelkou. Farmakologický ústav LF MU je rovněž národním koordinátorem s napojením na ECRIN-ERIC, což přispělo k úspěšné participaci na projektech klinického výzkumu financovaných z H2020. Farmakoekonomika: k zajištění bezpečné a účinné farmakoterapie (jeden z cílů klinické farmakologie) je nezbytně nutné hodnocení zdravotnických technologiích (HTA), včetně léčiv. Na Farmakologickém ústavu LF MU funguje pracovní skupina podílející se na farmakoekonomice a HTA, 2 postgraduální 35 studenti obhájili práci v této oblasti, ústavem jsou pořádány každoroční konference k těmto tématům. Postgraduální výuku v oblastech uvedených výše realizujeme i díky úspěšné edukační platformě PharmAround, která byla v letech 2011 – 2014 realizována z Operačního programu Vzdělávání pro konkurenceschopnost (OP VK). Výsledkem této fáze je ucelený systém odborných kurzů pro studenty, lékaře, pedagogy a výzkumné pracovníky, kterým dosud chyběla možnost komplexního vzdělávání v oblasti vývoje léčiv. Od roku 2014 pokračuje PharmAround ve své úspěšné činnosti za podpory nadačního fondu a nadále se zaměřuje prioritně na oblast vzdělávání a vzájemné spolupráce se zaměřením na celý životní cyklus léčiva a jeho dopadu na pacienty. Vzdělávací aktivity projektu PharmAround pokrývají všechny vývojové fáze léčiva a jejich absolventi získají pro svou praxi užitečný soubor znalostí. 36 5. ZÁVĚR Farmakologie je vědeckým i klinickým oborem, ve kterém se možná více než jinde potvrzuje, že uplatnění principů individualizované medicíny v kontextu pokroků dosažených v biologii, diagnostice a farmakoterapii onemocnění, zvyšuje šance na dlouhodobé přežití a lepší kvalitu života našich pacientů. Personalizovaný přístup hraje a bude v tomto směru hrát stále významnější roli. Farmakologie je velmi živým oborem s řadou výzkumných otázek i klinických aplikací. V takto dynamickém prostředí – kde jsme průběžně svědky zavádění nových farmakologických poznatků a inovativních léčiv – musí i výuka na lékařské fakultě toto reflektovat. Přes všechna nová technologická řešení databázových či jiných informačních systémů dnešní doby i nadále zůstává stále lékař tím, kdo farmakoterapii indikuje a řídí. Schopnost reagovat správně na nové poznatky a efektivně je uplatňovat v péči o pacienty je dána ve velké míře i znalostmi, jež lékař získává při svém vzdělávání. Na druhou stranu je potřeba akceptovat, že dnešní doba s obrovským množství informací nás posouvá více od znalostí (memorování – jež ovšem má své nepodkročitelné znalostní základy) do polohy schopnosti najít a správně vyhodnotit/pochopit. V oblasti farmakologie je tedy zásadním cílem naučit lékaře správně identifikovat, vyhodnocovat a používat informace ve vztahu k léčbě pacienta. 37 6. REFERENCE 1. Rubin RP. 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Duchnowska R, Wysocki PJ, Korski K, Czartoryska-Arłukowicz B, Niwińska A, Orlikowska M, Radecka B, Studziński M, Demlova R et al (CEEOG). Immunohistochemical prediction of lapatinib efficacy in advanced HER2positive breast cancer patients. Oncotarget. 2016 Jan 5;7(1):550-64. 4. Demlova R, Valik D, Obermannova R, Zdrazilova-Dubska L. The safety of therapeutic monoclonal antibodies: implications for cancer therapy including immuno-checkpoint inhibitors. Physiol Res. 2016 Dec 21;65(Suppl. 4):455-S462 5. Bilek O, Bohovicova L, Demlova R, Poprach A, Lakomy R, ZdrazilovaDubska L. Non-Small Cell Lung Cancer-from Immunobiology to Immunotherapy. Klin Onkol. 2016;29(Suppl. 4):78-87. 6. Klinické hodnocení fáze I/II „Kombinovaná protinádorová terapie s ex vivo manipulovanými dendritickými buňkami produkujícími IL-12 u dětských, adolescentních a mladých dospělých pacientů s progredujícími, relabujícím nebo primárně metastatickými malignitami vysokého rizika” EudraCT Number:2014-003388-39. Zadavatel LF MU Brno. https://www.clinicaltrialsregister.eu/ctr-search/trial/2014-003388-39/CZ 7. Zdrazilova-Dubska L, Fedorova L, Pilatova K, Mudry P, Hlavackova E, Matoulkova E, Flajsarova L, Demlova R, Valik D, Sterba J. TKI pazopanib impaires immunostimulatory properties of monocytes: Implication for monocyte-derived DC-based anti-cancer vaccine preparation. Annals of Oncology. 2016;27(Suppl.8): Meeting Abstract 18P 43 8. Merhautová J, Vychytilová-Faltejsková P, Demlová R, Slabý O. Systemic administration of miRNA mimics by liposomal delivery system in animal model of colorectal carcinoma. Physiol Res. 2016 Dec 21;65(Suppl.4):481- 488. 9. Merhautova J, Demlova R, Slaby O. MicroRNA-Based Therapy in Animal Models of Selected Gastrointestinal Cancers. Fron Pharmacol. 2016 Sep; 7(329):1-21 10. Merhautova J, Hezova R, Poprach A, Kovarikova A, Radova L, Svoboda M, Vyzula R, Demlova R, Slaby O. miR-155 and miR-484 Are Associated with Time to Progression in Metastatic Renal Cell Carcinoma Treated with Sunitinib. Biomed Res Int. 2015; ID941980 11. Merhautova J, Hezova R, Poprach A, Svoboda M, Demlova R, Slaby O. MiRNA associated with time to progression in metastatic renal cell carcinoma patients treated with sunitinib. Eur J Cancer. Sep 2015; 51(Suppl.3): Meeting Abstract S505 12. Horska K, Ruda-Kucerova J, Drazanova E, Karpisek M, Demlova R, Kasparek T, Kotolova H. Horska K, Ruda-Kucerova J, Babinska Z, Karpisek M, Demlova R, Opatrilova R, Suchy P, Kotolova H. Neuropharmacology. 2017 Jun 13; 123:148-158 13. Horska K, Ruda-Kucerova J, Babinska Z, Karpisek M, Demlova R, Opatrilova R, Suchy P, Kotolova H. Olanzapine-depot administration induces time-dependent changes in adipose tissue endocrine function in rats. Psychoneuroendocrinology. 2016; (73):177-185 44 8. PŘÍLOHY Příloha č. I: Habilitační spis Koncepty klinické farmakologie v éře personalizované medicíny, UK Bratislava, obhájeno 18. 10. 2015 (není součástí svazku, tvoří samostatnou přílohu) Příloha č. II: Komentovaná práce 1. Potential clinical benefit of therapeutic drug monitoring of imatinib in oncology. Turjap M, Juřica J, Demlová R. Příloha č. III: Komentovaná práce 3. Immunohistochemical prediction of lapatinib efficacy in advanced HER2-positive breast cancer patients. Duchnowska R, Wysocki PJ, Korski K, CzartoryskaArłukowicz B, Niwińska A, Orlikowska M, Radecka B, Studziński M, Demlova R et al (CEEOG) Příloha č. IV: Komentovaná práce 4. The safety of therapeutic monoclonal antibodies: implications for cancer therapy including immunocheckpoint inhibitors. Demlova R, Valik D, Obermannova R, Zdrazilova-Dubska L. Příloha č. V: Komentovaná práce 5. Non-Small Cell Lung Cancer-from Immunobiology to Immunotherapy. Bilek O, Bohovicova L, Demlova R, Poprach A, Lakomy R, Zdrazilova-Dubska L. Příloha č. VI: Komentovaná práce 8. Systemic administration of miRNA mimics by liposomal delivery system in animal model of colorectal carcinoma. Merhautová J, Vychytilová-Faltejsková P, Demlová R, Slabý O. Příloha č. VII: Komentovaná práce 9. MicroRNA-Based Therapy in Animal Models of Selected Gastrointestinal Cancers. Merhautova J, Demlova R, Slaby O. Příloha č. VIII: Komentovaná práce 10. miR-155 and miR-484 Are Associated with Time to Progression in Metastatic Renal Cell Carcinoma Treated with Sunitinib. Merhautova J, Hezova R, Poprach A, 45 Kovarikova A, Radova L, Svoboda M, Vyzula R, Demlova R, Slaby O. Příloha č. IX: Komentovaná práce 12. Aripiprazole-induced adverse metabolic alteration in polyI:C neurodevelopmental model of schzophrenia in rats. Horska K, Ruda-Kucerova J, Drazanova E, Karpisek M, Demlova R, Kasparek T, Kotolova H. Příloha č. X: Komentovaná práce 13. Olanzapine-depot administration induces time-dependent changes in adipose tissue endocrine function in rats. Horska K, Ruda-Kucerova J, Babinska Z, Karpisek M, Demlova R, Opatrilova R, Suchy P, Kotolova H. Klin Onkol 2015; 28(2): 105–111 105 PŘEHLED Možný klinický přínos terapeutického monitorování hladin imatinibu v onkologii Potential Clinical Benefit of Therapeutic Drug Monitoring of Imatinib in Oncology Turjap M.1–3 , Juřica J.2,3 , Demlová R.2,4 1 Oddělení klinické farmacie, FN Ostrava 2 Farmakologický ústav, LF MU, Brno 3 Skupina experimentální a aplikované neuropsychofarmakologie, CEITEC – Středoevropský technologický institut, MU, Brno 4 Oddělení klinických hodnocení, Masarykův onkologický ústav, Brno Souhrn Imatinib mesylát je kompetitivním inhibitorem tyrozinkinázy BCR/ABL a současně také inhibitorem několika receptorových tyrozinkináz. Za dobu od svého uvedení na trh se ukázal jako velmi cenný v terapii Philadelphia chromozom (BCR/ABL) – pozitivní (Ph+) chronické myeloidní leukemie a Kit (CD117) pozitivních gastrointestinálních stromálních tumorů. Léčivo je biotransformováno cestou cytochromu P450 a je popsáno množství významných lékových interakcí. Onkologičtí pacienti často užívají současně množství dalších léčiv zvyšujících pravděpodobnost takové interakce a svou roli může sehrávat i adherence k léčbě při dlouhodobém podávání. Režimy vycházející z fixního dávkování imatinibu nerespektují interindividuální rozdíly ve farmakokinetice léčiva a je možné, že někteří nemocní tak nedosahují dostatečných plazmatických koncentrací. Na základě evidence z klinických studií lze usuzovat, že existuje vztah mezi plazmatickými koncentracemi imatinibu a klinickou odpovědí. Imatinib se proto jeví být vhodným kandidátem pro terapeutické monitorování jeho plazmatických koncentrací. Tento článek předkládá přehled o farmakokinetice, lékových interakcích imatinibu, sumarizuje aktuální stav problematiky stanovení plazmatických koncentrací pro účely optimalizace terapie a dále popisuje možnosti, limity a návrhy pro terapeutické monitorování imatinibu. Klíčová slova imatinib – farmakokinetika – lékové interakce – terapeutické monitorování léčiv – chronická myeloidní leukemie – gastrointestinální stromální tumory Summary Imatinib mesylate is a competitive inhibitor of BCR/ABL tyrosine kinase and inhibits also several receptor tyrosin kinases. Since its launch to the market, imatinib has proven to be very valuable in the treatment of Philadelphia chromosome (BCR/ABL) – positive (Ph+) chronic myeloid leukemia and Kit (CD117) positive gastrointestinal stromal tumors. The drug is metabolized by cytochrome P450, and there are many clinically important pharmacokinetic drug-drug interactions described in the literature. Frequent polypharmacy in oncological patients increases probability of such interactions, and also adherence may play its role during long-term treatment. Fixed dosing therapeutic regimens fail to respect known interindividual variability in pharmacokinetics of the drug and thus, some patients may not achieve sufficient plasma concentrations. Based on current evidence, there seems to be a relationship between plasma concentration and clinical response to imatinib. Therefore, imatinib appears to be suitable candidate for therapeutic drug monitoring. Here, we present an overview of pharmacokinetics, drug-drug interactions and current knowledge and suggestions on therapeutic drug monitoring of imatinib, its potential benefits and limitations. Key words imatinib – pharmacokinetics – drug interactions – therapeutic drug monitoring – chronic myeloid leukemia – gastrointestinal stromal tumors Autoři deklarují, že v souvislosti s předmětem studie nemají žádné komerční zájmy. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. Redakční rada potvrzuje, že rukopis práce splnil ICMJE kritéria pro publikace zasílané do biomedicínských časopisů. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers.  PharmDr. Miroslav Turjap Oddělení klinické farmacie FN Ostrava 17. listopadu 1790 708 52 Ostrava-Poruba e-mail: miroslav.turjap@fno.cz Obdrženo/Submitted: 8. 12. 2014 Přijato/Accepted: 4. 2. 2015 http://dx.doi.org/10.14735/amko2015105 106 MOŽNÝ KLINICKÝ PŘÍNOS TERAPEUTICKÉHO MONITOROVÁNÍ HLADIN IMATINIBU V ONKOLOGII Klin Onkol 2015; 28(2): 105–111 Úvod Imatinib mesylát (Glivec®) je z hlediska svého mechanizmu účinku selektivním kompetitivním inhibitorem tyrozinkinázy BCR/ABL (breakpoint cluster region/Abelson), tedy konstitutivně aktivované tyrozinkinázy vzniklé jako produkt transkripce Philadelphia chromozomu (reciproká translokace mezi dlouhými raménky 9. a  22. chromozomu). Imatinib interaguje s proteinem BCR/ABL na nukleotidovém vazebném místě tak, že zabraňuje vazbě adenosintrifosfátu (ATP) a tím stabilizuje tento protein v  jeho inaktivní konformaci. Výsledným efektem je zabránění účinku genu BCR/ABL na proteinové úrovni, vedoucí následně k  apoptóze a  zástavě proliferace buněčného klonu. Imatinib současně inhibuje i některé receptorové tyrozinkinázy – PDGFR-α a PDGFR-β (Platelet-derived growth factor receptor, -α, -β; glykoproteinový růstový faktor), c-KIT (transmembránový tyrozinkinázový receptor), CSF-1R (receptor pro kolonie stimulující faktor 1) [1]. V ČR je Glivec® dostupný již od roku 2001, přičemž zásadně přispěl k inovaci tehdy dostupných léčebných postupů a zlepšení prognózy léčených pacientů. Imatinib je dle guidelines NCCN (National Comprehensive Center Network) pro chronickou myeloidní leukemii (chronic myeloid leuke- mia – CML) [2] indikován k 1. linii léčby chronické fáze Philadelphia chromozom (BCR-ABL)  – pozitivní chronické myeloidní leukemie (Ph+ CML) s doporučenou kontrolou cytogenetické odpovědi a monitorace hladiny transkriptu v pravidelných intervalech. V případě suboptimální odpovědi lze volit zvýšení dávky imatinibu, v případě selhání léčby imatinibem je pak indikována změna léčby alternativním tyrozinkinázovým inhibitorem (nilotinib nebo dasatinib). Dle NCCN doporučení je imatinib dále indikován u pacientů s akcelerovanou nebo blastickou fází CML se současným zvážením možnosti provedení transplantace kostní dřeně [2]. Vrámciléčbysolidníchnádorůjsoudalší indikací imatinibu pacienti s Kit (CD117) pozitivními inoperabilními nebo metastazujícími gastrointestinálními stromálními nádory (GIST) nebo pacienti po resekci GIST s vysokým rizikem recidivy [3]. Obvyklé dávkování imatinibu u  hematoonkologických malignit je 400–800 mg denně. Dávka 400 mg se používá standardně jako dávka iniciální, dávky 600–800 mg denně jsou podávány u  pokročilejších fází CML nebo u  pacientů s  akcelerovanou fází onemocnění nebo v blastické krizi. U dospělých pacientů s  inoperabilním a/nebo metastatickým maligním GIST je doporučená dávka 400mg denně, s možným zvýšením dávky na 600 mg nebo 800 mg u  pacientů s  primární nebo sekundárně vzniklou rezistencí na imatinib. Všeobecně se při terapeutickém selhání iniciální dávky nebo při suboptimální léčebné odpovědi doporučuje dávku eskalovat na 800 mg/den v případě, že pacient denní dávku 400 mg dobře snášel. V současné době je diskutována možnost přerušení léčby imatinibem u nemocných s  CML v  molekulární remisi. Výsledky dosavadních studií naznačují, že přerušení léčby by mohlo být perspektivně možné u vybraných pacientů se stabilní kompletní molekulární odpovědí trvající alespoň dva roky, za současného pečlivého monitorování molekulární odpovědi a  časného řešení případného relapsu [2]. Pro standardizaci kritérií bezpečného přerušení podávání imatinibu jsou nicméně potřebné výsledky dalších, prospektivních studií s větším počtem pacientů a delším sledováním. Proto aktuální guidelines NCCN  [2] i  European LeukemiaNet  [4] doporučují dlouhodobé pokračování léčby imatinibem u všech pacientů, kteří na léčbu odpovídají; případné ukončení léčby imatinibem u těchto pacientů může být zvažováno pouze v rámci některého z  protokolů klinických studií. Doporučená dávka imatinibu v  adjuvantní léčbě vysoce rizikových pacientů po resekci GIST je 400mg. Obecně je imatinib dobře snášen a výskyt závažných nežádoucích účinků grade 3 nebo 4, s výjimkou hematotoxicity, je nízký. Klinicky nejdůležitější jsou nežádoucí účinky hematologické, zejm. neutropenie a trombocytopenie, a proto je nutné zejm. v prvních týdnech léčby pečlivé monitorování krevního obrazu. Kromě hematologických komplikací patří mezi možné nežádoucí účinky výskyt gastrointestinálních komplikací (nevolnost, dyspepsie, průjem), poruchy vnitřního prostředí (otoky víček, obličeje, dolních končetin, méně často pleurální výpotek nebo ascites), případně bolesti svalů, kloubů, kostí nebo únava. Tyto nežádoucí účinky jsou většinou mírné a odeznívají během několika dnů či týdnů po vysazení léčby. Potenciálně nebezpečná může být hepatotoxicita, vzácně vedoucí k selhání jater; doporučuje se sledovat jaterní testy a omezit podávání paracetamolu. Důvodem jsou obavy z  aditivního hepatotoxického efektu obou látek a uvažuje se o možné inhibici O-glukuronidace paracetamolu imatinibem [5]. Farmakokinetické vlastnosti Imatinib mesylát se po perorálním podání dobře absorbuje, biologická dostupnost je až 98  % a  není ovlivněna potravou. Maximálních plazmatických koncentrací dosahuje imatinib po 2–4  hodinách po podání, extenzivně se váže na proteiny plazmy (až 95 %, z většiny na α1 kyselý glykoprotein). Distribuční objem imatinibu je 252 L [6]. Hlavní biotransformační cesta vede přes N-demetylaci k  piperazinovému metabolitu (CGP74577), který je biologicky aktivní. Tuto reakci katalyzuje forma 3A4 cytochromu P450. Farmakokinetika imatinibu je lineární v rozmezí dávek 25–1 000mg p.o. 1× denně. Eliminační parametry jsou věkem nebo hmotností ovlivněny jen mírně – s nárůstem hmotnosti se zvyšuje clearance (8,5 L/h u pacientů pod 50kg, 11,3 L/h u pacientů nad 100 kg), s věkem roste distribuční objem. Větší vliv na farmakokinetické parametry mají spíše albumin, buňky bílé krevní řady a biliru- bin [7,8]. Eliminační poločas imatinibu je 18 hodin, v případě jeho aktivního metabolitu pak dokonce 40 hodin. Během sedmi dní se tak vyloučí okolo 80 % podané dávky, většina ve formě metabolitů, a to zejm. biliární cestou (poměr exkrece stolice : moč je 5 : 1); jen asi 25 % se vyloučí v  nezměněné formě. Mírná až středně závažná hepatální insuficience zpravidla nevyžaduje korekci dávkování [8]. Imatinib je středně silným inhibitorem metabolické aktivity CYP2C9 (neočeká- MOŽNÝ KLINICKÝ PŘÍNOS TERAPEUTICKÉHO MONITOROVÁNÍ HLADIN IMATINIBU V ONKOLOGII Klin Onkol 2015; 28(2): 105–111 107 sice potenciálně může zvrátit rezistenci k léčivům transportovaným tímto přenašečem, na druhou stranu však klinicky může až nebezpečně zvýšit expozici těmto léčivům – např. pazopanibu nebo topotekanu [8,14]. Poněkud problematická je případná kombinace imatinibu s  warfarinem u  nemocných vyžadujících antikoagulační léčbu. Biotransformace warfarinu vede přes CYP2C9 (účinnější S enantiomer) a také přes CYP3A4 (méně účinný R enantiomer). O interakcích imatinibu a warfarinu není dostatek spolehlivých dat; některé studie naznačují, že warfarin neovlivňuje plazmatické koncentrace a účinek imatinibu [15]. Opačná interakce je dokumentována  – imatinib by mohl zvýšit účinek warfarinu pravděpodobně inhibičním účinkem na metabolickou aktivitu CYP3A4 [16], jehož je imatinib silným inhibitorem, méně významná je (slabší) inhibice metabolické aktivity CYP2C9. S ohledem na terapeutické režimy zahrnující obvykle více současně podávaných léčiv je obtížné identifikovat původce a  rozsah těchto interakcí. Klinický dopad zůstává Při kombinaci s inhibitory metabolické aktivity CYP3A4 naopak dochází k opačným projevům – zvýšení AUC, nežádoucích účinků a  toxicity imatinibu, jako v případě zvýšení AUC o 41 % po jednorázovém podání imatinibu (200 mg) s ketokonazolem (400mg) [11]. Méně často se objevují informace o  vlivu současného podání inhibitoru aktivity transportérů pro organické kationty (hOCT1) na plazmatické koncentrace/účinek imatinibu. Tento transportér je zodpovědný za uptake imatinibu do cílových buněk. Inhibice aktivity hOCT1 se paradoxně projeví zvýšenou plazmatickou koncentrací imatinibu a současně jeho sníženou intracelulární expozicí [10]. Interakce na úrovni CYP2D6  nebo CYP2C9  mají teoreticky podobné důsledky, projevují se však pravděpodobně s daleko menší intenzitou (biotransformační cesta prostřednictvím CYP3A4 je dominantní), a nejsou tedy klinicky relevantní. Imatinib je také inhibitorem aktivity transportéru BCRP (breast cancer resistance protein); tato interakce vají se klinicky závažné interakce) a poměrně silným inhibitorem metabolické aktivity CYP2D6 (dle inhibiční konstanty Ki ; klinicky se zatím takto neprojevuje) a  CYP3A4, přičemž je dokumentována řada lékových interakcí, především se substráty CYP3A4  [9]. Imatinib je také substrátem P-glykoproteinu (Pgp, produkt genu ABCB1) a je transportován přenašečem pro organické kationty (hOCT1). Interakce s inhibitory metabolické aktivity CYP3A, jež současně inhibují aktivitu Pgp, je tedy zvláště nebezpečná a může vyústit ve výrazné zvýšení plazmatické koncentrace a toxicity imatinibu [7,8,10]. Lékové interakce imatinibu Lékové interakce mohou být v  zásadě dvojího typu  – jednak mohou jiná léčiva ovlivňovat účinek imatinibu a  na druhé straně může imatinib ovlivňovat účinky jiných léčiv, především svým inhibičním účinkem na metabolickou aktivitu CYP3A4 a CYP2D6. Pro jednoduchost a s ohledem na zaměření článku se zde věnujeme pouze vlivu jiných látek na plazmatické koncentrace/účinek imatinibu. Mezi lékovými interakcemi výrazně převažují farmakokinetické nad farmakodynamickými. V  naprosté většině se jedná o  ovlivnění metabolické aktivity CYP3A4, v menší míře pak CYP2D6  a  CYP2C9. Současným podáním inhibitoru nebo induktoru metabolické aktivity těchto forem CYP dochází ke změnám v  plazmatických koncentracích a AUC (area under curve – plocha pod křivkou plazmatických koncentrací v čase) imatinibu. Tyto změny mohou být i velmi výrazné a klinicky relevantní (změny v AUC řádově o desítky procent) [11]. Druhý typ farmakokinetické interakce spočívá v inhibici aktivity P-glykoproteinového transportéru a tím ve zvýšení biologické dostupnosti a AUC imatinibu. Léčiva zapříčiňující tyto interakce jsou uvedena v tab. 1 [8–10,12]. Důsledkem současného podání induktorů metabolické aktivity CYP3A4 je zrychlení presystémové eliminace i biotransformace imatinibu a snížení AUC, cmax a snížení účinku imatinibu. Příkladem je snížení AUC0-–24h o 54 % po souběžném pětidenním podání imatinibu (400mg, 1× denně) a rifampicinu v denní dávce 600mg [13]. Tab. 1. Léčiva, která mohou významně ovlivnit plazmatickou koncentraci nebo účinek imatinibu [8–10,12]. Induktor Inhibitor CYP3A4 karbamazepin, dexametazon, dabrafenib, mitotan, nevirapin, bosentan, barbituráty, fenytoin, rifabutin, rifampicin chloramfenikol, některé makrolidy, (klaritromycin, telitromycin), azolová antimykotika (itrakonazol, ketokonazol, posakonazol, vorikonazol), inhibitory virových proteáz (atazanavir, boceprevir, lopinavir, nelfinavir, telaprevir, saquinavir, ritonavir, indinavir, darunavir, fosamprenavir) Pgp karbamazepin, dexametazon, doxorubicin, fenobarbital, fenytoin, primidon, rifampicin, třezalka tečkovaná, tenofovir, vinblastin abirateron, amiodaron, atorvastatin, karvedilol, klaritromycin, crizotinib, cyklosporin, darunavir, dipyridamol, grepfruitová šťáva, itrakonazol, ketokonazol, lapatinib, lopinavir meflochin, nelfinavir, nilotinib, progesteron, ritonavir, saquinavir, simeprevir, sunitinib, takrolimus,tamoxifen, telaprevir, vemurafenib, verapamil hOCT1 není známo amiodaron, levofloxacin, ganciklovir, chlorochin, indinavir, saquinavir, lamivudin, ranitidin, midazolam, metformin, progesteron hOCT1 – human organic cation transporter 1, transportér pro organické kationty 1 108 MOŽNÝ KLINICKÝ PŘÍNOS TERAPEUTICKÉHO MONITOROVÁNÍ HLADIN IMATINIBU V ONKOLOGII Klin Onkol 2015; 28(2): 105–111 ciál pro klinické využití a informační hodnota vyšetření konkrétních léčiv může být různorodá. Aby přinášelo vyšetření žádajícímu lékaři validní informaci a bylo nákladově efektivní, je velmi účelné mít zpracovánu metodiku TDM v  daném zdravotnickém zařízení, která definuje jednotlivé kroky a předpoklady vyšetření pro každé měřené léčivo (jak, kdy a kolik vzorků odebírat, jak je uchovávat a transportovat, kdy je/není účelné žádat stanovení koncentrací vzhledem k fázi léčby, na jaké otázky může vyšetření dát odpověď, sledované parametry a jejich doporučovaná rozmezí, náležitosti žádanky, potřebná biochemická vyšetření, lhůty dodání výsledků atd.). Zároveň je důležitá dobrá spolupráce žádajícího lékaře s  laboratoří, resp. interpretujícím klinickým farmaceutem/farmakologem, neboť bez správné interpretace výsledku a následného doporučení nebo návrhu opatření ztrácí vyšetření mnoho ze svého potenciálu, či může být dokonce kontraproduktivní. Terapeutické monitorování imatinibu v indikacích CML a GIST Imatinib má předpoklady k tomu, aby se u něj TDM stalo validním nástrojem k  optimalizaci a  individualizaci léčby. Dostupná evidence naznačuje, že jednou z příčin primární rezistence k léčbě inhibitory tyrozinkináz (TKi) mohou být nedostatečné plazmatické koncentrace léčiva. Podobně příčinou suboptimální odpovědi na léčbu imatinibem mohou být mezi jinými také problémy s compliance či interindividuální variabilita ve farmakokinetice léčiva [2]. K interindividuální variabilitě mohou dále významně přispívat lékové interakce diskutované výše. Guidelines NCCN pro léčbu CML doporučují v rámci monitorování odpovědi na léčbu TKi pravidelně posuzovat compliance a případný vliv lékových interakcí, pokud není odpověď na léčbu optimální [2]. Stanovení údolní plazmatické koncentrace (ctrough ) 1–3 měsíce po zahájení léčby může sloužit jako individuální referenční hladina pro další průběh léčby a případně časně upozornit na výrazně odlišné plazmatické koncentrace, než lze očekávat při daném dávkování. V dalším průběhu léčby může (při porovnání s aktuálně naměřenou hodg) dostupná validovaná, dostatečně citlivá a ekonomicky přijatelná analytická metoda pro stanovení koncentrací. Přítomnost aktivního metabolitu, významný interakční potenciál, nelineární farmakokinetika léčiva již v  rozmezí běžně používaných dávek či častý výskyt non-adherence jsou další faktory, které lze pomocí TDM zachytit či s nimi pracovat. TDM může být obecně užitečné např. pro: • potvrzení efektivních koncentrací (jako referenční hodnota pro další průběh léčby); • optimalizaci dávky na podkladě dosažení terapeutického referenčního rozmezí (např. pokud je efekt stávající léčby částečný/neuspokojivý a zároveň naměříme plazmatické koncentrace pod dolní hranicí referenčního rozmezí); • potvrzení suspektní toxicity (jsou přítomny známky toxicity daného léčiva a zároveň naměříme výrazně vysoké plazmatické koncentrace); • podezření na nonadherenci či selhávání léčby z  různých důvodů (plazmatické koncentrace pod limitem detekce či výrazně neodpovídající danému dávkování, neobvyklý poměr plazmatických koncentrací mateřské látky a metabolitu, velmi vysoké/nízké plazmatické koncentrace při běžném dávkování a dobré adherenci); • korekce dávkování při změnách renálních funkcí (typicky např. u aminoglykosidových antibiotik, vankomycinu, digoxinu); • posouzení vlivu komedikace (aktuálně nasazena/vysazena/změněna dávka medikace, která může snížit/zvýšit plazmatickou koncentraci sledovaného léčiva a potažmo jeho efekt, vliv zanechání kouření u některých léčiv atd.); • posouzení vlivu stavů, kdy lze předpokládat významné změny v distribuci léčiva (gravidita, ascites, edémy, kritické stavy, některá onemocnění, věkové a hmotnostní extrémy atd.). Měřená léčiva zpravidla splňují pouze některé z výše uvedených charakteristik „optimálního” léčiva, a proto i potennejasný a  výsledek předvídat prozatím nedokážeme. Dalším komplikujícím faktorem může být zvýšené riziko krvácení spojené se samotným imatinibem. Pokud jsou léčiva užívána současně, je nutná opatrnost, častější monitorování INR a zvýšený klinický dohled. Vhodnou alternativou warfarinu může být použití nízkomolekulárního heparinu. Interakce farmakodynamického nebo neznámého typu jsou vzácnější  – příkladem je očekávatelná interakce s vakcinací nebo imunosupresivy, zvýšení rizika agranulocytózy při současné komedikaci s  klozapinem, zvýšení rizika agranulocytózy a pancytopenie při současném užívání metamizolu, snížení účinku imatinibu po kombinaci s imunostimulačními látkami (např. i echinacea, beta-glukany), popř. snížení účinku tyreotropních látek. Možnosti a přínosy terapeutického monitorování léčiv Terapeutické monitorování léčiv (therapeutic drug monitoring – TDM) se zabývá stanovováním a následnou farmakokinetickou interpretací koncentrací léčiv v biologických vzorcích, vždy v kontextu klinických údajů a příslušných dat. V ČR se TDM již dlouhou řadu let využívá k  optimalizaci farmakoterapie léčivy, jako jsou aminoglykosidová antibiotika, vankomycin, digoxin, teofylin, amiodaron, celá řada antiepileptik a nověji také některých imunosupresiv, antipsychotik, azolových antimykotik a dalších látek. Největší benefit přináší TDM u léčiv, kterámajínásledujícícharakteristiky [17]: a) absence snadno měřitelného, bezpečného a validního ukazatele efektu léčby; b) toxicita či neúčinnost léčiva může přímo ohrozit stav pacienta; c) absence či slabá korelace dávka – klinická odpověď; d) úzké terapeutické rozmezí; e) korelace mezi plazmatickou koncentrací a klinickým efektem a/nebo toxicitou; f) velká interindividuální a  optimálně nízká intraindividuální variabilita v  koncentracích léčiva po podání stejné dávky; MOŽNÝ KLINICKÝ PŘÍNOS TERAPEUTICKÉHO MONITOROVÁNÍ HLADIN IMATINIBU V ONKOLOGII Klin Onkol 2015; 28(2): 105–111 109 s ctrough  ≥ 1 000 ng/mL oproti nižším ctrough (p = 0,02) [27]. Vztah mezi plazmatickými koncentracemi a klinickou odpovědí u pacientů s CML byl pozorován také v observačních studiích v reálných klinických podmínkách s velkým počtem zařazených pacientů (n = 1 216 [28] a n = 2 478 [29]). V roce 2014  byla publikována praktická guidelines autorské skupiny Yu et al (Cancer Institute-Antoni van Leeuwenhoek, Amsterdam) pro terapeutické monitorování inhibitorů tyrozinkináz (TKi)  [30], která na základě dosavadní evidence z observačních studií navrhují u pacientů s CML léčených imatinibem ctrough  ≥ 1 000 ng/mL jako cílový farmakokinetický parametr. Publikovány byly výsledky zatím jediné dokončené prospektivní randomizované intervenční studie Imatinib COncentration Monitoring Evaluation (I-COME) [22]. Cílem studie bylo zjistit, zda rutinní TDM s individualizací dávkování (k dosažení cílové plazmatické koncentrace ctrough   =  1  000  ng/mL) může zlepšit klinickou odpověď u  pacientů s  CML oproti kontrolní skupině bez rutinního TDM během jednoletého sledování. Autoři uvádějí, že bohužel nebylo možné formálně prokázat benefit rutinního TDM, protože během dvouletého náboru nebylo dosaženo plánovaného počtu zařazených pacientů (předpoklad 300, celkem zařazeno pouze 56), a dále z důvodu překvapivě nízké adherence lékařů (50 %) k doporučeným úpravám dávkování v intervenční skupině. Autoři diskutují určitá pozitivní zjištění u pacientů, u kterých byly úpravy dávkování respektovány, nicméně tato nebyla statisticky hodnotitelná. Očekávají se výsledky další prospektivní intervenční randomizované studie OPTIM IMATINIB [31], která si klade jako hlavní cíl posoudit účinnost a  realizovatelnost individualizace dávkování imatinibu založené na měření údolních plazmatických koncentrací imatinibu ve vztahu k dosažení MMR po 12 měsících léčby. Guidelines NCCN pro léčbu CML [2] aktuálně rutinní TDM imatinibu (tedy rutinní individualizaci dávkování k dosažení cílových plazmatických koncentrací) nedoporučují. Důvodem je doposud chybějící a medián všech hodnot ctrough v souboru dosahoval 979 ± 530 ng/mL a 879 ng/mL (min.–max., 153–3  910  ng/mL). Z  důvodu vysoké interindividuální variability byli pacienti rozděleni na kvartily Q1–Q4 dle dosahovaných ctrough a tyto následně korelovány s odpovědí. Kumulativní relativní četnosti CCyR i MMR se statisticky významně lišily mezi jednotlivými kvartily (p = 0,01; resp. p = 0,02 celkově). Ctrough byly statisticky významně vyšší u pacientů, kteří dosáhli CCyR ve srovnání s  těmi, kteří CCyR nedosáhli (1 009 ± 544 ng/mL, vs. 812 ± 409 ng/mL, průměr ± SD; p = 0,01). Byl také pozorován trend ke kratšímu přežívání bez sledované události (event-free survi- val – EFS) u pacientů s nižšími ctrough . Očekávané relativní četnosti EFS po pěti letech léčby byly 78 %, 83 % a 89 % pro Q1, Q2–Q3 a Q4 (p = 0,16) [24]. Takahashi et al provedli soubornou analýzu dat šesti japonských studií s pacienty s CML (n = 254) [25]. Průměr a  medián ctrough celého souboru činily 1 011 ± 565 a 900 ng/mL (min.–max., 111–3 620 ng/mL). Autoři pozorovali statisticky významně vyšší ctrough u pacientů, kteří dosáhli MMR (1 107 ± 594 ng/mL vs. 873 ± 529 ng/mL; p = 0,002) a CCyR (1 058 ± 585 ng/mL vs. 835 ± 524 ng/mL; p = 0,033) oproti těm, kteří MMR a CCyR nedosáhli. Pravděpodobnost dosažení MMR byla statisticky významně vyšší u pacientů s ctrough  > 1 002 ng/mL oproti ctrough  < 1 002 ng/mL (p = 0,012) [25]. Koren-Michowitz et al ve své studii analyzovali plazmatické koncentrace u  191  pacientů s  CML léčených imatinibem a tyto porovnávali s dosažením CCyR [26]. Medián a průměr ctrough v celém souboru dosahoval 994 ng/mL, resp. 1 070 ± 686 ng/mL. Pacienti, kteří dosahovali CCyR, měli statisticky hraničně vyšší ctrough (1 078 ± 545 ng/mL) než pacienti bez CyR (827 ± 323 ng/mL; p  =  0,045). Při souborném hodnocení po rozdělení pouze do dvou skupin pacienti s CCyR/částečnou CyR dosahovali statisticky významně vyšších ctrough než pacienti s  minimální/žádnou CyR (1  066  ±  534  ng/mL vs. 814 ± 314 ng/mL; p = 0,002) [26]. Marin et al ve studii s 84 pacienty s CML zaznamenali statisticky významně vyšší procentuální četnost MMR u pacientů notou) pomoci posoudit vliv lékové interakce či adherence k léčbě. TDM může pomoci vyloučit nedostatečné plazmatické koncentrace jako možnou příčinu suboptimální odpovědi či primární rezistence nebo naopak vysoké plazmatické koncentrace jako možnou příčinu výskytu známek toxicity [18–21]. Tento model, tzv. rescue TDM (TDM v případě potřeby, při výskytu potíží s léčbou), je dostupný např. ve Švýcarsku [22]. CML V posledních přibližně sedmi letech byly v renomovaných onkologických časopisech publikovány klinické studie, které se zabývaly primárně vztahem mezi plazmatickými koncentracemi imatinibu a klinickou odpovědí. Definice cytogenetických a  molekulárních odpovědí v níže uvedených studiích odpovídá definicím dle European Leukemia Net [4]. Picard et al popisují studii s  68  pacienty s CML léčenými imatinibem v dávce 400  nebo 600 mg/den po dobu min. 12 měsíců [23]. Pacienti užívající 400mg, resp. 600mg/den dosahovali údolních (tj. před podáním další dávky) plazmatických koncentrací (ctrough ) 1 058 ± 557 ng/mL, resp. 1 444 ± 710 ng/mL (průměr ± SD), přičemž hodnoty vykazovaly vysokou interindividuální variabilitu (min.–max., 181–2 947 ng/mL). Ctrough byly retrospektivně korelovány s odpovědí. Statisticky významně vyšší ctrough byly pozorovány ve skupině pacientů s kompletní cytogenetickou odpovědí (complete cytogenetic response – CCyR, 1 123 ± 617 ng/mL vs. 694  ±  556  ng/mL, průměr  ±  SD; p = 0,03) a tzv. velkou molekulární odpovědí (major molecular response – MMR, 1 452 ± 649 ng/mL vs. 869 ± 427 ng/mL, průměr ± SD; p < 0,001) oproti skupině bezCCyR,resp.MMR,nezávislenaužívané denní dávce léčiva. ROC analýzou byla vypočtena prahová ctrough 1 002 ng/mL, která odlišovala respondéry/nonrespondéry dle dosažení MMR se senzitivitou 77 % a specificitou 71 % [23]. Larson et al potvrdili tyto výsledky v rozsáhlejší retrospektivní studii zahrnující 351 pacientů s CML [24]. Pacienti užívali 400mg imatinibu denně. V této studii byly ctrough stanovovány ve fázi steady state a  korelovány s  odpovědí během pětiletého sledování. Průměr 110 MOŽNÝ KLINICKÝ PŘÍNOS TERAPEUTICKÉHO MONITOROVÁNÍ HLADIN IMATINIBU V ONKOLOGII Klin Onkol 2015; 28(2): 105–111 je koncept terapeutického monitorování léčiv a úprava dávky během léčby na základě naměřených údolních plazmatických koncentrací (ctrough ; koncentrace před podáním další dávky) ve fázi steady-state. Dalším možným a i v praxi využívaným postupem „targeted therapy“ je tzv. koncept TAD (toxicity-adjusted dosing) a úprava dávky na základě klinicky pozorovaných nežádoucích účinků, kdy jejich absence je považována za projev nedostatečného dávkování a naopak významné nežádoucí účinky (grade 3–4) za projev „předávkování“. Třetím zmiňovaným postupem je tzv. ramp-dosing (varianta TAD), kdy v klinické praxi postupně zvyšujeme dávku až k projevům toxicity. Nežádoucí účinky jsou v  druhém a třetím případě využívány jako náhradní cílový parametr. První varianta, tedy TDM s klinicky ověřeným vztahem mezi plazmatickou koncentrací a účinkem léčiva (kdy je známa „cut-off“ plazmatická koncentrace nebo ještě lépe terapeutické rozmezí, stanovené ideálně na základě randomizovaných prospektivně vedených intervenčních klinických studií) s následným standardním zavedením do klinické praxe se jeví jako optimální. Lze uvést příklady zemí, ve kterých má terapeutické monitorování plazmatických koncentrací protinádorových léčiv dlouhou tradici a kde je i TDM perorálních TKi (ve formě rescue TDM) zavedeno do praxe řady klinických pracovišť. K těmto zemím patří zejm. Francie, Nizozemí nebo Švýcarsko. V  květnu 2014 byla v European Journal of Cancer publikována kolektivem autorů právě z  těchto zemí práce s  názvem „Therapeutic drug monitoring in cancer – Are we missing a trick?“, která řeší příspěvek TDM k možnému vylepšení terapeutických postupů u řady cílených protinádorových léčiv, nikoliv však specificky pouze u imatinibu [34]. Jak již bylo uvedeno výše, v případě imatinibu jsou známy výsledky observačních studií, které popisují korelaci plazmatických koncentrací a odpovědi na léčbu u pacientů s CML a GIST. Publikováno bylo systematické review na toto téma [21] a v případě CML také výsledky observačních studií v  reálindividuální variabilitu ve vztahu k terapeutickým účinkům i účinkům nežádoucím. V klinické praxi vidíme pacienty, kteří při doporučeném dávkování neodpovídají na léčbu dle předpokladů, kteří mají řadu nežádoucích účinků či naopak u kterých žádné nežádoucí účinky nezaznamenáváme. Z výzkumného hlediska jsou tyto variability v  onkologii, zejm. ve vztahu k  účinku, zkoumány především z hlediska rozdílností cílové struktury či signální dráhy na úrovni tumoru s využitím dnes již celkem bezproblémově dostupných molekulárně-genetických nebo proteomických metod; publikované práce s  touto tematikou lze počítat dnes již v tisících. Mnohem méně prací se však zabývá variabilitou na úrovni samotného„nositele“ tumoru, tedy pacienta. K základním postulátům farmakologie patří fakt, že léčivo ovlivní onemocnění konkrétního pacienta a  jeho nádor, ale i  konkrétní pacient (resp. organizmus) ovlivní chování léčiva po jeho podání. Fakt, který bývá opomíjen, resp. nebývá zdůrazňován, je, že „personalizovaně“ musíme vnímat nejen strukturu, na kterou léčivo působí – tedy „target“, ale i hostitele – tedy jedince, kterému je léčivo podáváno – tedy„host-related factors“. Tento typ personalizace má vycházet ze znalosti genetického pozadí (farmakogenetiky), souběžných komorbidit, lékových interakcí, u perorální léčby i adherence pacienta a podobně. Nesmíme zapomenout, že „personalizovaná terapie“ se vždy týká pacienta – neléčíme nádor, ale konkrétního jedince s nádorem. Obdobnějetomui u tzv.targetedtherapy, kdy tato molekulárně cílená léčba např. inhibitory tyrozinkináz vychází ve svém standardním dávkování z principu zahájení léčby fixními dávkami, které nerespektují významné a již publikované interindividuální rozdíly ve farmakokinetice těchto léčiv. Podávání fixních dávek může rezultovat v suboptimální plazmatické koncentrace nedostačující k inhibici cílové struktury, jak je patrné z výsledků klinických studií uvedených výše. Konkrétně u imatinibu je diskuze stran optimální dávky a délky podávání stále živá. Je otázkou, jakým způsobem upravit dávkování v klinické praxi. Navržených přístupů je několik a jedním z nich evidence z prospektivně vedených intervenčních studií, které by prokazovaly benefit rutinního TDM ve vztahu ke klinické odpovědi. Uvádí však užitečnost TDM např. při posuzování adherence k léčbě. GIST Demetri et al zkoumali u 73 pacientů s pokročilým GIST vztah mezi plazmatickými koncentracemi imatinibu a odpo- vědí [32]. Pacienti byli randomizováni do skupin užívajících 400 nebo 600mg imatinibu denně. Ctrough v ustáleném stavu (prooběskupiny)vykazovalyvýznamnou interindividuální variabilitu (min.–max., 414–4 182 ng/mL). Mezi skupinami pacientů s  rozdílnou odpovědí na léčbu (kompletní nebo parciální odpovědí nebo stabilizací onemocnění) nebyl pozorován statisticky významný rozdíl mediánů ctrough (p = 0,25). Když byli pacienti rozděleni do kvartilů dle dosahovaných ctrough , pacienti v  dolním kvartilu Q1 (< 1 100 ng/mL)dosahovalistatisticky významně kratšího času do progrese onemocnění (time to progression – TTP, medián 11,3 měsíce) než pacienti ve vyšších kvartilech: medián TTP 30,6  měsíce v  Q2–Q3  (1  100–2  040  ng/mL), resp. 33,1 měsíce v Q4 (> 2 040 ng/mL) (p = 0,0105 celkově, p = 0,0029 pro Q1 vs. Q2–Q4). Statisticky signifikantní rozdíl mezi jednotlivými kvartily nebyl pozorován ve vztahu k mediánu celkového přežití (overall survival – OS) (p = 0,16) [32]. Praktická guidelines autorské skupiny Yu et al (Cancer Institute-Antoni van Leeuwenhoek, Amsterdam) navrhují u pacientů s GIST léčených imatinibem cílovou ctrough   ≥  1  100  ng/mL  [30]. Zatím jediná prospektivní intervenční studie SARC019 [33], která měla za cíl zjistit, zda eskalace dávky imatinibu k dosažení ctrough  ≥ 1 100 ng/mL zlepší klinickou odpověď u nemocných s metastazujícím GIST, byla ukončena předčasně kvůli pomalému náboru pacientů, přičemž data nebylo možno analyzovat. Evidence z prospektivních studií tedy aktuálně chybí a v současnosti rovněž nelze na základě principů evidence-based medicine (EBM) rutinní TDM imatinibu v léčbě GIST doporučit. Diskuze a závěr V klinických studiích i v reálné klinické praxi lze pozorovat významnou inter- MOŽNÝ KLINICKÝ PŘÍNOS TERAPEUTICKÉHO MONITOROVÁNÍ HLADIN IMATINIBU V ONKOLOGII Klin Onkol 2015; 28(2): 105–111 111 22. Gotta V, Widmer N, Decosterd LA et al. Clinical usefulness of therapeutic concentration monitoring for imatinib dosage individualization: results from a randomized controlled trial. Cancer Chemother Pharmacol 2014; 74(6): 1307–1319. doi: 10.1007/s00280-014-2599-1. 23. Picard S, Titier K, Etienne G et al. Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia. Blood 2007; 109(8): 3496–3499. 24. Larson RA, Druker BJ, Guilhot F et al. Imatinib pharmacokinetics and its correlation with response and safety in chronic-phase chronic myeloid leukemia: a subanalysis of the IRIS study. Blood 2008; 111(8): 4022–4028. doi: 10.118 2/blood-2007-10-116475. 25. Takahashi N, Wakita H, Miura M et al. Correlation between imatinib pharmacokinetics and clinical response in Japanese patients with chronic-phase chronic myeloid leukemia. Clin Pharmacol Ther 2010; 88(6): 809–813. doi: 10.1038/clpt.2010.186. 26. Koren-Michowitz M, Volchek Y, Naparstek E et al. Imatinib plasma trough levels in chronic myeloid leukaemia: results of a multicentre study CSTI571AIL11TGLIVEC. Hematol Oncol 2012; 30(4): 200–205. doi: 10.1002/hon.2005. 27. Marin D, Bazeos A, Mahon FX et al. Adherence is the critical factor for achieving molecular responses in patients with chronic myeloid leukemia who achieve complete cytogenetic responses on imatinib. J Clin Oncol 2010; 28(14): 2381–2388. doi: 10.1200/JCO.2009.26. 3087. 28. Bouchet S, Titier K, Moore N et al. Therapeutic drug monitoring of imatinib in chronic myeloid leukemia: experience from 1216 patients at a centralized laboratory. Fundam Clin Pharmacol 2013; 27(6): 690–697. doi: 10.1111/fcp.12007. 29. Gotta V, Bouchet S, Widmer N et al. Large-scale imatinib dose-concentration-effect study in CML patients under routine care conditions. Leuk Res 2014; 38(7): 764–772. doi: 10.1016/j.leukres.2014.03.023. 30. Yu H, Steeghs N, Nijenhuis CM et al. Practical guidelines for therapeutic drug monitoring of anticancer tyrosine kinase inhibitors: focus on the pharmacokinetic targets. Clin Pharmacokinet 2014; 53(4): 305–325. doi: 10.1007/s40262-014-0137-2. 31. OPTIM IMATINIB. A prospective randomized phase II study evaluating the monitoring of imatinib mesylate (Gliveec®) plasmatic through level in patients newly diagnosed with chronic phase chronic myelogenous leukaemia (CP-CML) [cited 2015 Jan 25]. Available from: https://www.clinicaltrialsregister.eu/ctr-search/trial/2010 -019568-35/FR. 32. Demetri GD, Wang Y, Wehrle E et al. Imatinib plasma levels are correlated with clinical benefit in patients with unresectable/metastatic gastrointestinal stromal tumors. J Clin Oncol 2009; 27(19): 3141–3147. doi: 10.1200/JCO.2008.20.4818. 33. SARC019. A randomized, phase 3 study of dose escalation versus no dose escalation of Imatinib in metastatic GIST patients with Imatinib trough levels less than 1100 nanograms/mL [cited 2015 Jan 27]. 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Drug interactions with the tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib. Blood 2011; 117(8): E75–E87. doi: 10.1 182/blood-2010-07-294330. 11. Dutreix C, Peng B, Mehring G et al. Pharmacokinetic interaction between ketoconazole and imatinib mesylate (Glivec) in healthy subjects. Cancer Chemother Pharmacol 2004; 54(4): 290–294. 12. White DL, Saunders VA, Dang PO et al. OCT-1-mediated influx is a key determinant of the intraceflular uptake of imatinib but not nilotinib, (AMN107): reduced OCT-1 activity is the cause of low in vitro sensitivity to imatinib. Blood 2006; 108(2): 697–704. 13. Bolton AE, Peng B, Hubert M et al. Effect of rifampicin on the pharmacokinetics of imatinib mesylate (Gleevec, STI571) in healthy subjects. Cancer Chemother Pharmacol 2004; 53(2): 102–106. 14. Houghton PJ, Germain GS, Harwood FC et al. Imatinib mesylate is a potent inhibitor of the ABCG2 (BCRP) transporter and reverses resistance to topotecan and SN-38 in vitro. Cancer Res 2004; 64(7): 2333–2337. 15. Breccia M, Santopietro M, Loglisci G et al. Concomitant use of imatinib and warfarin in chronic phase chronic myeloid leukemia patients does not interfere with drug efficacy. Leuk Res 2010; 34(8): e224–e225. doi: 10.1016/j. leukres.2010.03.015. 16. Lin AM, Rini BI, Derynck MK et al. A phase I trial of docetaxel/estramustine/imatinib in patients with hormone-refractory prostate cancer. Clin Genitourin Cancer 2007; 5(5): 323–328. 17. MacKichan JJ. Interpretation of serum drug concentrations. In: Lee M (ed.). Basic skills in interpreting laboratory data. 5th ed. Bethesda: American Society of Health-System Pharmacists 2013: 71–117. 18. Baccarani M, Dreyling M, Group EG. Chronic myeloid leukaemia: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2010; 21 (Suppl 5): v165–167. doi: 10.1093/annonc/mdq201. 19. Teng JF, Mabasa VH, Ensom MH. The role of therapeutic drug monitoring of imatinib in patients with chronic myeloid leukemia and metastatic or unresectable gastrointestinal stromal tumors. Ther Drug Monit 2012; 34(1): 85–97. doi: 10.1097/FTD.0b013e31823cdec9. 20. Gao B, Yeap S, Clements A et al. Evidence for therapeutic drug monitoring of targeted anticancer therapies. J Clin Oncol 2012; 30(32): 4017–4025. doi: 10.1200/JCO.2012.43.5362. 21. Gotta V, Buclin T, Csajka C et al. Systematic review of population pharmacokinetic analyses of imatinib and relationships with treatment outcomes.Ther Drug Monit 2013; 35(2): 150–167. doi: 10.1097/FTD.0b013e318284ef11. ných klinických podmínkách  [28,29]. Cílené prospektivní intervenční randomizované studie [22,33] zatím nerozšířily naše poznatky; další cílená prospektivní studie s pacienty s CML [31] stále běží. V případě, že se podaří potvrdit pozorování a poznatky z observačních studií v robustních prospektivních intervenčních klinických studiích, může se rutinní TDM (s  úpravou dávkování k  cílovým plazmatickým koncentracím) zařadit mezi cenné nástroje sloužící k optimalizaci a individualizaci léčby imatinibem. Ve světle současné evidence a  při zachování principů EBM nelze rutinní TDM imatinibu aktuálně doporučit. Nicméně poznatky ze zahraničí napovídají, že tzv. rescue TDM, tedy využití stanovení plazmatických koncentrací imatinibu individuálně ve specifických případech (posouzení adherence k léčbě, vlivu lékové interakce, v případě suboptimální klinické odpovědi, selhání léčby či výskytu neobvyklých/závažných nežádoucích účinků) může být užitečné již nyní, což je v souladu s guidelines NCCN v případě obou diagnóz, tedy CML i GIST. Literatura 1. Klener P, Klener P Jr. ABL1, SRC a další nereceptorové tyrozinkinázy jako nové cíle specifické protinádorové léčby. Klin Onkol 2010; 23(4): 203–209. 2. National Comprehensive Cancer Network [homepage on the Internet]. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines): Chronic Myelogenous Leukemia, Version 1.2015 [cited 2015 Jan 18]. Available from: http://www.nccn.org/professionals/physician_ gls/pdf/cml.pdf. 3. National Comprehensive Cancer Network [homepage on the Internet]. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines): Soft Tissue Sarcoma, Version 2.2014 [cited 2015 Feb 1]. Available from: http://www. nccn.org/professionals/physician_gls/pdf/sarcoma.pdf. 4. Baccarani M, Deininger MW, Rosti G et al. European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013. Blood 2013; 122(6): 872–884. doi: 10.1182/blood-2013-05-501 569. 5. Liu Y, Ramírez J, Ratain MJ. Inhibition of paracetamol glucuronidation by tyrosine kinase inhibitors. Br J Clin Pharmacol 2011; 71(6): 917–920. doi: 10.1111/j.1365-212 5.2011.03911.x. 6. Schmidli H, Peng B, Riviere GJ et al. Population pharmacokinetics of imatinib mesylate in patients with chro- Oncotarget550www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 1 Immunohistochemical prediction of lapatinib efficacy in advanced HER2-positive breast cancer patients Renata Duchnowska1 , Piotr J. Wysocki2 , Konstanty Korski3 , Bogumiła Czartoryska- Arłukowicz4 , Anna Niwińska5 , Marlena Orlikowska6 , Barbara Radecka7 , Maciej Studziński8 , Regina Demlova9 , Barbara Ziółkowska10 , Monika Merdalska11 , Łukasz Hajac12 , Paulina Myśliwiec13 , Dorota Zuziak14 , Sylwia Dębska-Szmich15 , Istvan Lang16 , Małgorzata Foszczyńska-Kłoda2 , Bożenna Karczmarek-Borowska17 , Anton Żawrocki18 , Anna Kowalczyk18 , Wojciech Biernat18 , Jacek Jassem18 , for the Central and East European Oncology Group (CEEOG)  1 Military Institute of Medicine, Oncology Department, Warsaw, Poland  2 West Pomeranian Cancer Center, Szczecin, Poland  3 Greater Poland Cancer Center, Poznań, Poland  4 Białystok Oncology Center, Białystok, Poland  5 The Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland  6 Warmia and Masuria Oncology Center, Olsztyn, Poland  7 Opole Oncology Center, Poland  8 Oncology Center, Bydgoszcz, Poland  9 Masaryk Memorial Cancer Institute, Brno, Czech Republic 10 Chemotherapy Department, Regional Hospital, Wrocław, Poland 11 Oncology Center, Kielce, Poland 12 Oncology Center, Wrocław, Poland 13 Oncology Center, Zielona Góra, Poland 14 Oncology Center, Bielsko-Biała, Poland 15 Medical University of Łódź, Łódź, Poland 16 Department of Medical Oncology, National Institute of Oncology, Budapest, Hungary 17 Department of Chemotherapy, Subcarpathian Oncology Center, Rzeszów, Poland 18 Medical University of Gdańsk, Gdańsk, Poland Correspondence to: Renata Duchnowska, e-mail: rdtt@wp.pl Keywords: breast cancer, epidermal growth factor receptor type 2, lapatinib, mTOR, p-MAPK Received: May 19, 2015 Accepted: November 13, 2015   ­   Published: November 24, 2015 ABSTRACT Molecular mechanisms of lapatinib resistance in breast cancer are not well understood. The aim of this study was to correlate expression of selected proteins involved in ErbB family signaling pathways with clinical efficacy of lapatinib. Study group included 270 HER2-positive advanced breast cancer patients treated with lapatinib and capecitabine. Immunohistochemical expression of phosphorylated adenosine monophosphate-activated protein (p-AMPK), mitogen-activated protein kinase (p-MAPK), phospho (p)-p70S6K, cyclin E, phosphatase and tensin homolog were analyzed in primary breast cancer samples. The best discriminative value for progression-free survival (PFS) was established for each biomarker and subjected to multivariate analysis. At least one biomarker was determined in 199 patients. Expression of p-p70S6K was independently associated with longer (HR 0.45; 95% CI: 0.25–0.81; p = 0.009), and cyclin E with shorter PFS (HR 1.83; 95% CI: 1.06–3.14; Oncotarget551www.impactjournals.com/oncotarget p = 0.029). Expression of p-MAPK (HR 1.61; 95% CI 1.13–2.29; p = 0.009) and cyclin E (HR 2.99; 95% CI: 1.29–6.94; p = 0.011) was correlated with shorter, and expression of estrogen receptor (HR 0.65; 95% CI 0.43–0.98; p = 0.041) with longer overall survival. Expression of p-AMPK negatively impacted response to treatment (HR 3.31; 95% CI 1.48–7.44; p = 0.004) and disease control (HR 3.07; 95% CI 1.25–7.58; p = 0.015). In conclusion: the efficacy of lapatinib seems to be associated with the activity of downstream signaling pathways – AMPK/mTOR and Ras/Raf/ MAPK. Further research is warranted to assess the clinical utility of these data and to determine a potential role of combining lapatinib with MAPK pathway inhibitors. INTRODUCTION The introduction of trastuzumab, a monoclonal antibody directed against the epidermal growth factor 2 receptor (HER2) has led to major improvement in the treatment of patients with HER2-positive breast cancer [1–5]. The therapeutic mechanisms of trastuzumab involve both the inhibition of HER2-dependent signaling pathways and the engagement of immune responses via antibody-dependent cellular cytotoxicity [6]. Despite impressive clinical efficacy of trastuzumab, many patients are refractory to this agent or develop secondary resistance. The postulated mechanism of trastuzumab resistance include the expression of the truncated-active form of the HER2 receptor (p95HER2), the cross-talk between HER2 and insulin-like growth factor-1 receptor, the deficiency of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and activating mutations in the p110-alpha subunit of phosphoinositide- 3-kinase (PI3K), and activity of Rac1 – a Ras-like small GTPase affecting trastuzumab-mediated endocytosis of the HER2 receptor [7–19]. A small-molecule HER2 kinase inhibitor – lapatinib entered the clinical practice later than trastuzumab and has been mostly used as a second-line therapy [20]. Due to its different mode of action, the molecular resistance mechanisms of lapatinib can not be simply extrapolated from those of trastuzumab [21, 22]. The resistance to this compound may be caused by mechanisms occurring at various levels within a cancer cell: the outer/inner leaflet of the plasma membrane, cytoplasm or nucleus [14, 23–30]. Normally, activation of growth factor-associated signaling cascades is initiated at the plasma membrane in response to receptor activation (homo-, or heterodimerization) [31]. Subsequently, the signal is transmitted downstream towards the nucleus via a signaling network, which comprises multiple kinases. Signal transduction pathways in cancer cells may become activated regardless of the receptor status. Spontaneously activated signal transduction elements may be responsible for resistance to receptor-targeted therapies, since crucial pathways remain active despite receptor blockade. Hence, the activity of signal transduction molecules may potentially correlate with the resistance to lapatinib. This study investigated the immunohistochemical (IHC) expression of selected molecules involved in the important signaling pathways associated with the family of epidermal growth factor (ErbB) receptors: phosphorylated adenosine monophosphate-activated protein alpha 1 (p-AMPK-Ser486), the mitogen-activated protein kinase (p-MAPK-T185 + Y187 + T202 + Y204), phospho (p)-p70S6K, the hypoxia-inducible factor 2 alpha (HIF2 alpha), PTEN, and cyclin E. Their status was retrospectively correlated with the clinical efficacy of lapatinib. Our aim was to shed new light on the molecular mechanisms involved in the resistance of breast cancer to lapatinib. RESULTS Patient characteristics Tumor samples from 270 patients were subjected to analysis, of which in 199 at least one biomarker was determined (Figure 1, Table 1). Eighty-four percent of the tumors were invasive ductal cancers (no special type), 67% were estrogen receptor (ER)-negative and 77% progesterone receptor (PR)-negative. Eleven percent of patients presented with metastatic disease at initial breast cancer diagnosis. Radical surgery was performed in 91% of patients; 98% received chemotherapy in (neo)adjuvant and/or a metastatic setting, 36% received endocrine therapy and all were administered trastuzumab in an adjuvant or a metastatic setting, usually in combination with chemotherapy. In 69% of patients, the first manifestation of progression was distant metastasis, with viscera being the most common dominant metastatic site. Forty-three percent of patients developed brain metastases during the course of their disease. Clinical outcomes The median duration of lapatinib and capecitabine therapy was 6 months (range 0–52). In 82% of patients, treatment was terminated due to disease progression. Other reasons were toxicity (7%), patient refusal (2%), death (3%), others (5%) and unknown (1%). The best response to a combination of lapatinib and capecitabine were CR (5%), PR (31%), stable disease (42%) and progression (16%); in the remaining 6% of patients response was unknown or not evaluated. Oncotarget552www.impactjournals.com/oncotarget The duration of follow-up from breast cancer diagnosis varied from 6.7 to 242 months. The median PFS from the start of lapatinib therapy was 6.2 months (range 0–54). PFS was significantly longer in patients with response to treatment (median 10.4 months; hazard ratio [HR] 0.44, 95% confidence interval [CI] 0.35–0.56, p < 0.01) or disease control (median 8.1 months; HR 0.27; 95%CI 0.20–0.35; p < 0.01), compared to those with refractory disease (median 2.3 months). The status of p-AMPK alpha1, p-MAPK, p-p70S6K, HIF-2 alpha, cyclin E and PTEN was determined in 176, 184, 190, 188, 180 and 176 cases, respectively (CONSORT Diagram, Figure 1). The immunostained sections of all studied proteins are shown on Figure 2. In all cases staining was heterogeneous. For cyclin E the staining was nuclear, for HIF-2 cytoplasmic and for p-AMPK alpha1, p-MAPK, p-p70S6K, and PTEN nuclear and cytoplasmic. Two of the examined biomarkers: p-p70S6K and cyclin E proved predictive for PFS, with the best discriminating H-scores of 10 and 200, respectively. The expression of p-p70S6K (HR 0.47; 95%CI 0.26–0.86; p = 0.014) was associated with longer, and the expression of cyclin E (HR 1.71; 95%CI 1.00–2.93; p = 0.05) with shorter PFS. The predictive value of these two biomarkers was confirmed in the multivariate analysis (HR 0.45; 95% CI 0.25–0.81; p = 0.009 and 1.83; 95%CI 1.06–3.14; p = 0.029, respectively; Figure 3A–3B and Table 2). Negative prognostic factors for OS included the expression of p-MAPK (HR 1.68; 95%CI 1.18–2.40; p = 0.007) and cyclin E (HR 2.86; 95%CI 1.23–6.66; p = 0.015; Figure 4A–4B and Table 2), in addition to regional vs. local type of first progression (HR 3.39; 95%CI 1.38–8.28; p = 0.008), whereas ERα expression positively impacted OS (HR 0.60; 95%CI 0.39–0.92; p = 0.033; Figure 4C and Table 2). The significance of these biomarkers was confirmed in the multivariate analysis (Table 2). The expression of p-AMPK alpha1 negatively impacted response to treatment (HR 3.31; 95%CI 1.48– 7.44; p = 0.004) and disease control (HR 3.07; 95%CI 1.25–7.58; p = 0.015) in the multivariate analysis. A subset analysis considering ER status showed that p-MAPK expression in the ER-positive cohort was associated with significantly shorter PFS (HR 3.14; 95%CI 1.59–6.20; p = 0.001) and OS (HR 2.53; 95%CI 1.05– 6.11; p = 0.038), whereas no such correlation was seen in the ER-negative cohort (Table 3). Another biomarker with adverse impact in the ER-positive cohort was HIF-2 alpha (HR for OS 3.38; 95%CI 1.13–10.08; p = 0.029). In turn, the expression p-p70S6K in the ER-positive cohort was associated with longer PFS (HR 0.22; 95%CI 0.06–0.75; p = 0.016; Table 3). The expression of cyclin E was more common in the ER-negative cohort (p = 0.003) and in this subset associated with shorter PFS (HR 1.78; 95%CI 1.02–3.09; p = 0.041) and OS (HR 2.38; 95%CI 1.09– 5.18; p = 0.029). No such impact of cyclin E was found in the ER-positive subgroup. The significance of these biomarkers was confirmed in the multivariate analysis. Figure 1: CONSORT Diagram. Origin of patients analyzed for p-AMPK alpha1, p-MAPK, p-p70S6K, cyclin E, HIF2 alpha and PTEN. Oncotarget553www.impactjournals.com/oncotarget Figure 2: Immunohistochemical intensity scoring of p-AMPK alpha1, p-MAPK, p-p70S6K, HIF-2 alpha, cyclin E and PTEN (magnification, x20). A. weak; B. moderate; C. strong. Oncotarget554www.impactjournals.com/oncotarget DISCUSSION Despite spectacular progress in the treatment of HER2-positive breast cancer, overcoming primary and acquired resistance to anti-HER2 agents remains a critical challenge [32, 33]. In contrast to trastuzumab, the anti-tumor activity of lapatinib is based solely on the intracellular inhibition of cell-signaling by competing with ATP for the ATP-binding domain in the cytoplasmic tail of the tyrosine kinase receptor – mostly HER2 and EGFR [34, 35]. Accordingly, the postulated mechanisms underlying lapatinib resistance differs from those reported for trastuzumab. Previous studies have shown up-regulated ER-associated signaling genes, including FOXO3a and caveolin-1, or Akt pathway transcripts (AKT1, MAPK9, HSPCA, IRAK1, CCND1) in lapatinib resistant cells [36]. Other factors contributing to lapatinib resistance include dominant activating mutations in PIK3CA, E545K and Figure 3: Kaplan-Meier progression free survival curves. A. p-p70S6K ≥ 10 staining H-score (HR 0.47; p = 0.014); B. cyclin E ≥ 200 staining H-score (HR 1.71; p = 0.05). Oncotarget555www.impactjournals.com/oncotarget Table 1: Patient characteristics Variable n = 199 Mean age at diagnosis (range) 50 (23–81) Menopausal status  Premenopausal  Postmenopausal 78 (43) 102 (57) Histology  Ductal  Lobular  Other   Not determined   Ductal and lobular 168 (84) 15 (8) 5 (3) 8 (4) 2 (1) Grade  1  2  3 3 (2) 74 (46) 82 (52) Estrogen receptor  Negative  Positive 134 (67) 65 (33) Progesterone receptor  Negative  Positive 153 (77) 46 (23) Clinical stage at diagnosis  I  IIA  IIB  IIIA  IIIB  IIIC  IV 16 (8) 22 (12) 30 (16) 44 (23) 42 (22) 16 (8) 21 (11) Breast cancer surgery  No  Mastectomy   Breast conserving surgery 17 (9) 157 (79) 24 (12) Radiotherapy  No  Adjuvant   Definitive  Palliative   Combination thereof 44 (23) 70 (36) 18 (9) 24 (12) 40 (20) Chemotherapy  No  Neoadjuvant  Adjuvant   For advanced disease   Combination thereof 3 (2) 86 (43) 32 (16) 36 (18) 128 (64) (Continued ) Oncotarget556www.impactjournals.com/oncotarget H1047R or overexpression of AXL, a membrane-bound tyrosine kinase receptor with resulting crosstalk between HER, AXL and ER receptor pathways [37]. There are two major signaling pathways controlled by receptors belonging to the ErbB-receptors family – Ras/Raf/MAPK, regulating cell division and proliferation, and PI3K/ Akt/mTOR, regulating cell growth and survival [38]. Hence, particular impairments in these pathways result in improper activation of signaling cascades and may influence the clinical efficacy of lapatinib [39, 40]. Our study suggests that another key element involved in regulation of mTOR1 complex – phosporylated AMPK alpha 1 protein kinase – may negatively impact response to lapatinib. AMPK acts as a crucial regulator of cell growth, proliferation and autophagy [41–42]. Intensive cellular energy-consuming processes, such as glucose deprivation, hypoxia, oxidative stress, hyperosmotic stress, or tissue ischemia, result in increased concentration of AMP, which leads to AMPK activation. Subsequently, activated AMPK, via phosphorylation of raptor or TSC2, inhibits activity of mTORC1, leading to general blockade of cellular anabolic processes and simultaneously activating catabolic processes [43]. The direct phosphorylation of raptor by AMPK leads to mTORC1 disruption and cell cycle arrest induced by energy stress [44–47]. Alteration of mTOR signaling networks, which is a common phenomenon in human cancers, may result from impairment of upstream regulatory mechanisms [48]. We showed that p-70S6K phosphorylation, reflecting the PI3K/AKT/mTOR pathway activity, was associated with improved PFS in lapatinib-treated patients, particularly in those with ER-positive tumors. Phosphorylation of p-70S6K depends solely on the activation of mTORC1, whereas p-70S6K exerts a negative feedback loop that inhibits PI3K/Akt via IRS-1 [47]. It is possible, that the favorable impact of p70S6K is associated with its inhibitory activity against various (not only ErbB-family members) membrane receptor complexes. However, a genuine predictive value of p-70S6K for lapatinib would necessitate testing this biomarker also in lapatinib-untreated patients, to exclude its possible favorable prognostic impact shown previously in early ERα-positive breast cancer [49]. In our study a member of Ras/Raf/MAPK pathway – p-MAPK, appeared to be a negative prognostic factor, mainly in patients with ER-positive tumors. This may indicate that in advanced HER2-positive breast cancer patients treated with lapatinib, phosphorylation of p-70S6K reflects significant activity of the Ras/Raf/MAPK pathway, particularly when accompanied by p-AMPK up-regulation. However, since p-MAPK is a distant downstream element of the Ras/Raf/ MAPK signaling pathway, its activity may result from crosstalk with various distinct signaling pathways. This Variable n = 199 Trastuzumab therapy  No  Adjuvant 0 (0) 48 (24)   For advanced disease   Combination thereof 136 (68) 15 (8) Endocrine therapy  No  Neo(adjuvant)   For metastatic disease   Combination thereof 128 (64) 36 (18) 17 (9) 18 (9) Type of first progression  Local  Regional  Distant   Combined local,   regional and/or distant 20 (10) 19 (10) 138 (69) 22 (11) Dominant site of metastatic disease   Soft tissue  Bone  Visceral 34 (18) 26 (14) 125 (68) Brain metastases  No  Yes 113 (57) 86 (43) Oncotarget557www.impactjournals.com/oncotarget observation may also suggest a potential role of combining lapatinib with MAPK pathway inhibitors. Previous studies have shown that, unlike trastuzumab, lapatinib affects cell cycle kinetics through Ras/MAPK, and had less effect on cell survival [14]. Our results suggest that the potential anti-tumor role of AMPK activators, such as metformin, may be limited in lapatinib-treated patients and requires further research [50]. A phase I trial evaluating a combination of lapatinib with metformin or sirolimus (mTOR inhibitor) in advanced cancer patients is currently ongoing (www.clinicaltrials.gov; NCT01087983). Similarly to previous studies [14, 22], we have not found a correlation between the PTEN status and clinical efficacy of lapatinib. Indeed, the activation of the PI3K/Akt/mTOR pathway resulting from mutations of PIK3CA or loss/mutations of PTEN has been attributed to the development of resistance to trastuzumab [7–12, 14] but not to lapatinib [14, 22]. As expected, in this series a high expression of cell cycle-regulating protein – cyclin E was more common in the ER-negative tumors, and in this subset was associated with apparently shorter PFS and OS. This biomarker was earlier shown to confer a poor clinical outcome in breast cancer [51]. Recent study demonstrated that cyclin E levels decrease upon HER2 down-regulation and HER2 inhibition, suggesting that HER2 regulates cyclin E function [52]. Finally, in a small group of HER2-positive breast cancer patients treated with trastuzumab-based therapy, cyclin E amplification or overexpression was associated with significantly impaired clinical outcomes [52]. Taken together, these data indicate that cyclin E may represent another potential therapeutic target in overcoming lapatinib resistance. In our study expression of HIF-2 alpha was associated with poor OS in the subset of ER-positive tumors. HIF-2 alpha is a key regulatory factor in tumor growth and its adverse prognostic impact has been previously reported [53, 54]. Not surprisingly, our study showed impaired survival in patients with ER-negative tumors. A negative prognostic impact of ER-negativity in HER2-positive breast cancer patients, was earlier reported by other authors [55, 56]. Indeed, the clinical behavior (including tumor kinetics and sites of recurrence) of ER-positive/HER2 positive subtype (HER2-positive luminal B breast cancer) differs from that of ER-negative HER2 enriched subtype [55–58]. A recent study suggested that the clinical benefit of first-line trastuzumab in advanced breast cancer may be predictive for the efficacy of second- and later lines of anti-HER2 therapies [59]. As our series included patients exposed to trastuzumab in both adjuvant and metastatic setting, we were unable to include this variable in the analysis. In conclusion, our study suggests that the clinical efficacy of lapatinib may be associated with the activity of downstream signaling pathways – AMPK/mTOR and Ras/Raf/MAPK. These data may indicate a potential role of combining lapatinib with MAPK pathway inhibitors Table 2: Hazard ratios for progression free (PFS) and overall survival (OS): univariate and multivariate analyses Univariate analysis Variable PFS HR (95%CI); p OS HR (95%CI); p p-p70S6K Cyclin E p-MAPK HIF-2 alpha PTEN p-AMPK Type of first progression* ER negative vs positive 0.47 (0.26–0.86); 0.014 1.71 (1.00–2.93); 0.05 0.89 (0.60–1.29); 0.51 1.23 (0.60–2.51); 0.57 1.04 (0.66–1.62); 0.88 0.94 (0.23–3.79); 0.92 1.30 (0.91–1.86); 0.15 0.95 (0.76–1.19); 0.65 0.82 (0.43–1.56); 0.545 2.86 (1.23–6.66); 0.015 1.68 (1.18–2.40); 0.007 0.77 (0.39–1.52); 0.45 1.08 (0.69–1.70); 0.728 0.96 (0.31–3.03); 0.95 3.39 (1.38–8.28); 0.008 0.60 (0.39–0.92); 0.033 Multivariate analysis Variable PFS HR (95%CI); p OS HR (95% CI); p p-p70S6K Cyclin E p-MAPK Type of first progression* ER negative vs positive 0.45 (0.25–0.81); 0.009 1.83 (1.06–3.14); 0.029 NC NC NC NC 2.99 (1.29–6.94); 0.011 1.61 (1.13–2.29); 0.009 1.87 (1.02–3.44); 0.044 0.65 (0.4–0.98); 0.041 PFS: progression free survival; OS: overall survival; ER: estrogen receptor; * regional vs. local; NC: not calculated (P > 0.1 in univariate analysis and not included in the stepwise and Cox regression multivariate analysis Oncotarget558www.impactjournals.com/oncotarget (Continued ) Oncotarget559www.impactjournals.com/oncotarget and justify further research on combinations of lapatinib with mTOR inhibitors, such as everolimus. We are aware of several limitations of this study. First, our investigations included only downstream signaling pathways, and not the underlying molecular alterations. Second, although our series was homogeneous, i.e. all patients were treated with lapatinib and capecitabine, a nonrandomized study design did not allow to test the predictive value of particular markers. Notably, a proportion of samples was excluded from analysis due to analytical problems, and there was no lapatinib-untreated control group. Finally, this analysis used material obtained from the primary tumor, whereas several studies showed phenotypic instability of metastatic sites, particularly in relation to hormone receptors [60, 61]. The question of whether biomarkers analyzed in this study are also a subject of such conversions, and whether this impacts response to lapatinib, remains to be answered. Hence, these results should be considered preliminary and only hypothesis generating. Further investigations are warranted to verify the clinical utility of our findings. MATERIALS AND METHODS Patients This study was approved by the Institutional Review Board of the coordinating center (the Military Institute of Medicine in Warsaw, Poland). An initial study population included HER2-positive advanced breast cancer patients treated in 31 oncology centers in Poland, Hungary, Czech Republic, Lithuania and Romania between 2004 and 2013. The patients should have received a combination of lapatinib at an initial dose of 1250 mg per day continuously and capecitabine at a dose of 2000 mg/m2 of body-surface area on days 1 through to 14 of a 21day cycle for at least 6 weeks. Patients must have earlier received trastuzumab and 1–3 lines of chemotherapy for advanced disease. Other eligibility criteria included age above 18 years, no previous or concomitant malignant disease except for basal cell carcinoma of the skin, tumor lesions evaluable for therapeutic response to lapatinib and the availability of formalin-fixed, paraffin embedded (FFPE) tumor tissue specimens for analysis. The following information was extracted from the medical records: the date of breast cancer diagnosis, previous local and systemic therapy, the date and type of the first progression (local, regional, distant), the dominant site of metastatic disease (soft tissue, bone, viscera), the date of brain metastasis diagnosis, the dates on which lapatinib and capecitabine were administered, the date of the first progression while on lapatinib and capecitabine therapy, and the date of death or the last follow-up visit. For tumors involving more than one category, the dominant site of distant disease was classified by the category associated Figure 4: Kaplan-Meier overall survival curves. A. p-MAPK ≥ 50 staining H-score (HR 1.68; p = 0.0007); B. cyclin E ≥ 250 staining H-score (HR 2.86; p = 0.0015); C. estrogen receptor: positive vs. negative (HR 0.60; p = 0.033). Oncotarget560www.impactjournals.com/oncotarget with the worst prognosis, irrespective of the extent of involvement, in the following order of increasing gravity: soft tissue, bones, and viscera. Due to the retrospective nature of this study, tumor staging was performed using the American Joint Committee on Cancer/the Union for International Cancer Control classification from 1997. Follow-up information was extracted from medical records and tumor registries. All data were coded to secure full protection of personal information. Alive patients had to provide written informed consent for the use of their archival tumor samples for analysis, according to regulations in particular countries. Pathology The starting material from each patient was an archival formalin-fixed, paraffin embedded (FFPE) specimen(s) from the primary breast cancer obtained at surgery or by tissue biopsy. A pre-cut section of each tumor, stained with hematoxylin and eosin, was reviewed by two board-certified pathologists (WB and KK) to confirm breast cancer diagnosis and determine whether a sufficient invasive breast cancer component was present (1 cm2 invasive tissue; ≥ 30% tumor cells). In each case, assuming potential intratumoral heterogeneity, 2 tissue cores (1.5 mm in diameter) were punched out from the FFPE tissue blocks containing primary breast cancer (“donor”) and transferred into a “recipient” paraffin block using Manual Tissue MicroArrayer (MTA I, Beecher Instrument Inc.) A total of 10 tissue microarrays (TMAs) were constructed. Immunohistochemistry (IHC) staining IHC analysis was performed in tumor tissue in accordance with standard protocols, on 5 μm histological slides derived from the TMA blocks. The tumor-associated stromal cells were not analyzed. The staining was performed according to manufactures’ protocols with the use of the following antibodies: p-AMPK alpha1 (ab39400 rabbit polyclonal, Abcam, Cambridge, UK; dilution 1:300), p-MAPK (ab50011 mouse monoclonal, Abcam, Cambridge, UK; dilution 1:100), p-p70S6K (ab32359 rabbit monoclonal, Abcam, Cambridge, UK; dilution 1:25), HIF2alpha Table 3: Hazard ratios for progression free (PFS) and overall survival (OS): a subset univariate and multivariate analyses considering ER status Univariate analysis Variable N PFS HR (95%CI); p OS HR (95%CI); p ER-positive  p-p70S6K   Cyclin E  p-MAPK   HIF-2 alpha  PTEN  p-AMPK 61 54 57 58 60 54 0.22 (0.06–0.75); 0.016 1.71 (0.53–5.55); 0.37 3.14 (1.59–6.20); 0.001 1.86 (0.58–6.02); 0.299 0.88 (0.41–1.88); 0.733 0.54 (0.17–1.78); 0.313 0.45 (0.18–1.09); 0.077 1.21 (0.29–5.12); 0.795 2.53 (1.05–6.11); 0.038 3.38 (1.13–10.08); 0.029 1.67 (0.70–4.01); 0.251 0.41 (0.12–1.35); 0.142 ER-negative  p-p70S6K   Cyclin E  p-MAPK   HIF-2 alpha  PTEN  p-AMPK 129 126 127 130 116 122 0.66 (0.31–1.44); 0.299 1.78 (1.02–3.09); 0.041 0.89 (0.59–1.41); 0.611 0.87 (0.35–2.15); 0.768 1.14 (0.65–1.99); 0.64 1.80 (0.44–7.32); 0.41 0.65 (0.31–1.34); 0.24 2.38 (1.09–5.18); 0.029 0.87 (0.54–1.38); 0.548 0.73 (0.30–1.80); 0.494 0.79 (0.46–1.34); 0.375 1.17 (0.37–3.71); 0.786 Multivariate analysis Variable N N PFS HR (95%CI); p HR (95%CI); p ER-positive  p-p70S6K  p-MAPK   HIF-2 alpha 61 57 58 0.10 (0.02–0.38); 0.001 4.48 (1.97–10.18); 0.001 1.51 (0.73–3.13); 0.263 0.23 (0.06–0.81); 0.023 3.91 (1.71–8.90); 0.001 4.74 (1.49–15.07); 0.008 ER-negative   Cyclin E 126 1.78 (1.02–3.09); 0.041 2.38 (1.09–5.18); 0.029 PFS: progression free survival; OS: overall survival; ER: estrogen receptor; N: number of cases Oncotarget561www.impactjournals.com/oncotarget (ab20654 rabbit monoclonal, Abcam, Cambridge, UK; dilution 1:250), cyclin E (HE12 mouse monoclonal, Thermo Sci, Waltham, MA, USA; dilution 1:50) and PTEN (6H2.1 mouse monoclonal, DAKO Denmark; dilution 1:100). Positive controls were used according to manufacturer's recommendations and negative controls included standard staining procedures with the omitting of the primary antibody step. TMA sections were deparaffinized in xylene and rehydrated through graded alcohols. Antigen retrieval procedure was performed using Target Retrieval Solution, with pH depending on monoclonal antibody in electric pressure cooker, followed by 20 min cooling before further immunostaining. Endogenous reactivity was blocked with normal goat serum. Following the preliminary stages, incubation with the primary antibody was carried out for 30 minutes. The binding of the monoclonal antibody was detected with biotin-labeled goat anti-mouse or anti-rabbit immunoglobulin G (IgG) and horseradish peroxidase-labeled avidin – biotin complex. IHC stains were scored manually according to staining intensity (0 – negative, 1 – weak, 2 – moderate, 3 – strong) and the percentage of positive tumor cells. Each tissue core was assessed separately and the core with the highest staining intensity was considered representative for the particular case. To accurately describe the extent of immunohistochemical staining of a tumor and to potentially increase the predictive information, expression of particular biomarkers was assessed using the staining H-score. The H-score was calculated for each biomarker by the formula: 3 x percentage of strong cellular (cytoplasmic or nuclear wherever appropriate) staining plus 2 x percentage of moderate cellular staining plus percentage of weak cellular staining, giving a range of 0 to 300. The cutoff values for each biomarker were optimized using Cox regression model to maximize the hazard ratio (HR) between patients with expression levels above vs. below the cutoff. Statistical analysis All statistical analyses were performed using STATA software version 11. Statistical significance was defined as p < 0.05. Categorical variables were compared using Pearson’s chi-squared test (χ2 ) and Spearman’s R rang. The primary endpoint was progression free survival (PFS), defined as the time from the date of the lapatinib start to the date of the disease progression or death, whichever occurred first. The secondary endpoints were an objective response, defined as a complete response (CR) or a partial response (PR) and disease control, defined as CR, PR and stable disease combined, determined according to Response Evaluation Criteria in Solid Tumors (RECIST) v 1.0 criteria. Survival curves were plotted using the Kaplan-Meier method, starting from the first day of lapatinib therapy to the date of death or the last follow-up. Univariate analyses were performed with a log-rank test, Wilcoxon test and Cox proportional hazard and logistic regression. Multivariate analysis used a stepwise forward selection of univariate model with p ≤ 0.10. ACKNOWLEDGMENTS Central and East European Oncology Group members who contributed to this study: Wojciech Olszewski Oncology Center-Institute Warsaw, Poland; Anna Surus-Hyla, Jolanta Żok and Wojciech Rogowski, Warmia and Masuria Oncology Center, Olsztyn, Poland; Ewa Chmielowska, Oncology Center, Bydgoszcz, Poland; Rostislav Vyzula and Renata Horova, Masaryk Memorial Cancer Institute, Brno, Czech Republic; Jolanta Smok-Kalwat, Oncology Center, Kielce, Poland; Tomasz Jankowski, Lublin Oncology Center, Poland; Agata Sałek, Subcarpathian Oncology Center, Rzeszów, Poland; Alexander Eniu and Gabriela Morar Bolba, Oncology Cancer Institute I. Chiricuta, ClujNapoca, Romania; Dorota Półchłopek, Oncology Center, Brzozów, Poland; Elżbieta Nowara, Oncology Institute, Gliwice, Poland; Dorota Bogus and Andrzej Kałmuk, Regional Hospital, Częstochowa, Poland; Eduardes Aleknavicius, Oncology Institute, Vilnius University, Lithuania; Iwona Rynkiewicz-Zander and Piotr Wiosek, District Hospital, Elbląg, Poland; Łukasz Głogowski, Regional Hospital, Bytom, Poland; Ida Cedrych and Aleksandra Grela-Wojewoda, Oncology Institute, Kraków, Poland; Monika Kulma-Kreft, Gdynia Oncology Center, Poland; Piotr Sawrycki, Regional Hospital, Toruń, Poland; Zsuzsanna Kahan, Department of Oncology, University of Szeged, Hungary; Małgorzata Ploch-Glapińska, Regional Military Hospital, Wrocław, Poland. Special thanks to the CEEOG Database Manager Anita Zakrzewska and the Statistician Barbara Zaborek for invaluable assistance. CONFLICTS OF INTEREST The author(s) indicated no potential competing interests. 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PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online)  2016 Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic Fax +420 241 062 164, e-mail: physres@biomed.cas.cz, www.biomed.cas.cz/physiolres Physiol. Res. 65 (Suppl. 4): S455-S462, 2016 REVIEW The Safety of Therapeutic Monoclonal Antibodies: Implications for Cancer Therapy Including Immuno-Checkpoint Inhibitors R. DEMLOVA1,2 , D. VALÍK1,2 , R. OBERMANNOVA1,2 , L. ZDRAŽILOVÁ-DUBSKÁ1,2 1 Department of Pharmacology, Faculty of Medicine, Masaryk University Brno, Czech Republic, 2 Masaryk Memorial Cancer Institute, Brno, Czech Republic Received September 6, 2016 Accepted October 24, 2016 Summary Monoclonal antibody-based treatment of cancer has been established as one of the most successful therapeutic strategies for both hematologic malignancies and solid tumors. In addition to targeting cancer antigens antibodies can also modulate immunological pathways that are critical to immune surveillance. Antibody therapy directed against several negative immunologic regulators (checkpoints) is demonstrating significant success in the past few years. Immune checkpoint inhibitors, ipilimumab, pembrolizumab and nivolumab, have shown significant clinical benefit in several malignancies and are already approved for advanced melanoma and squamous NSCLC. Based on their mechanism of action, these agents can exert toxicities that are unlike conventional cytotoxic chemotherapy, whose nature is close to autoimmune diseases - immune related adverse events (irAEs). In this review we focus on the spectrum of irAEs associated with immune checkpoint antibodies, discussing the pharmacological treatment strategy and possible clinical impact. Key words Cancer treatment • Monoclonal antibodies • Immune checkpoint inhibitors • Immune related adverse events Corresponding author R. Demlova, Department of Pharmacology, Faculty of Medicine, Kamenice 5, 625 00 Brno, Czech Republic. Email: demlova@med.muni.cz Monoclonal antibodies in cancer Monoclonal antibodies (mAbs) are a rapidly growing class of human therapeutics representing approximately 25 % of drugs under development (Datamonitor: Pipeline insights 2009). By 2014, 34 therapeutic mAbs are predicted to be on the market for treating cancer, autoimmune diseases, and infectious diseases (Datamonitor: Pipeline insights 2009). Since their discovery in 1975, mAbs have been described as "magic bullets" with the potential to seek out and bind targets with high affinity and specificity (Kohler et al. 1975). Cancer diseases are one of the major groups where monoclonal antibodies are used in clinical practice. There have been twelve antibodies that have received approval from the FDA for the treatment of a variety of solid tumors and hematological malignancies in 2014 (Scott et al. 2012). In addition, there are a large number of additional therapeutic antibodies that are currently being tested in early- and late-stage clinical trials. The most common type of mAbs used to treat cancer are “naked mAbs”. Most naked mAbs attach to antigens on cancer cells, but some work by binding to antigens on other, non-cancerous cells, or even free-floating proteins. We can simplify three major mechanism of actions of naked mAbs. One principle is boosts a person’s immune response against cancer cells by attaching to them and acting as a marker for the body’s immune system to destroy them. An example is alemtuzumab, which binds to the CD52 antigen on lymphocytes and is used to treat some patients with chronic lymphocytic leukemia (CLL). Another naked mAbs work mainly by attaching to and blocking antigens on cancer cells that help cancer cells grow or spread. For example, trastuzumab is an antibody against the HER2 protein. Currently the most researched mAbs are naked mAbs which boost the immune response S456 Demlova et al. Vol. 65 by targeting immune system checkpoints like anti-CTLA-4 monoclonal antibody ipilimumab or nivolumab and pembrolizumab targeting programmed cell death 1 receptor (PD-1). Adverse effects of this immune checkpoint inhibitors will be discussed in this review in more detail. In cancer therapy apart from the naked Mabs we can use also conjugated monoclonal antibodies, mAbs joined to a chemotherapy drug or to a radioactive particle. Conjugated mAbs are also sometimes referred to as tagged, labeled, or loaded antibodies. Ibritumomab tiuxetan is an example of a radiolabeled mAb. Antibodydrug conjugates (ADCs) like brentuximab vedotin targets the CD30 antigen, trastuzumab emtansine (also called TDM-1) is an antibody that targets the HER2 protein attached to a chemo drug called DM1. It’s used to treat some breast cancer patients whose cancer cells have too much HER2. Bispecific monoclonal antibodies are made up of parts of 2 different mAbs, meaning they can attach to 2 different proteins at the same time. An example is blinatumomab, which is used to treat some types of acute lymphocytic leukemia (ALL). One part of blinatumomab attaches to the CD19 protein, which is found on some leukemia and lymphoma cells. Another part attaches to CD3, a protein found on immune cells called T cells. By binding to both of these proteins, this drug brings the cancer cells and immune cells together, which is thought to cause the immune system to attack the cancer cells. Compared with chemotherapy drugs, monoclonal antibodies tend to have fewer serious side effects, however, as with all agents, administration of mAbs should be associated with adverse events (AEs) as a result of enhancing/inhibiting the activity of the target molecule on the target tissue, or due to interactions of the mAb with target molecules on tissues other than the intended ones (Catapano et al. 2013). Classification of adverse effects of biological agents Traditionally, adverse reactions should be subclassified according to their action (Naisbitt et al. 2000) (Table 1): so-called type A reactions correspond to the pharmacological activity of the drug, and are thus predictable (Hoigne et al. 1993). About 16 % of side effects are type B reactions (Naisbitt et al. 2000), which are not related to the pharmacological activity of the drug and are nonpredictable. The majority of type B reactions are immune-mediated side-effects like hypersensitivity reactions. Types C, D, and E are not mechanisms but characteristics of their manifestations; they are not referred to frequently in the literature. The letter C refers to continuous, chronic. Type D refers to delayed in appearance, making them difficult to diagnose. Type E refers to end of use, F means failure of therapy (Edwards et al. 2000). Table 1. Classification of adverse drug reactions (Naisbitt et al. 2000). Type A (augmented) reactions Predicted from the known pharmacology of the drug. These reactions are dose-dependent: examples are bleeding with anticoagulants Type B (bizarre) reactions Reactions are not predicted from the known pharmacology of the drug. They appear (but actually are not) relatively dose-independent, as very small doses might already elicit symptoms. They include immune-mediated side-effects like maculopapular exanthema, but also other hypersensitivity reactions, like aspirin-induced asthma Type C (chronic) reactions Which are related to the chemical structure and its metabolism, e.g. paracetamol hepatotoxicity Type D (delayed) reactions Which appear after many years of treatment, e.g. bladder carcinoma after treatment with cyclophosphamide Type E (end of treatment) reactions Occur after drug withdrawal, e.g. seizures after stopping phenytoin Biological agents differ from most drugs as they are not small chemical compounds (xenobiotics) but are proteins produced in a way to make them as similar to human proteins as possible. They are not metabolized like drugs but are processed like other proteins, with the differences in pharmacokinetic as well as pharmacodynamic properties. Thus, adverse reactions to biological agents might differ from those elicited by 2016 mAbs and Their iAE S457 classical drugs. More appropriate classification of mAbs adverse effects is a subclassification published by Pichler et al. (2006) based on mechanism of action and structure, as illustrated in Figure 1. To distinguish it from the classification of side-effects to chemicals/drugs (Table 1), the Greek alphabet is used for the five types (type α, β, γ, δ and ε). Type α (high cytokine and cytokine release syndrome) are side-effects connected to the systematic application of cytokines in relatively high doses or to high concentrations of cytokines released into the circulation (Vasquez et al. 1995). Type β reactions can be termed as hypersensitivity. Thereby basically three forms of allergies can be differentiated: IgE-, IgG- and T cellmediated reactions. Type γ immune (cytokine) imbalance syndromes have immunological features, but cannot be explained by high cytosine levels or typical hypersensitivity reactions. As illustrated in Figure 1, these reactions can be further subdivided in, impaired functions, and unmasking or causing an immune imbalance leading to autoimmune, auto-inflammatory or allergic reactions. Type δ (cross-reactivity) might be that antibodies generated to an antigen expressed on tumour cells might also cross-react with normal cells, which express this structure as well, albeit to a lower degree (Perez-Soler et al. 2005). Type ε (non-immunological side-effects) may elicit symptoms not directly related to the immune system, sometimes revealing unknown functions of the biological agents given or targeted. Fig. 1. Type of adverse effects of biological agents. (Pichler 2006). Immuno-checkpoints inhibitors and immune related adverse events (irAEs) Cancer therapy based on monoclonal antibodies against checkpoints of immune reaction is today considered a breakthrough method in oncology. Results with the anti-CTLA-4 (cytotoxic T lymphocytic antigen-4) antibody ipilimumab in the treatment of advanced malignant melanoma have represented a revolution in anti-tumour therapy and been a catalyst for developing new antibodies focusing on other control molecules (checkpoints) (Lakomy et al. 2015) What is especially worth mentioning in this context are the antibodies against programmed cell death receptor (PD-1) – nivolumab and pembrolizumab and against its ligand (PD-L1), BMS-936559, MPDL3280A or lambrolizumab ((Weber et al. 2015, Hamid et al. 2013)). The development and use of immune therapy have achieved their greatest progress so far in advanced melanoma, but soon we will be similarly treating non-small-cell pulmonary carcinoma and other tumour types. These antibodies prevent an inhibition of interaction between PD-1 and PD-L1/L2, on the other hand cause secondary potentiation of the effector component of immunity on the “peripheral” level directly in the tumour tissue. Break of tolerance towards the tumour may, however, be accompanied by unwanted break of tolerance towards “normal tissues”, which leads to adverse reactions whose nature is close to autoimmune diseases – immune related adverse events (irAEs). IrAEs include dermatologic, GI, hepatic, endocrine, and other less common inflammatory events. Because irAEs likely arise from general immunologic enhancement, temporary immunosuppression with corticosteroids, tumor necrosis factor α antagonists or other agents is often necessary and should follow established algorithms (Postow et al. 2015). Due to the high frequency of irAEs with the risk of life-threatening complications, sufficient education of S458 Demlova et al. Vol. 65 patients, their family members and medical specialists is necessary. Warnings and recommendations have been prepared about how to proceed suspected irAEs. Early commencement of immunosuppressive therapy with corticosteroids is the key step towards management of the incident, reduction of morbidity and potentially also mortality. If corticoids are not sufficiently effective, other immunosuppressants are added, such as infliximab or mycophenolate mofetil (Lakomy et al. 2015). Side effects of ipilimumab Ani-CTLA-4 monoclonal antibody ipilimumab was the first immune checkpoint receptor used in daily clinical practice. The frequency of adverse effects of ipilimumab is relatively high; in a pre-authorization study of the dose of 3 mg/kg it ranged between 80 – 90 %. Luckily the toxicity was mild to moderate in the vast majority of the cases (grade 1 and 2 toxicity). Serious (grade 3) and life-threatening (grade 4) toxicity pursuant to NCI- CTCAE v3.0 (National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0) was reported for about 20 – 25 % of the study population (Hodi et al. 2010). Adverse effects may already occur in the course of or after the infusion (nausea, vomiting, febrility, pain and vertigo, rash and pruritus), but these reactions are singular and mostly non-serious. The general recommendation is to suspend the infusion until symptoms retreat, with potential administration of antihistaminics or antipyretics, and subsequently to restart at a slower (about half) speed with patient monitoring (Fecher et al. 2013). Premedication with antihistaminics or antipyretics before the following administrations is recommended for consideration in those cases. In the case of serious adverse reaction of levels 3 and 4 (bronchospasm, hypotension, anaphylaxis) the procedure is identical to other hypersensitivity reactions, with the permanent discontinuation of therapy recommended. The irAEs are a bigger concern, though, as they are most common and grow with the dose. In the case of the dose of 3 mg/kg they appear in up to 60 % of patients, while severe irAE toxicity of levels 3 and 4 has been reported for 10 – 15 % of the study subjects (Hodi et al. 2010, Wolchok et al. 2010). The most frequent irAEs include dermal toxicity (rash, pruritus), enterocolitis and diarrhea, endocrinopathy (hypophysitis, thyroiditis) and liver test elevation. The immune system may, however, attack any part of the anatomy (heart, lungs, kidneys, nervous system, eyes, hematopoietic system etc.). Biopsy then most often reveals tissue infiltration with T lymphocytes or with neutrophils (Fecher et al. 2013). Over time, we can first expect dermal toxicity (after three weeks), followed by colitis and diarrhea (after five weeks), hepatic toxicity (after 6 – 7 weeks) and finally endocrinopathy (after 7 – 8 weeks from therapy commencement) (Weber et al. 2012). We should also remember that IrAEs may, however, also appear a couple of weeks or months after therapy completion. The text below will deal with the most frequent irAEs and how to address them. Immune-conditioned dermal toxicity Dermal toxicity is one of the most common irAEs (40 – 45 %) usually first occurring after week 3 of therapy and culminating in week 6 (Lakomy et al. 2015, Hodi et al. 2010, Weber et al. 2012). This is mostly level 1 toxicity (affecting <10 % of the body surface) or toxicity level 2 (with an effect on between 10 and 30 % of the body surface). Severe toxicity (levels 3 and 4) is luckily rare, only ranging between 1 – 2 % in the preauthorization study. Usual manifestations include maculopapular exanthema (20 %) and/or pruritus (25 %). Another manifestation may be vitiligo, which is rather cosmetic issue, but the patient must be educated about protection against UV radiation. Blisters are already a manifestation of a severe reaction. Stevens-Johnson syndrome and toxic epidermal necrolysis occur in <1 % patients and are a reason for permanent discontinuation of therapy. Immune-conditioned colitis and diarrhea Diarrhea induced by ipilimumab is another of the most common autoimmune-conditioned adverse reactions. This effect appears after week 5 of therapy. In the case of an ipilimumab dose of 3 mg/kg, this AE occurs in about 30 % patients, and incidence rises with the dose – 44 % at 10 mg/kg i.v. (Hodi et al. 2010, Weber et al. 2012). Severe diarrhea or colitis levels 3 or 4 occur in the case of a 3 mg/kg dose in about 5 – 8 % of patients, colitis most often affecting the descending colon (Wolchok et al. 2010). A big threat is potential intestinal perforation, with potential fatal consequences. Prophylactic administration of oral corticoids (budesonide) did not change the incidence of diarrhea (Weber et al. 2009). The so far published results suggest that immunosuppressive corticoid therapy administered in 2016 mAbs and Their iAE S459 the course of the immunotherapy does not substantially affect the therapeutic effect of ipilimumab (Weber et al. 2009, Harmankaya et al. 2011). Immune-conditioned hepatic toxicity Hepatic toxicity caused by ipilimumab, even though rarer in comparison to the previously mentioned immune-conditioned adverse effects, may also represent a life-threatening reaction. The onset of this irAE usually occurs after week 6 of ipilimumab therapy, becoming very rare after week 14 (Weber et al. 2012). If all recommendations for diagnosis and treatment are observed, the prognosis for patients with this type of irAE is excellent; the liver test results usually normalize very quickly after commencement of the appropriate therapy – in about two weeks (Della Scarpati et al. 2014). A patient with hepatic toxicity may be asymptomatic (laboratory results usually show elevated ALT and/or AST and/or bilirubin), but frequent complaints include febrility, fatigue, nausea, jaundice, changed stool color or urine colour, and sometimes pruritus. Patient examination must include taking a careful medical history to exclude other causes – fungi, alcohol, contact with chemicals or the effect of concurrent medication (such as paracetamol and others). Clinical examination may reveal, in addition to icterus, also hepatomegaly, the patient may be exhausted, or the find may be completely negative. An infectious cause must always be excluded in level 2 and higher of hepatic toxicity, and antibodies against nuclear antigen and mitochondrial antigen must be sampled (ANA, SMA). Exclusion of progression of the malignant disease by imaging methods is also necessary. In the case of level 3 and 4 toxicity, liver biopsy is to be considered (finds typical of hepatic toxicity include infiltration of the liver parenchyma with T lymphocytes, sometimes with necrosis of hepatocytes) (Lakomy et al. 2015). Immune-conditioned endocrinopathy Like the immune-conditioned hepatopathy endocrinopathy is also less frequent than dermal or gastrointestinal toxicity, but again represents a potentially life-threatening condition. The deceptiveness of endocrinopathy lies in its late onset (7 – 8 weeks after ipilimumab therapy commencement, with the highest incidence between weeks 12 and 24), and in the fact that, unlike hepatic toxicity, the probability of occurrence does not decrease in time from completion of therapy; instead, the appearance of the curve is rather like a plateau (Weber et al. 2012). Possible endocrinopathies include hypopituitarism (with or without hypophysitis), hypofunction of the adrenals, hypo- or hyperfunction of the thyroid gland and hypofunction of the gonads. In the case of a suspected adverse effect of this kind, the medical history of the patient is again important (incidence of endocrinopathies in the patient's personal and family history), the patient may complain of non-specific symptoms such as fatigue, weakness, febrilia, abdominal pain, vomiting, diarrhea, headaches or sensory disorders. Clinical warning symptoms include signs of dehydration, hypotension or other signs of commencing systemic inflammatory response syndrome. Laboratory examinations, apart from basic internal environment parameters (often hyperkalemia, hyponatremia and hypoglycemia) and blood count, include endocrinopathy screening: sampling of free T3 and T4, TSH, antibodies against thyroidal peroxidase – anti TPO, morning serum cortisol, levels of corticotropin (ACTH), and in males testosterone levels, and in females FSH and LH levels. For differential diagnosis, an ACTH stimulation test may be considered. Indicated imaging methods include MRI of the brain, with a focus on hypophysis and also on the exclusion of brain metastases. It must be mentioned at this point, that there does not exist any general consensus about the therapy and monitoring, and the literature shows conflicting data on the therapy of this adverse effect and the possibility of returning to ipilimumab therapy (Weber et al. 2012, Della Scarpati et al. 2014, O´Day et al. 2010). Repeated consultations and cooperation with an endocrinologist are a priority. If corticoids need to be administered (toxicity levels 3 and 4), then corticoids with mineralocorticoid activity are preferred with slow discontinuation (Lakomy et al. 2015). Less frequent immune-conditioned adverse effects Other possible, although rare (less than 1 % of cases), immune-conditioned adverse effects of ipilimumab include: meningitis, uveitis, pneumonitis, pancreatitis, pericarditis, myocarditis, nephritis, various angiopathies, hemolytic anemias, thrombocytopenia etc. Straightforward therapeutic procedures are not known, where such adverse effects occur therapy is managed like it is for other autoimmune diseases with corticoids as the S460 Demlova et al. Vol. 65 therapy of choice. Generally, level 3 and 4 toxicity is a reason for discontinuing ipilimumab therapy. Details are in the ipilimumab SmPC (EMA 2012). Immune-conditioned adverse effects of antiPD-1/PD-L1 antibodies and their combination with ipilimumab Toxicity of these antibodies is generally lower than of ipilimumab Topalian et al. 2012, Wolchok et al. 2015). Severe dermal toxicity levels 3 and 4 are exceptional (2 %). Also, level 3 or 4 diarrhea is uncommon (1 – 2 %), as is hepatic toxicity (below 3 %) and severe endocrinopathy (<1 %). Unlike ipilimumab, pneumonitis occurs more often after anti-PD-1 antibodies (<5 %). Therapy of severe pneumonitis is again based on corticoids in high doses (methylprednisolone 2 mg/kg i.v. 1 – 2 times daily) or infliximab. Other toxicities are sporadic, but cannot be neglected either. The principles of therapy of irAEs caused by anti-PD-1/PD-L1 antibodies are the same as with ipilimumab. A considerably higher incidence of irAEs has been described in reports from studies of combinations of anti-PD- 1 and anti CTLA-4 antibodies (nivolumab + ipilimumab). Here the occurrence of severe irAEs of levels 3 and 4 ranged around 50 %, with significant representations of gastrointestinal toxicity (15 %) and hepatic toxicity (19 %). A number of these severe toxicities, however, only met the laboratory criteria NCI-CTCAE (such as the elevation of amylase and lipase without clinical signs of pancreatitis). The combination resulted in no new toxicity (Wolchok et al. 2015). Conclusions The development of immune checkpoint inhibitors targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death-1 (PD-1) has significantly improved the treatment of a variety of cancers. Although these agents can lead to remarkable responses, their use can also be associated with unique adverse effects. The spectrum of AEs associated with immune checkpoint antibodies are termed immunerelated AEs (irAE's). The underlying pathophysiology relates to the immune-based mode of action of these agents, leading to T-cell inflammatory infiltration of solid organs, and increased serum inflammatory cytokines. IrAEs are frequent side effects of checkpoint inhibitors, they are possibly life-threatening and there is no known predictive biomarker for their occurrence. The most frequent irAEs include dermal toxicity (exanthema, pruritus), GIT toxicity (diarrhea, colitis), endocrine toxicity (hypopituitarism, hypophysitis, hypothyreosis, adrenal insufficiency), liver toxicity (elevation of transaminase, hepatitis), and in connection with anti-PD-1 antibodies, pneumonitis. IrAEs have their onset and duration (kinetics) well described, but since they can even occur several months after therapy completion (endocrinopathy), the safe interval after therapy completion is not known. Incidence of irAEs is higher for ipilimumab (dose-dependent) in comparison to anti-PD-1/PD-L1 antibodies, and even higher for the combination of ipilimumab and nivolumab. Retreat of toxicity may be slower in the case of anti-PD-1/PD-L1 antibodies; therefore long-term follow up is recommended (Weber et al. 2015). The important is the early medication of immunosuppressant with corticoids and their slow discontinuation, if the corticoids show insufficient or no effect, other immunosuppressants must be prescribed. These recommendations for therapy of irAEs are universal for all checkpoint inhibitors, although they are mainly based on experience from clinical studies with ipilimumab. New adverse effects may occur with the introduction of new antibodies and their combinations. The success of therapy with checkpoint inhibitors is conditional not only upon the erudition of the clinician, but also upon having an educated and cooperating patient (Lakomy et al. 2015). 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WOLCHOK JD, CHIARION-SILENI V, GONZALEZ R, RUTKOWSKI P, GROB JJ, COWEY CL, LAO CD, SCHADENDORF D, FERRUCCI PF, SMYLIE M, ET AL.: Efficacy and safety results from a phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment- naive patients (pts) with advanced melanoma (MEL) (CheckMate 067). J Clin Oncol 33 (Suppl: abstr. LBA1), 2015. WOLCHOK JD, NEYNS B, LINETTE G, NEGRIER S, LUTZKY J, THOMAS L, WATERFIELD W, SCHADENDORF D, SMYLIE M, GUTHRIE T, ET AL.: Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomized, double-blind, multicentre, phase 2, dose-ranging study. Lancet Oncol 11: 155-164, 2010. PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online)  2016 Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic Fax +420 241 062 164, e-mail: physres@biomed.cas.cz, www.biomed.cas.cz/physiolres Physiol. Res. 65 (Suppl. 4): S481-S488, 2016 Systemic Administration of miRNA Mimics by Liposomal Delivery System in Animal Model of Colorectal Carcinoma J. MERHAUTOVÁ1,2 , P. VYCHYTILOVÁ-FALTEJSKOVÁ1 , R. DEMLOVÁ2 , O. SLABÝ1 1 Molecular Oncology II – Solid Cancer, CEITEC, Masaryk University, Brno, Czech Republic, 2 Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic Received August 28, 2016 Accepted September 12, 2016 Summary MiRNAs are important regulators of gene expression and changes in their levels are linked with various pathological states, including solid tumors. MiR-215 has been identified as a tumor suppressor in colorectal cancer (CRC). Following our previous in vitro and in vivo experiments, the aim of this project was to study the possibility of increasing the levels of miR-215 in tumor cells by systemic administration of miRNA mimics in liposomal delivery system in vivo. By subcutaneous xenotransplantation of human cancer cells to NSG mice, CRC model was established. The treatment [miR-215 mimics in liposomes (20 and 40 μg/mouse), control oligonucleotide in liposomes, or saline] was administered repeatedly by i.v. injection via tail-vein. Animals were sacrificed, tumor were dissected and measured by a caliper. Expression of miR-215 in tumors, lungs and liver was quantified by RT-PCR. There was no significant differences in tumor volume and miR-215 expression between all three treatment groups. Therefore, the decrease in tumor volume was not achieved. By comparing the levels of miR-215 in lungs, liver and tumors after the treatment, we suggest that the liposomes are accumulated in the lungs and do not concentrate sufficiently in the tumor site to exert significant tumor-suppressive effect. Key words Colorectal neoplasms • microRNAs • Liposomes • Mice Corresponding author O. Slabý, Molecular Oncology II – Solid Cancer, CEITEC, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic. E-mail: ondrej.slaby@ceitec.muni.cz Introduction Colorectal carcinoma (CRC) represents the second most frequent cancer disease in men and women in the Czech Republic. For the last 10 years, the incidence of CRC is stable ranging around 80 cases per 100,000 persons a year and the mortality is slowly decreasing (Dušek et al. 2007, Ferlay et al. 2013). Nevertheless, a large proportion of patients is diagnosed with advanced or metastatic disease (stages III, IV). Clinical outcomes of therapy in metastatic stage are quite worse in comparison with early (I, II) stages and the pharmacotherapy is mostly palliative. Treatment of advanced and metastatic CRC consists of chemotherapy regimens based on 5-fluorouracil (5-FU) and leucovorin combined with irinotecan, or oxaliplatin. 5-FU could be also replaced with capecitabine (Fínek et al. 2016). Cytostatics are nowadays frequently combined with targeted therapy. There are more target molecules: epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and tyrosine kinases associated with various receptors including VEGFR, PDGFR, FGFR, KIT, RET etc. The most frequently used targeted therapy agents are monoclonal antibodies against EGFR (cetuximab, panitumumab) and VEGF (bevacizumab). Anti-EGFR therapy is designated for patients with confirmed wildtype RAS oncogene (KRAS, NRAS), as mutations in RAS are associated with poor anti-EGFR therapy outcomes (Lin et al. 2011, Adelstein et al. 2011). Multikinase inhibitor regorafenib, and fusion protein (“soluble VEGF receptor”) aflibercept are designated for patients with progression on standard therapy, or intolerance of it S482 Merhautová et al. Vol. 65 (Van Cutsem et al. 2016, Fínek et al. 2016). Even in this more targeted and moderately personalized treatment settings, a group of patients does not have clinical benefit from the therapy, or their tumor gains secondary resistance. Besides finding new predictive biomarkers to distinguish more precisely patients with or without clinical benefit from the therapy in advance, there is a constant need to bring new therapeutics with new mechanisms of action into the clinical practice. MiRNA-based therapeutics, that emerged in the drug research and development with increasing knowledge about the roles of miRNAs in the cell and in the cancer cell specifically, could represent such new targeted substances and some of them are heading towards the clinical practice. Besides growing number of in vitro and in vivo studies that aim to discover potential therapeutic properties of miRNA mimics, inhibitors, or expression vectors, there are several ongoing clinical trials in oncology, e.g. with miR-34a mimics (Adams et al. 2015) or miR-16 mimics (Kao et al. 2015, Quinn et al. 2015). MiRNAs are endogenous small non-coding RNAs. They attenuate mRNA translation posttranscriptionally by binding to the 3’-untranslated region of mRNA. The matching is guided by specific “seed” sequence of 6-8 nucleotides of a miRNA and results in the inhibition of translation, destabilization and subsequent degradation of target mRNA (Krol et al. 2012). One miRNA can regulate many genes which could be functionally different, or linked in a specific intracellular pathway. While miRNAs are supposed to regulate more than 50 % of human genes, their network interferes with most cellular processes like metabolic and energetic maintenance, differentiation, cell cycle and proliferation, survival, or death (Esquela-Kerscher et al. 2006). Therefore, dysregulation of miRNAs’ expression is linked with various pathological states, including solid tumors (Ruan et al. 2009). Quantity of in vitro and in vivo evidences indicate, that change in pathological miRNAs levels is capable of transforming the cancer cell phenotype. Significant changes in miRNA expression could be found in tumors compared with relevant healthy tissue. By large-scale miRNA profiling and following validation, our group has previously identified miRNAs significantly altered in CRC. Among others, miR-215 was identified as decreased, and in vitro functionally characterized as tumor suppressor (Faltejsková et al. 2012). It was also found down-regulated in patients with CRC relapse (Karaayvaz et al. 2011). MiR-215 influences apoptosis, cell cycle, viability, and migration of CRC cells in vitro (Faltejsková et al. 2012). It is tightly associated with protein p53, since p53 is induced by miR-215, and miR-215 is regulated by p53 in a feedback loop. By induction of p21, miR-215 is able to stop cell cycle, and it also inhibits expression of thymidylate synthase, dihydrofolate reductase (Braun et al. 2008) and BMI1 gene, that plays a role in cancer cells self-renewal ability (Jones et al. 2015). Transcription of miR-215 is activated by transcription factor CDX1 (caudal-type homeobox 1), which regulates differentiation of enterocytes and is frequently downregulated due to hypermethylation of promotor in CRC (Jones et al. 2015). Down-regulation of this miRNA is suggested to be one of the early steps in colorectal neoplastic transformation, as tumors initiated both by APC gene mutations, and chronic inflammation share this specific molecular pathology (Necela et al. 2011). Moreover, our group has recently found, that overexpression of miR-215 in colorectal cancer cell line leads to significant decrease in tumor volume in tumorigenicity assay in vivo (Vychytilová-Faltejsková data not yet published). Altogether, these findings suggest possible therapeutic roles of miR-215 in CRC. Therefore, the aim of this work was to study the possibility of increasing the intracellular level of miR-215 in tumor by systemic administration of miRNA mimics in liposomal delivery system in vivo. Methods Subcutaneous xenotransplantation All animal procedures were performed in accordance with the Czech legislation (Act No. 246/1992) and with approval of both local and national Committees for Animal Welfare. NSG mice (18-27 g, 8-14 weeks old) were housed and monitored in an individually ventilated cage system (Techniplast, Buguggiate, Italy) with ad libitum access to water and feeding. The subcutaneous xenotransplantation of human cancer cells was performed according to the protocol described by Morton and Houghton (2007) with minor changes. Briefly, mice were anesthetized with etomidate (30 mg/kg) by i.p. injection. 2.5 × 106 human CRC cells HCT-116+/+ suspended in 100 μl of phosphate-buffered saline (PBS) were injected subcutaneously on dorsal site of a mouse. On Day 7 postinoculation, palpable tumor 2016 miRNA Mimics in Animal Model of Colorectal Carcinoma S483 with approximate volume of 500 mm3 were present in each animal (Fig. 1). Fig. 1. Mouse with subcutaneous tumor seventh day postinoculation. Preparation of miRNA mimics encapsulated in liposomes MaxSuppressor™ In Vivo RNA-LANCEr II (Bioo Scientific, Austin, USA) was used as a liposomal delivery system. MiRNA mimics (mirVanaTM miRNA mimic hsa-miR-215, Thermo Fischer Scientific, Waltham, USA) and negative control oligonucleotide (mirVanaTM miRNA mimic Negative Control #1, Thermo Fischer Scientific, Waltham, USA) were encapsulated into the liposomes according to the manufacturer’s protocol. Briefly, RNA substances were diluted in water for injections (B.Braun Medical, Hessen, Germany) to 10 mg/ml and stored in -80 °C. 11 μl of RNA solution [10 μl (0.1 mg) + 10 % excess], 55 μl of PBS 10× and 434 μl RNase-free water were added into 1 bottle of MaxSupressor™ which consists of 50 μl of neutral lipid emulsion (NLE). The mixture was sonicated for 5 min in room temperature. Experimental treatment Two doses [20 μg (1 nmol)/mouse, and 40 μg (2 nmol)/mouse] of miRNA mimics encapsulated in liposomes were tested in animal model of CRC, which was established by subcutaneous xenotransplantation. For the dose of 20 μg/mouse, 1 bottle of MaxSupressor™ mixture was dived into 5 doses of 100 μl volume, and 2.5 doses in the case of higher dosing, while the volume of injection was increased to 200 μl. Intravenous injection into the tail vein started on Day 7 postinoculation. In the Experiment A, 12 NSG mice were randomly divided into 3 groups: a) active treatment (miRNA mimics in liposomes, 20 μg/mouse), b) negative control (negative control oligonucleotide in liposomes), and c) saline. Tail-vein injections were repeated after 48 h for total 4 times. 24 h after the last dose, mice were sacrificed by anesthetic overdosing. In the Experiment B, 12 NSG mice were randomly divided into 3 groups: a) active treatment (40 μg/mouse), b) negative control, and c) saline. Following procedures were the same as in Experiment A. In the Experiment C, 10 NSG mice were randomly divided into 2 groups: a) active treatment (20 μg/mouse), b) control (saline). Tail-vein injections were repeated after 48 h for total 3 times. 24 h after the last dose, mice were sacrificed. At the end of all experiments, a necropsy was performed, tumors were extirpated, and measured by Vernier caliper. Lungs and liver were removed and washed with sterile saline. All tissues were stored in RNAlater (Sigma-Aldrich, Waltham, USA) for further analysis. Determination of miR-215 expression in animal tissues Tissue specimens were homogenized (MM301, Retsch GmbH & Co. KG, Germany) and total RNA was isolated using mirVana miRNA Isolation Kit (Ambion, Austin, USA) according to the manufacturer’s protocol. Concentration and purity of the isolated RNA were determined spectrophotometrically using Nanodrop ND-1000 (Thermo Fisher Scientific, Waltham, USA). Reverse transcription was performed using gene-specific primer (hsa-miR-215-5p) according to the TaqMan MicroRNA Assay protocol. TaqMan Universal PCR Master Mix (NoUmpErase UNG; Thermo Fisher Scientific, Waltham, USA) was used for RT-PCR quantification, which was performed on QuantStudio 12K Flex Real-time PCR System (Applied Biosystems, Foster City, USA). Data normalization and statistical analysis Tumor volume was calculated using a mathematical approximation V=0.5 × (L × W)2 , where L and W stand for length and width of a tumor, S484 Merhautová et al. Vol. 65 respectively. Tumor volumes were analyzed using Kruskal-Wallis test in the case of three groups of treatment, or Mann-Whitney test when comparing two groups (both GraphPad Prism 5.03). Normalized (2-(Ct-40) ) miR-215 expression data were analyzed by KruskalWallis test or Mann-Whitney test, respectively (GraphPad Prism 5.03). p-values ≤0.05 were considered statistically significant. Results Effect of intravenous administration of miR-215 mimic on tumor volume Experiments A and B assessed the effect of intravenous administration of miR-215 mimics on tumor growth in mice model of CRC. The model was established by subcutaneous xenotransplantation of human CRC cells HCT-116+/+ . Tail-vein injections started on Day 7 postinoculation and were repeated 4 times. In Experiment A, miRNA mimics were administered in the dose of 20 μg/mouse and were encapsulated in liposomes. Interestingly, there were no statistically significant changes in tumor volume measured after 4 administrations of 20 μg miR-215 mimics compared with negative control (negative control oligonucleotide in liposomes), or saline (Kruskal-Wallis test, p>0.05, Fig. 2A). Expression of miR-215 in tumor tissue was quantified by RT-PCR. In accordance with the results related to the tumor volume, miR-215 level was not significantly higher in the group of mice treated with miR-215 mimics compared with the negative control and saline (Kruskal-Wallis test, p>0.05), however a slight trend in increase could be seen here (Fig. 2B). As the results of Experiment A were negative, we stated two hypotheses of responsible issues. The first was potentially inappropriate dose. To test this, we increase the dose of miR-215 to 40 μg/mouse. The design of Experiment B was the same. Again, there were no significant changes in tumor volume measured after 4 administrations of miR-215 mimics compared with negative control and saline (Kruskal-Wallis test, p>0.05, Fig. 3A). We have also measured the level of miR-215 in tumor tissue by qRT-PCR and the results were quite similar as in Experiment A, i.e. non-significant trend in increase of miR-215 in tumors of animal treated with miRNA mimics (Kruskal-Wallis test, p>0.05, Fig. 3B). The second hypothesis was related to extratumoral accumulation of liposomes. In order to verify this, we removed lungs and liver from 1 animal per group from Experiment B and analyzed the levels of miR-215. In these pilot settings, we obtain interesting comparison suggesting potential accumulation in lungs, although this result was quite preliminary because of minimal number of samples (data not shown). Potential extratumoral accumulation with liposomes containing miRNA mimics cargo Experiment C was intended to elucidate the potential undesired accumulation of the liposomal delivery system. Number of treatment groups was reduced to the active treatment by liposomal miR-215 mimics (20 μg/mouse), and saline. Tumors, lungs and liver were gathered from all animals (N=10). As was expected, there were no statistically significant changes in tumor volume measured after 3 administrations of miR-215 mimics compared with saline (Mann-Whitney test, p>0.05, Fig. 4A). An increase in the levels of Fig. 2. Tumor volume (A) and miR-215 expression (B) in Experiment A after four administrations of miR-215 mimics (20 μg/mouse, MIM), negative control oligonucleotide (NC), or saline (SAL). Each group consisted of 4 mice. Kruskal-Wallis test, NS not significant (p>0.05). 2016 miRNA Mimics in Animal Model of Colorectal Carcinoma S485 miR-215 in tumors of mimics-treated mice was not statistically significant probably due to higher variability of measured levels (Mann-Whitney test, p>0.05, Fig. 4B). We have observed low expression of miR-215 in lungs and tumors of control animals treated with saline. MiR-215 levels was significantly higher in lungs of mimics-treated animals than in tumors of the same animals, and also than in tumors and lungs of control saline-treated mice (Kruskal-Wallis test, p=0.0277, Fig. 5A). Although the result suffered with high variability of miR-215 lungs expression levels, the hypothesis of lung accumulation seems to be a possible explanation. The expression of miR-215 in liver was high both in active treatment and in control group, and the groups did not significantly differ (Mann-Whitney test, p>0.05, Fig. 5B). Thus, we can assume no liver accumulation. Discussion MiR-215 was proved as tumor suppressor in CRC both descriptively in human tumor tissue, and functionally in stable cell lines. As it is down-regulated in tumor, increase of miR-215 levels is associated with decreased proliferation, viability, and migration of cancer cells in vitro, and decreased tumor volume in vivo (Braun et al. 2008, Faltejsková et al. 2012). The aim of our study was to increase miR-215 intracellular levels by systemic administration of miRNA mimics. These substances are oligonucleotides with various chemical modifications in the structure made in order to grant higher effect than native mature miRNAs through higher affinity to the target mRNA. We have chosen liposomal delivery system, since liposomes have already been a part of standard pharmacotherapy (e.g. liposomal doxorubicin, amphotericin B etc.). Liposomes could be accumulated in tumor site by enhanced permeability and retention effect caused by imperfect neoangiogenesis and delayed lymphangiogenesis (Matsumura and Maeda 1986). The most important benefit of the liposomal reagent MaxSuppressor™ In Fig. 3. Tumor volume (A) and miR-215 expression (B) in experiment B after four administrations of miR-215 mimics (40 μg/mouse, MIM), negative control oligonucleotide (NC), or saline (SAL). Each group consisted of 4 mice. Kruskal-Wallis test, NS not significant (p>0.05). Fig. 4. Tumor volume (A) and miR-215 expression (B) in experiment C after three administrations of miR-215 mimics (20 μg/mouse, MIM), or saline (SAL). Each group consisted of 5 mice. Mann-Whitney test, NS not significant (p>0.05). S486 Merhautová et al. Vol. 65 Vivo RNA-LANCEr II is the use of charge neutral lipids (NLE). While cationic lipids have enhanced capacity for binding negatively charged oligonucleotides, they exert electrostatic interactions with various molecules on the cell surface and could decrease proliferation, or alter gene expression (Wu et al. 2001). Liposomes originating from positively charged lipids could also form aggregates with plasma proteins which leads to elimination by mononuclear phagocyte system and accumulation in liver and spleen (Zhang et al. 2012). By choosing charge neutral lipid emulsion, we assumed to avoid these issues and retain positive aspects of liposomal delivery system. MaxSuppressor™ In Vivo RNA-LANCEr II has been previously used in animal models of cancer diseases to delivery miRNA-based experimental therapeutics. Most of this experiments used intratumoral delivery of liposomes, but doses similar to ours were also used intravenously in subcutaneous xenografts, orthotopic models and metastasis models of lung, breast, and prostate carcinoma, multiple myeloma, Ewing’s sarcoma etc. (Trang et al. 2011, DeVito et al. 2011, Amodio et al. 2012, Imam et al. 2012, DiMartino et al. 2013, Hatano et al. 2015). Unfortunately, we were not able to significantly increase intracellular levels of miR-215 in tumor by intravenous administration of miR-215 mimics encapsulated in NLE liposomes. Not surprisingly with this finding, the tumor volume also did not differ between the treatment groups. In a search for a possible explanation, we have stated two hypotheses. The first was related to the appropriate dose. Doses ranging from 20 to 30 μg/mouse were frequent in the literature. Although we have assessed two doses, they were not proportionally different because we were limited with the tolerable volume of intravenous injection in mice and from the other side with encapsulation capacity of NLE liposomes guaranteed by the manufacturer. Moreover, a trend in increase of miR-215 in the group of mice treated with 20 μg miR-215 mimics, which was not statistically significant, was later repeated but not augmented after administration of 40 μg mimics/mouse assuming no dose-dependent observable changes. The dose remains an important point that need further research, however we have focused on the second hypothesis – a possibility of extratumoral accumulation. Almost all new delivery systems have to deal with plasma protein interactions and possible recognition by circulating or organ immune cells followed by elimination. Trang et al. (2011) described the situation 10 min after intravenous injection of 20 μg of miR-214 mimics in NLE liposomes. They found highest level in blood (approx. 4 mil. copies/10 ng RNA), while in perfused lung, there were approx. 34,000 copies/10 ng RNA, and in the liver approx. 4,000/10 ng RNA. They also assessed distribution of NLE liposomes with oligonucleotide cargo into orthotopic lung tumors by in vivo bioimaging 48 h after administration and successfully used miR-34 and let-7b mimics in NLE formulation in orthotopic animal model of lung carcinoma (Trang et al. 2011). Together with our findings, it could be concluded, that lung accumulation probably prevails over time in NLE liposomes, which makes them suitable for distribution of drugs or experimental substances into the lung tissue. Our study is limited primarily with variability of some of the measurements originating probably from small number (4-5) of mice in the treatment groups, which influences statistical significance of some of the Fig. 5. Comparison of miR-215 expression in lungs and tumor (A) and in the liver (B) in experiment C after three administrations of miR-215 mimics (20 μg/mouse, MIM), or saline (SAL). Each group consisted of 5 mice, L stands for lungs, LIV liver, T tumors. Kruskal-Wallis test (A), or Mann-Whitney test (B), NS not significant (p>0.05), * p=0.0277. 2016 miRNA Mimics in Animal Model of Colorectal Carcinoma S487 results. Another limitation lies in the use of subcutaneous model. As this setting is relatively easy to produce, monitor and maintain, and it is generally the most frequently used approach, it has quite limited translational potential. Our results cannot be applied for an estimation of intestinal or colonic distribution of liposomal dosage form. Orthotopic model of CRC, created by implantation of human cancer cells into the intestinal wall of caecum in mice under general anesthesia would produce more complex picture. Conclusions The application of NLE liposomes in animal model of CRC likely requires substantial optimization mainly in terms of the model type, route of administration and posology. It could not also be excluded that it represents an impass. Nevertheless, miR-215 remains an auspicious therapeutic target in CRC for its tumor suppressor effects, even though there are still some obstacles to deal with in order to bring this miRNA closer to clinical practice. Conflict of Interest There is no conflict of interest. Acknowledgements The authors thank Jan Verner for his advice and everyday care for the animals, and Jaroslav Nádeníček and Kamila Součková for technical assistance in the Animal Facility. 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REVIEW published: 27 September 2016 doi: 10.3389/fphar.2016.00329 Frontiers in Pharmacology | www.frontiersin.org 1 September 2016 | Volume 7 | Article 329 Edited by: Gautam Sethi, National University of Singapore, Singapore Reviewed by: Dhiraj Kumar, National Centre for Cell Science, India Subash Gupta, Banaras Hindu University, India *Correspondence: Ondrej Slaby ondrej.slaby@ceitec.muni.cz Specialty section: This article was submitted to Cancer Molecular Targets and Therapeutics, a section of the journal Frontiers in Pharmacology Received: 28 June 2016 Accepted: 06 September 2016 Published: 27 September 2016 Citation: Merhautova J, Demlova R and Slaby O (2016) MicroRNA-Based Therapy in Animal Models of Selected Gastrointestinal Cancers. Front. Pharmacol. 7:329. doi: 10.3389/fphar.2016.00329 MicroRNA-Based Therapy in Animal Models of Selected Gastrointestinal Cancers Jana Merhautova1, 2 , Regina Demlova2 and Ondrej Slaby1, 3 * 1 Molecular Oncology II – Solid Cancer, Central European Institute of Technology, Masaryk University, Brno, Czech Republic, 2 Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic, 3 Masaryk Memorial Cancer Institute, Brno, Czech Republic Gastrointestinal cancer accounts for the 20 most frequent cancer diseases worldwide and there is a constant urge to bring new therapeutics with new mechanism of action into the clinical practice. Quantity of in vitro and in vivo evidences indicate, that exogenous change in pathologically imbalanced microRNAs (miRNAs) is capable of transforming the cancer cell phenotype. This review analyzed preclinical miRNA-based therapy attempts in animal models of gastric, pancreatic, gallbladder, and colorectal cancer. From more than 400 original articles, 26 was found to assess the effect of miRNA mimics, precursors, expression vectors, or inhibitors administered locally or systemically being an approach with relatively high translational potential. We have focused on mapping available information on animal model used (animal strain, cell line, xenograft method), pharmacological aspects (oligonucleotide chemistry, delivery system, posology, route of administration) and toxicology assessments. We also summarize findings in the field pharmacokinetics and toxicity of miRNA-based therapy. Keywords: microRNA, gastric cancer, pancreatic cancer, gallbladder cancer, colorectal cancer, animal model, mice, preclinical testing INTRODUCTION Research in the field of non-coding nucleic acids has advanced extensively in the last 15 years. It is now well known, that dysregulation of miRNAs, powerful regulators of gene expression, is associated with many diseases. MiRNAs are investigated thoroughly in cancer biology and oncology and the number of published articles is growing (Figure 1). Last 10 years brought us an immense amount of information about the roles of miRNAs in cancer cell pathophysiology. All described hallmarks of cancer (Hanahan and Weinberg, 2011) are in relation with some miRNA imbalance (Ruan et al., 2009). Attempts to therapeutically interfere with miRNAs levels in pathologic cells are moving forward to preclinical and clinical phases of new therapies development. Although there are severe limitations and barriers facing miRNA-based therapy, more and more studies are performed with auspicious results. The purpose of this review is to analyze preclinical studies carried out on animal models of selected gastrointestinal cancer (gastric, pancreatic, gallbladder, and colorectal). We have focused primarily on pharmacological aspects of miRNA-based therapy with the emphasis on delivery systems, and also on the type of animal model, and on toxicity assessments. Eventually, we summarize important findings in the field pharmacokinetics and toxicity of miRNA-based therapy to make the picture comprehensive. Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers FIGURE 1 | Number of new publications found by the term “miRNA AND cancer” in SCOPUS database. The Biogenesis of Endogenous miRNAs MiRNAs are endogenous small (∼22 nt) single-stranded non-coding RNAs. Their main role in the cell lies in post-transcriptional attenuation of mRNA translation. The biosynthesis of miRNAs begins in the nucleus. Long double-stranded transcripts (pri-miRNAs) are formed by RNA-polymerases II and III. Pri-miRNAs are cleaved by the ribonuclease Drosha and DGCR8 protein to form pre-miRNAs, double-stranded chains ∼70 nt long. Pre-miRNAs are then transported from the nucleus to the cytoplasm via Exportin-5 protein. In the cytoplasm, mature miRNAs are created through the interaction with endonuclease Dicer and TRBP protein. Double-stranded formation is rearranged, the guide strand forms a complex with Argonaut and other proteins forming miRISC complex, and plays an active role in the gene expression attenuation. The other strand, called passenger strand, is usually degraded in the cytoplasm, or persists and may exert its own biological activity. For detailed information on miRNA biogenesis see a recent review by Romero-Cordoba et al. (2014). There are also number of proofs of mature miRNAs’ presence and activities in the nucleus (Hwang et al., 2007; Park et al., 2010; Jeffries et al., 2011; Li et al., 2013). It seems that these miRNAs could transfer from cytoplasm to nucleus and nucleolus via Exportin-1 and Importin-8 (Li et al., 2013; Wei et al., 2014) and influence expression of other miRNAs, or of its own (Tang et al., 2012; Zisoulis et al., 2012; Wei et al., 2014). Mechanism of Action MiRNAs bind mainly to the 3′-untranslated region of mRNA (3′-UTR), although there are several evidences that miRNAs could bind to the 5′-UTR, or to the coding sequence itself (Ott et al., 2011; Gu et al., 2014). In the case of imperfect matching, the duplex mRNA:miRNA is not translated, or it is translated incompletely and the polypeptide chain is subsequently degraded. Binding of miRNA to mRNA target also activates deadenylation of 3′-poly(A) end of mRNA through deadenylases, which is a first step of mRNA destabilization and later degradation by 3′- and 5′-exonucleases (Figure 2). Perfect matching leads to direct cleavage of the target mRNA. Imperfect matching is more common in animal cells, while perfect matching is typical for plant cells (Axtell et al., 2011). The binding specificity is ensured by the seed sequence of miRNA, which contains 6–8 nt and which is very often conservative through the species (Hogg and Harries, 2014). One miRNA can regulate many different genes, and more than 50% of all genes are suggested to be regulated by miRNAs. Thus, miRNA network affect most of cellular processes from the basic metabolic maintenance, through differentiation, cell division and proliferation, to the death (Calin and Croce, 2006; Esquela-Kerscher and Slack, 2006; Garzon et al., 2006; Zhang et al., 2013). In cancer tissues, a lot of changes in miRNA levels could be found. MiRNAs decreased in cancer cells are termed tumor suppressors and reversely, oncogenic miRNAs are those abundant in cancer tissue. There have already been signs of miRNAs that function both as tumor suppressors, and oncogenes depending on the cell type and state (contextdependent miRNAs) (Esquela-Kerscher and Slack, 2006; Kasinski and Slack, 2011). To reverse the pathologic imbalance of miRNAs mature miRNAs, miRNA-mimics, precursors, or expression vectors are administered to increase the level of a specific tumorsuppressor miRNA, and miRNA inhibitors are administered to decrease the level of oncogenic miRNA (Figure 3). Promising results of in vitro studies are nowadays being verified on animal models and first preclinical, or even clinical trials are under way. SEARCH STRATEGY Web of Science database was searched for in vivo studies published in the last 5 years (2010–2015) that were focused on colorectal, pancreatic, gallbladder and gastric cancer. Searching formulas miRNA AND vivo AND colorectal/pancreatic/ gallbladder/gastric in article topic (title, abstract and keywords) was used. The search was finished by the end of February 2016. About 430 articles were found and further analyzed to select the specific experimental design: at first, induction of a tumor by transplantation of human or murine tumor cells, or tumor tissue, then followed by the administration of miRNA mimic, precursor, expression vector, or inhibitor. The bulk of the studies found by the searching formula were excluded because of using different methods, e.g., influencing the expression level of miRNA in cancer cells before transplantation into the animal body, or administration of other substances that affect miRNA levels and processes like natural compounds, siRNAs etc. 26 studies included in this review matched the aforementioned criteria. Both the articles themselves and the supplemental materials were scrutinized with accent on animal model used (animal strain and gender, xenograft method, cancer cell line, or source), pharmacological aspects (oligonucleotide chemistry, delivery system, posology, route of administration), toxicology assessments (methods and findings), and eventually the experimental therapy effect. Some of the information could not be obtained from articles or supplements, as they lacked e.g., animal gender specification. Frontiers in Pharmacology | www.frontiersin.org 2 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers FIGURE 2 | Biosynthesis and mechanism of action of miRNAs. The biosynthesis begins in the nucleus by transcription of miRNA genes by RNA polymerase II (Pol II). Long transcripts, pri-miRNAs, are cleaved by Drosha and DGCR8 protein creating pre-miRNA with hairpin structure. Exportin 5 transfers pre-miRNA into the cytoplasm, where it is processed by Dicer into miRNA duplex. Mature single-strand miRNA forms RISC complex with Argonaut (Ago) and other proteins and attenuates mRNA translation and leads to the destabilization of mRNA by deadenylation. OVERVIEW OF THE SELECTED STUDIES All selected studies assorted by the organ of cancer cells’ origin are summarized in Tables 2–5. Visual summary of therapeutic strategy, type of animal model and routes of administration of miRNA-based therapy is demonstrated in Figures 4–6. We have analyzed 26 studies, 20 of them used the miRNA replacement therapy regimen, and others were miRNA inhibitions. Two studies combined miRNA replacement therapy with chemotherapy, two studies combined miRNA inhibition with either chemotherapy, or immunotherapy. Subcutaneous xenograft model was used in 23 cases, orthotopic xenotransplantation was performed in two experiments, and combination of both was done in one study. In 17 studies, miRNA-based therapeutics were administered locally, i.e., injected intratumorally. Five studies involved systemic administration by tail-vein, or intraperitoneal injection, while four studies combined both routes of administration in a separate substudies, or combined systemic administration of e.g., chemotherapy, with local administration of miRNA-based therapy. MiRNAs studied in the selected articles were both known tumor suppressors, or oncogenes, and also context-dependent miRNAs whose effect varies according to the type of cancer cell. All of them influence the main hallmarks of cancer such as uncontrolled tumor cell proliferation, impaired process of apoptosis, defects in the control of cell cycle, increased migration and invasivity, or tumor angiogenesis (Table 1). Some miRNAs were tested in combination with cytostatic agents (doxorubicin, gemcitabine, or oxaliplatin) to achieve sensitization of chemotherapy-resistant cells and tumors, e.g., by decreasing the expression of efflux proteins such as ABCB1 (P-glycoprotein). The main result of all in vivo studies was inhibition of tumor xenograft growth, at least in a transient manner. All results and references could be found in Tables 2–5. Toxicity assessment was part of 11 studies. It was performed at least as animal body weight control but usually was followed by animal behavior observation, histopathology examination of tissue dissections of various organs (brain, heart, liver, lungs, spleen, kidney), or blood biochemistry with regard to liver and kidney functions (blood urea nitrogen, liver enzymes, bilirubin). There were two declared deaths of experimental Frontiers in Pharmacology | www.frontiersin.org 3 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers FIGURE 3 | Strategies in miRNA-based therapy. The most frequently used animal model of cancer is immunodeficient mouse bearing a subcutaneous tumor created from cells of human origin. In miRNA-based therapy, two concepts are adopted nowadays, which is the replacement therapy (left) and inhibition therapy (right). Tumor suppressors MiRNAs are decreased in cancer cells and to increase their levels mature miRNAs, miRNA-mimics, precursors, or expression vectors are administered. Oncogenic miRNAs are abundant in cancer tissue and to silence their effects, various types of miRNA inhibitors could be administered. animals in the selected studies. These mice were administered cholesterol-conjugated oligonucleotide, but both were from the negative control group. The cause of death was not determined (Ye et al., 2013). One study declared slight but statistically significant elevation of blood urea nitrogen in the group of mice treated systemically with mature miRNA conjugated with carbonate apatite nanoparticles (Hiraki et al., 2015). Transient hepatotoxicity was found in mice systemically treated with adenoviral vector Ad-L5-8miR148aT, but the symptoms were milder than those produced by administration of Adwt (Bofill-De Ros et al., 2015). No immune response to the RNA-based treatment was reported in the selected studies, as they used immune deficient strains. Depending on the specific genotype, nude or severe immunodeficient (SCID) mice lack normal cytokine production together with other immune impairments. The selected studies utilized various types of administered miRNA-based substances and different delivery systems. These issues and their fine tuning are the main points in the successful development of a miRNA-based therapy. Ability to overcome natural barriers that face transferring an oligonucleotide into the cell has to be balanced with the extent of toxicity, as systems with good cell penetration are usually more cytotoxic in a non-specific manner. To bring a complex sight on the development of miRNA-based therapy in gastrointestinal cancer, we gathered relevant information about the type of substances, delivery systems and routes of administration used in the selected 26 studies and we discuss them in detail. The issue of toxicity is described for each delivery system and later on also for the concept of miRNA-based therapy itself. IMPORTANT ISSUES IN THE FIELD OF miRNA-BASED THERAPY PRECLINICAL TESTING Routes of Administration and Delivery Systems MiRNA-based therapeutics in animal studies summarized in this review were administered either systemically, or locally. In systemic delivery, the intravenous (tail-vein) and intraperitoneal injections were used (Figure 6). Local administration was performed as intratumoral injection into the subcutaneous tumors. Delivery systems employed in the presented studies include viral vectors, biocompatible cationic polymers and copolymers, inorganic nanoparticles, atelocollagen, and liposomes. Frontiers in Pharmacology | www.frontiersin.org 4 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers TABLE 1 | Examples of studied miRNAs in association with some of the hallmarks of cancer and other cancer cells attributes (I, inhibition strategy; R, replacement strategy). Cancer cell attribute Studied miRNA Replacement or inhibition strategy References Uncontrolled cell proliferation miR-21 I Sicard et al., 2013 miR-27a R Bao et al., 2014 miR-33a R Ibrahim et al., 2011 miR-145 R Ibrahim et al., 2011 miR-218 R He et al., 2012 miR-429 R Sun Y. et al., 2014 Impaired apoptosis let-7 I Geng et al., 2011 miR-20a I Chang et al., 2013; Wang et al., 2013 miR-21 I Frampton et al., 2011; Sicard et al., 2013 miR-27a R Bao et al., 2014 miR-145 R Ibrahim et al., 2011 miR-4689 R Hiraki et al., 2015 Dysfunction in cell cycle control miR-133a R Dong et al., 2013 miR-200a R Cong et al., 2013 miR-218 R He et al., 2012 miR-1266 R Chen et al., 2014 Cell migration and invasivity miR-27a R/I Frampton et al., 2011; Bao et al., 2014 miR-200a R Cong et al., 2013 miR-429 R Sun Y. et al., 2014 miR-1207-5p R Chen et al., 2014 Neoangiogenesis miR-27a I Frampton et al., 2011 miR-27b R Ye et al., 2013 Resistance to cytostatic agents miR-103 R Zhang et al., 2015 miR-107 R Zhang et al., 2015 Viral Vectors Viral vectors could be administered both locally, and systemically and they include lentiviruses, adenoviruses, and adeno-associated viruses (Chen et al., 2015). Viral delivery of antisense construct expression vectors was used to inhibit miR-21 and miR-148a in animal models of pancreatic cancer (Bao et al., 2014; Bofill-De Ros et al., 2015), while expression vector for miR-1266/1207- 5p was examined in replacement therapy in gastric carcinoma (Chen et al., 2014). Viruses are able to effectively deliver miRNA therapeutics (precursors, mimics, genes, or inhibitors) into the tumor cell, but their use could be associated with the risk of insertional mutagenesis, gain of replication competency of viral particles, or immune activation. Nucleic acid of adenoviruses (dsDNA viruses) and adeno-associated viruses (ssDNA viruses) usually do not integrate into the host cell genome, while lentiviral (ssRNA viruses) integrates (Soriano et al., 2013; Chen et al., 2015). Adeno-associated viruses are generally less immunogenic, but adenovirus-based delivery system could produce at least transient hepatotoxicity (Broderick and Zamore, 2011; Aslam et al., 2012) as was also observed by Bofill-De Ros et al. in animal model of pancreatic ductal adenocarcinoma (Bofill-De Ros et al., 2015). Cationic Polymer Polyethylenimine In the selected studies, the most frequently used synthetic polymer was polyethylenimine (PEI). It was utilized to deliver mimics or expression vectors of miR-34a, miR-206, and miR- 217 in animal model of pancreatic cancer, or miR-33a and miR-145 in the model of colorectal carcinoma (Tables 4, 5). PEI is cationic polymer able to produce nanoparticles. It has linear or branched structure and different molecular weight according to the reaction conditions during the synthesis. Due to the positive charge, PEI has high capacity for negatively charged oligonucleotides and nucleic acids which are moreover condensed after complexation with PEI, and thus protected from nucleases. The charge of PEI also facilitate cellular uptake by electrostatic interaction with negatively charged surface molecules (e.g., heparin sulfate proteoglycans), after which the particles enter the cell by endocytosis. PEI is able to disrupt the endosome and release the cargo into the cytoplasm, which grants this method high transfection efficacy. The disruption of endosome is achieved by protonization of PEI and buffering of acidic environment of the vesicle. These processes are followed by osmolarity changes and water intake which leads to the swelling and burst of the endosome (Höbel and Aigner, 2013; Zhang et al., 2013). Better capacity and efficacy is achieved by branched PEI but at the cost of higher nonspecific cytotoxicity. In the presented studies, mostly linear PEI is used, like commercially available transfection reagents ExGen500TM (Euromedex, Mundolsheim, France) and in vivo- jetPEITM (Polyplus Transfection, Illkirch, France) assigned for in vivo experiments. Other issues associated with PEI delivery are an aggregation of created nanoparticles, or opsonization in the plasma recognized by phagocytes. PEI with high density of positive charge could also trigger erythrocyte aggregation and thrombosis (Kanasty et al., 2012). PEI particles could be conjugated with various molecules [e.g., polyethylene glycol (PEG), or antibodies] to resolve such difficulties (Malek et al., 2009). PEI is not a biodegradable polymer, thus its toxicity is intensively discussed. It depends strongly on molecular weight and branching (Fischer et al., 1999) and also on the cargo, as it may neutralize the charge of PEI. There are studies describing immune activation in vivo, (Beyerle et al., 2011) hepatotoxicity and lethality in mice (Chollet et al., 2002) and increased apoptosis in vitro (Merkel et al., 2011), and also those that proved no immune response, or hepatotoxicity in mice (Bonnet et al., 2008). Inorganic Nanoparticles—Iron Oxide, and Carbonate Apatite Sun et al. used iron oxide nanoparticles to deliver miR- 16 and overcome doxorubicin resistance in animal model of gastric adenocarcinoma (Sun Z. et al., 2014). Iron oxide nanoparticles (IONPs) are biocompatible and biodegradable particles with magnetic properties. They are composed of magnetite [Fe3O4, iron (II,III) oxide], or maghemite (Fe2O3, Frontiers in Pharmacology | www.frontiersin.org 5 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers ferric oxide) and are usually coated with various other molecules (PEI, PEG, chitosan etc.) to improve their properties (Kievit and Zhang, 2011). IONPs could also serve as theranostics (i.e., substances with both diagnostic and therapeutic purpose). As well as other nanoparticles, IONPs protect nucleic acids from being cleaved by nucleases (Kievit et al., 2009) but could also be opsonized in the plasma and recognized by phagocytes, mainly by the reticuloendothelial system (RES). Non-coated IONPs are distributed in heart, liver, spleen, lungs, kidney, brain, stomach, small intestine, and bone marrow, while the highest concentration are reached in the liver and spleen due to the elimination by RES and macrophages (Wang et al., 2010). IONPs enter the cell by endocytosis and are degraded in the endosomes (Xie et al., 2009). Particles between 10 and 60 nm are the most effective, as they undergo limited kidney and liver/RES uptake, and are absorbed by tumor cells (Kievit and Zhang, 2011). Cytotoxicity of IONPs coated with PEI occurs in vitro in higher concentration than is needed for sufficient transfection (Lellouche et al., 2015). The administration in vivo could increase blood iron and intracellularly increase oxidative stress (Mahmoudi et al., 2011). Non-coated particles could produce hepatotoxicity, and lung or kidney damage (Hanini et al., 2011). Study of Hiraki et al. describes utilization of different inorganic nanomaterial, carbonate apatite nanoparticles. They used these particles as a delivery system for mature miR- 4689 in animal model of colorectal adenocarcinoma (Hiraki et al., 2015). Carbonate apatite [Ca10(PO4)6−X(CO3)X(OH)2] is composed of calcium cations and phosphate and carbonate anions in defined ratios. It was firstly described as a transfection reagent and a delivery system for plasmid DNA by Chowdhury et al. (2006). Nanoparticles of carbonate apatite are stable in plasma (pH = 7.4), protect nucleic acids from nuclease cleavage, but in acidic environment of endosomes, they are quickly degraded. Their cargo is then released and could probably escape from endosomes, as high effectivity of this transfection method was proved for DNA (Wu et al., 2015) and RNA (Hossain et al., 2010). In mice, these nanoparticles are accumulated in tumor probably due to the EPR effect (discussed below), but slight accumulation was found also in the liver (Wu et al., 2015). As this method arose from calcium phosphate co-precipitation, which is known to produce certain level of cytotoxicity in vitro, adverse effects in animals were inquired. In mice, Wu et al. declared no mortality, weight loss, or histological damage in liver, kidney, and spleen after administration of common dose, and also after 2.5 and 5-fold higher doses. They also do not observed any urinary calculi in a mouse model of repeated administration. The team advanced to the evaluation of the delivery system on monkeys (macaques Macaca fascicularis, formerly M. cynomolgus). Monkeys received repeated i.v. infusions during a movement restraint. Equivocal results were obtained, as some animals had reversible increase in AST, ALT, LDH, and CPK enzymes, but from further analyses of isoenzymes, authors suggested that these increments might arise from the stress associated with body restriction rather from heart or liver damage (Wu et al., 2015). Atelocollagen For direct intratumoral treatment, atelocollagen was used in the study of Frampton et al. in pancreatic ductal adenocarcinoma to deliver miR-21, miR-23a, and miR-27a (Frampton et al., 2011). Atelocollagen is a biocompatible and biodegradable polymer. It was developed and tested in vivo for gene (plasmid) delivery with controlled release by Ochiya et al. (Ochiya et al., 1999; Hao et al., 2016). Atelocollagen is prepared from collagen extracted from bovine dermis. Natural collagen contains specific amino acid sequences on both C- and N-terminus (“telopeptides”), which are highly immunogenic. By digestion with pepsin, these telopeptides are cleaved. The polymer is liquid at low temperatures, but solidifies at temperatures above 30◦C (Ochiya et al., 2001; Komatsu et al., 2016). After intramuscular injection of plasmid DNA in complex with glucose and atelocollagen in mice, the transfection was efficient and last more than 60 days. No apparent toxicity, or hematologic changes were observed in this study, (Ochiya et al., 1999) as well as Frampton et al. described neither changes in mice body weight, nor serious adverse effects (Frampton et al., 2011). Cationic Lipids and Liposomes For intratumoral administration, several studies used lipidbased transfection reagent Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, USA) designed originally for in vitro experiments, e.g., Dong et al. in animal model of colorectal adenocarcinoma to deliver miR-133a (Dong et al., 2013). Pramanik et al. utilized DOTAP (N-[1-(2,3-dioleoyloxy)propyl]N,N,N-trimethylammonium methyl-sulfate) with co-lipids formula to deliver plasmid expression vector of miR-34a, and miR-143/145 cluster systemically in animal model of pancreatic ductal adenocarcinoma (Pramanik et al., 2011). Both delivery systems are composed of cationic lipids that form liposomes, vesicles with lipophilic bilayer and aqueous core able to encapsulate hydrophilic molecules (Mallick and Choi, 2014). By electrostatic interaction, cationic lipids have increased capacity for negatively charged nucleic acids (Xue et al., 2015). They enter the cells by endocytosis and are able to destabilize and breach endosomal membrane by interaction with its phospholipids (Zelphati and Szoka, 1996). Cationic lipids exert detergent effect on lipid membranes and interact also with enzymes, thus could irritate cells, decrease proliferation, alter gene expression, and even trigger cell lysis (Wu et al., 2001). They share the same advantages and disadvantages that account for positive charge as cationic polymers. They form aggregates with plasma proteins leading to RES elimination and accumulation in spleen and liver (Nchinda et al., 2002; Zhang et al., 2012). With decrease of positive charge, RNA encapsulation and transfection efficacy is decreasing. PEGylation increases blood circulation time of liposomes, (Pathak et al., 2011; Suk et al., 2016) but could lead to the formation of anti-PEG IgM antibodies (Ishida et al., 2006). Liposomes are generally less immunogenic than cationic polymers. But after processing of liposomes, some RNA molecules might remain on the surface of a particle. In the studies using siRNA, these residues lead to the significant immune activation (Xue et al., 2015). Inflammatory response in the liver followed by hepatotoxicity and with higher doses Frontiers in Pharmacology | www.frontiersin.org 6 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers TABLE2|Invivostudiesinanimalmodelsofgastricadenocarcinoma. Xenografted cellline MiRNA(s) ofinterest Therapeutic strategy Animal model Animal strain (gender) Deliverysystemand chemical modifications Routeof administration DosageFrequencyof administration ResultsToxicity, undesired effects References SGC-7901miR-17- 5p/20a ITSXBALB/cmiceAntagomiR-17-5pand antagomiR-20a (RiboBio) Intratumoralinj.25µmolTwiceweeklyfor2 weeks Inhibitionoftumor growth,increasein thepercentageof apoptoticcellsin tumortissue NotassessedWangetal.,2013 SGC-7901miR-200aRTSXBALB/c-A mice miRNA-mimics (GenePharma)with Lipofectamine2000 (Invitrogen) Intratumoralinj.10µlTwiceafter2daysInhibitionoftumor growth NotassessedCongetal.,2013 SGC-7901miR- 1266/1207- 5p RTSXNudemice (females) Lentiviralvector (Lv-miR-166/1207-5p, GeneChem Management) Intratumoralinj.0.1ml107 PFU/ml SingledoseInhibitionofTumor growthandtumor cellsproliferation NotassessedChenetal.,2014 Doxorubicin resistant SGC- 7901/ADRfluc miR-16RT+ chemotherapy SXBALB/cmice (females) miRNA oligonucleotides (GenePharma)bound onPEG-coatedFe3O4 nanoparticles Tail-veininj.5mg/kg (1nmol) Seventimes(days 0,3,7,10,14,17, 21postinocul) Tumorsize reduction,increase ofnumberof apoptoticnuclei, increased sensitivityto doxorubicin Nonoticeable damagein histologyanalysis ofheart,liver, spleenandkidney SunZ.etal.,2014 DoxorubicinIntraperitoneal inj. 2.5mg/kgOnceaweekfor4 weeks(days0,7, 14,21) Doxorubicin resistant SGC- 7901/ADR miR- 103/107 RT+ chemotherapy SXBALB/cmiceCholesterol-conjugated 2′-O-methyl-modified agomiR-103/107 (RiboBio) Intratumoralinj.1nmolEvery4daysfor seventimes Delayedtumor growth,reduction intumorvolume, lowerproliferative potential, increased sensitivityto doxorubicin Noobvioussigns oftoxicitysuchas weightlossover thecourseofthe treatment Zhangetal.,2015 DoxorubicinIntraperitoneal inj. 2mg/kgEveryotherday Ad-wt,wildtypeadenovirus;ALT,alaninetransaminase;AST,aspartateaminotransferase;BUN,bloodureanitrogen;DOTAP,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl-sulfate;DSPE-PEG,1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000];GEM,gemcitabine;incl.,including;inj.,injection;IT,inhibitiontherapy;mAb,monoclonalantibody;NOD/SCID,non-obesediabetic/severecombinedimmunodeficiency;OD, opticaldensity;OX,orthotopicxenograft;PEG,polyethyleneglycol;PEI,polyethylenimine;PFU,plaque-formingunits;postinocul.,postinoculation;RT,replacementtherapy;SCID,severecombinedimmunodeficiency;SX,subcutaneous xenograft;TNF-α,tumornecrosisfactorα;VP,viralparticles. Frontiers in Pharmacology | www.frontiersin.org 7 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers TABLE3|Invivostudyinanimalmodelsofgallbladdercarcinoma. Xenografted cellline MiRNA(s) ofinterest Therapeutic strategy Animal model Animal strain (gender) Deliverysystemand chemical modifications Routeof administration DosageFrequencyof administration ResultsToxicity, undesired effects References GBC-SD initially transfected withmiR-20a antagomir (200nM)for3 days miR-20aITSXNudemiceAntagomiR-20aIntratumoralinj.5nmolTwiceweekly for2weeks Inhibitionof tumor growth NotassessedChangetal.,2013 even lethality was described (Tan and Huang, 2002; Zhang et al., 2005). Pharmacokinetics of Therapeutic Oligonucleotides Chemistry, Physico-Chemical Properties, and Absorption Pharmacokinetics of therapeutically administered oligonucleotides is strongly driven by their physico-chemical properties. Generally, these properties are not sequence-specific in qualitative point of view, but can be quantitatively different from sequence to sequence, and could differ also between chemistries. Native oligonucleotides are small, negatively charged molecules, which means that the transfer through lipophilic membranes necessary for the absorption into systemic blood circulation and also later into the intracellular space is quite problematic. The most common change in oligonucleotide chemistry is the replacement of phosphodiester bond with phosphorothioate bond in the backbone. This change goes usually hand in hand with chemical modification of the 2′ functional group on ribose in the nucleotide (2′-hydroxyl could be substituted e.g., to 2′-O-methyl, 2′-O-methoxyethyl, or 2′-fluoro group), and conjugation with cholesterol. These modifications either increase the stability of oligonucleotides modifying their susceptibility to RNAse cleavage (phosphorothioate bonds, 2′-O-modifications), or increase cellular uptake of the molecule (cholesterol conjugation). Cholesterol-conjugated 2′-O-methyl/methoxyethyl-modified oligonucleotides are sometimes termed “agomiRs” or “antagomiRs” depending on their mechanism of action, and they were utilized in some of the studies focused on gastrointestinal cancer presented in this article (Chang et al., 2013; Wang et al., 2013; Zhang et al., 2015; Zou et al., 2015). Modification on 2′ position could also change the affinity of oligonucleotides to plasma proteins which has a high impact on pharmacokinetics, most importantly on distribution and excretion (Crooke, 2007). Another possibility to change oligonucleotide structure is chemical modification of the ribose forming a 2′,4′-bicyclic structure, which is termed locked nucleic acid (LNA) (Kumar et al., 1998). The most common type of LNA is oligonucleotide with one or more 2′-O-4′-methylene-β-D-ribosyl structure. This bicyclic bridge locks ribose in one of its conformation increasing binding affinity and decrease the susceptibility to nuclease cleavage (Braasch and Corey, 2001). Various chemical modifications in the oligonucleotide structure are now available owing to the development of commercially available miRNA mimics. According to the information provided by manufacturers, miRNA mimics should possess higher affinity to miRISC and thus to the mRNA of interest. MiRNA-mimics should have no off-target biological activities due to the passenger strand, and should exert higher effect than native mature miRNAs. The chemistry modifications differ between passenger and guide strand, and the molecules could also be triple-stranded (e.g., Exiqon, Vedbaek, Denmark). Detailed information about the specific chemistry of the Frontiers in Pharmacology | www.frontiersin.org 8 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers miRNA mimic are usually not released. Some evidences were published last year, that bring the commercially available miRNA mimics into focus because of non-specific dampening effect on overall gene expression, accumulation of non-endogenous high molecular weight RNA species and unintentional passenger strand loading into the RISC discovered after transient transfection of human cell lines. Søkilde et al. describe variations even between batches of a commercially available miRNA mimic obtained from one manufacturer (Søkilde et al., 2015). The authors emphasize the issue of a proper dosage of miRNA mimic and its optimization, and suggest to prefer viral and genetic approaches, as the created transcripts follow the physiological biosynthesis pathway and their mechanism of action could be considered as the very same as endogenous miRNAs (Jin et al., 2015). Undesirable physico-chemical properties of oligonucleotides could be attenuated by delivery systems mentioned before, which subsequently influence pharmacokinetic processes of miRNAbased therapeutics. Distribution, Protein Binding, and Tissue Accumulation After being absorbed or injected into systemic circulation, charged molecules of oligonucleotides bind to various plasma proteins, above all on albumin and α2-macroglobulin (Cheng et al., 2013). The binding and the distribution is nonlinear, saturable, changes slightly with length and sequence of oligonucleotides and is different in rodents and in human. Distribution to the tissues is very quick and prevails over metabolic degradation (Levin, 1999). Naked oligonucleotides accumulate in the liver, kidneys, spleen, bone marrow and lymphatic nodes, while they do not cross the blood-brain barrier, placental barrier and they are not present in testes. In the treatment of cancer, accumulation of a drug in the tumor tissue or in the metastasis site is a desirable state. MiRNA-based therapeutics could achieve this due to enhanced permeability and retention (EPR) effect of a tumor. Enhanced permeability of new vessels and relative lack of lymphatic vessels in the tumor site was firstly described by Matsumura and Maeda (1986). Charge-neutral small particles complexed or loaded with miRNA-based therapeutics have enhanced extravasation and could accumulate in the tumor. Metastatic sites are generally less accessible, as their EPR effect is not so significant (Maeda, 2015). According to the technology of the delivery system used, miRNA-based therapeutic could accumulate also extratumorally in various tissues. All cells capable of phagocytosis accumulate naked oligonucleotides, liposomes, or nanoparticles, e.g., RES cells present in the liver (Kupfer cells) and in the circulation, tissue monocytes and macrophages, and proximal tubular cells (Chen et al., 2015). In this case, the delivery system alone as a protection could be insufficient, because in plasma, these particles get coated by proteins recognized by the RES. The most common defense against RES is PEGylation, binding of polyethylene glycol substituents on the surface of a nanoparticle or liposome, which prevent binding of opsonization proteins and became very common. Contrarily, the excess of PEG on the surface of a delivery system particle could diminish cellular uptake, therefore the process of PEGylation should be optimized (Seto, 2010). Oligonucleotides, liposomes and polymer-based nanocarriers enter the cell by active mechanism, endocytosis. Escape from endosomes is desired to reach the interaction of miRNA with mRNA, however, this is another obstacle in miRNA-based therapy. Some of the carriers could enhance endosomal escape by steric or osmotic effects. pH sensitive molecules could change structure in relatively acidic environment due to electrostatic interactions, which is leading to the mechanical disruption of the vesicle and release of miRNA into cytoplasm (Ju et al., 2014). Other molecules are accepting H+ (proton sponges) and by alteration of ion homeostasis cause swelling and burst of the endosome (Akinc et al., 2005; Chen et al., 2015). Metabolism of miRNA-Based Therapeutics Ubiquitous nucleases begin to degrade oligonucleotides shortly after administration. According to the chemistry changes, free oligonucleotides are metabolized by 3′- and 5′-exonucleases or by endonucleases, and the rate of metabolism depends on the chemical modifications. Endonuclease cleavage is slower and takes place only when 3′ and 5′ end of oligonucleotide is protected by methoxyethyl-modified nucleotides. As was mentioned before, modifications on 2′-hydroxyl on ribose or structural changes in the backbone such as LNA structure can decrease the affinity of nucleases to cleave miRNAbased therapeutics. Also the complexes of oligonucleotides with nanoparticles or liposomes have modified susceptibility to nuclease cleavage. The metabolites of nuclease cleavage are weakly bound to the plasma proteins and therefore are rapidly excreted in urine. Oligonucleotides do not undergo liver oxidation by cytochrome P450, or conjugation processes (Levin, 1999; Crooke, 2007). Excretion Oligonucleotides not bound to proteins are excreted in the urine, while binding to plasma proteins, or other delivery systems like liposomes and nanoparticles of specific parameters (e.g., hydrodynamic diameter up to 5–6 nm) results in protection from being urinary excreted (Crooke, 2007; Cheng et al., 2013). As oligonucleotides accumulate also in the liver, they could be excreted by both these organs. About 10% of the administered dose of naked oligonucleotides, and 80% of the metabolites are urinary excreted. The remaining are excreted by faeces, or endure bound to the tissue, or inside the cells. The elimination halflife of oligonucleotides is 1–30 days depending on the type of tissue. This attribute allows designing a therapeutic regimen comfortable for potential patients with one dose administration for a week, 2 weeks, or a month (Crooke, 2007). Toxicity of miRNA-Based Therapy The toxicity of oligonucleotide administration was largely studied in the field of antisense therapy. In miRNA-based therapy specifically, toxicity assessments are not a part every in vivo study and we have very limited information from the first phases of clinical research. For antisense oligonucleotides not targeted to miRNAs, there are evidences from rodent and non-rodent Frontiers in Pharmacology | www.frontiersin.org 9 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers TABLE4|Invivostudiesinanimalmodelsofpancreaticductaladenocarcinoma. Xenografted cellline miRNA(s)of interest Therapeutic strategy Animal model Animal strain (gender) Deliverysystem andchemical modifications Routeof administration DosageFrequencyof administration ResultsToxicity, undesired effects References MiaPaCa-2 LuciaF1 miR-21ITOXSCIDCB17 mice Lentiviralvector producinghairpins antisenseto miR-21 [LV(a/miR-21)] Intratumoral inj. 150ngSingledoseInhibitionof tumor growthand proliferation, inductionof apoptosis, activationof angiogenesis Nochangesin bodyweight Sicardetal., 2013 IT+ chemotherapy Lentiviralvector producinghairpins antisenseto miR-21 [LV(a/miR-21)] Intratumoral inj. 150ngSingledosesynergic effect,strong inhibitionof tumor growth GemcitabineIntraperitoneal inj. 125mg/kgTwiceweekly for14days MiaPaCa-2miR-34a, miR-143/145 cluster RTSX OX CD-1miceLiposomal nanoparticlesfrom cationic amphiphileDOTAP andco-lipids (cholesterol, DSPE-PEG-OMe) withplasmide pMSCV-puro expression constructof miR-34aand miR-143/145 (Clontech Laboratories) Tail-veininj.50µgThreetimes perweekfor3 weeks Inhibitionof tumor growth, more widespread apoptosis No histopathology orbiochemical evidenceof toxicity(incl. hematology, liverandrenal function) Pramanik etal.,2011 Capan-1, Capan-2 miR-219-1-3pRTSXSCIDCB17 mice (males) Plasmide pcDNA6.2-miR- 219withExGen 500transfection reagent (Euromedex) Intratumoral inj. 20µgSingledoseDecreaseof tumor growthand proliferation NotassessedLahdaoui etal.,2014 (Continued) Frontiers in Pharmacology | www.frontiersin.org 10 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers TABLE4|Continued Xenografted cellline MiRNA(s)of interest Therapeutic strategy Animal model Animal strain (gender) Deliverysystem andchemical modifications Routeof administration DosageFrequencyof administration ResultsToxicity, undesired effects References PANC-1miR-34aRTSXBALB/c mice (females) Cationicpolymer fromPEIand β-cyclodextrin conjugatedwith CC9peptide Tail-veininj.15µmol(20 µg) Twiceweekly for2weeks Inhibitionof tumor growthand decreasein size, inductionof cancercell apoptosis NotassessedHuetal.,2013 PANC-1miR-217RTSXBALB/c mice (males) miR-217 expressionvector invivo-jetPEITM (201-50G; Polyplus) Intratumoral inj. 100µgtwiceDecreaseof tumor growth NotassessedZhaoetal., 2010 RWP-1 miR-148aITSX Athymic nu/nu mice (males) Engineered oncolytic adenovirus Ad-L5-8miR148aT (insertionof8 targetsitesfor miR-148a) Intratumoral inj. 5×1010 VP/tumor SingledoseSignificant inhibitionin tumor growthand reduced tumorweight Lessinduction ofALT,AST andbilirubin indicativeof attenuated viraltoxicity thanAd-wt, hepatotoxicity istransient Bofill-DeRos etal.,2015 Patient derived CP13 Intratumoral inj. Patient derived CP15 Intratumoral inj. Tail-veininj. MIA PaCa-2, PANC-1 miR- 21/23a/27a ITSXBALB/c mice (females) AntimiR-21with atelocollagen Intratumoral inj. 12µmolOnceweekly for3weeks followedby 3-weekpause andagain onceweekly for3weeks Suppression oftumor growth,the effectwas lostafter3 weeks Nodeath,loss ofbody weight,or grossadverse effects occurredinthe mice Frampton etal.,2011 (Continued) Frontiers in Pharmacology | www.frontiersin.org 11 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers TABLE4|Continued Xenografted cellline MiRNA(s)of interest Therapeutic strategy Animal model Animal strain (gender) Deliverysystem andchemical modifications Routeof administration DosageFrequencyof administration ResultsToxicity, undesired effects References MIA PaCa-2, PANC-1 miR- 21/23a/27a ITSXBALB/c mice (females) AntimiR- 21/23a/27awith atelocollagen Intratumoral inj. 4µmolfor eachantimiR Reductionin tumor volume sustainedto theendof the experiment despitea 3-weekrest period Nodeath,loss ofbody weight,or grossadverse effects occurredinthe mice Frampton etal.,2011 PANC-1, PANC10.05 miR-206RTSXSCIDmicemirVanamiR-206 mimics(Ambion) withinvivo-jetPEI (Polyplus)in 0.015% collagenaseII (Sigma)solution Intratumoral inj. 10µgThreetimes (day1,8,13) Increased tumor necrosis,no changesin totaltumor burden NotassessedKeklikoglou etal.,2015 Gemcitabine- resistant MIA PaCa-2R miR-205RTSXathymic nudemice (males) Gemcitabine conjugated miR-205 polyplexesfrom amphiphilic copolymerwith PEG Intratumoral inj. 1mg/kg (GEM40 mg/kg) Threetimesa weekfor2 weeks Reductionin tumor growthrate andweight, reductionin cell proliferation, increasein apoptosis Nosignificant changein bodyweight Mittaletal., 2014 Capan-2, MiaPaCa-2 miR-29a, 330-5p RTSXSCID CB-17mice (males) miRNAscloned intothe pCDNA6.2emGFP vector administeredwith Exgen500 (Euromedex) reagentand glucose5%(v/v) Intratumoral inj. 20µgSingledoseSignificant decreaseof tumor growthand weight NotassessedTréhouxetal., 2015 Hs766t-L2 initially transfected witha miR-29c agomir (200nM) miR-29cRTOXnudemiceagomiR-29cIntraperitoneal inj. 5nmolTwiceweekly for2weeks Reduced liver metastasis NotassessedZouetal., 2015 Frontiers in Pharmacology | www.frontiersin.org 12 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers TABLE5|Invivostudiesinanimalmodelsofcolorectaladenocarcinoma. Xenografted cellline MiRNA(s)of interest Therapeutic strategy Animal model Animal strain (gender) Deliverysystem andchemical modifications Routeof administration DosageFrequencyof administration ResultsToxicity, undesired effects Reference LS174TmiR-33a RTSX Athymic nudemice (Hsd:Athymic Nude- Foxn1nu) PEI complexes (PEIF25- LM/miRNA) Intraperitoneal inj. 0.77nmol(10 µg) Threetimes perweekfor 25days Reduction intumor proliferation and growth Nochangesin bodyweight, behavioral alterations,or othersignsof discomfort,no changesin ALTandAST, noinductionof TNFα Ibrahimetal., 2011 HCT-116miR-145intratumoral inj. 0.3nmol (4µg) HCT-116miR-218RTSXNudemice (females) miR-218orcontrol miRpreincubated withLipofectamine 2000(Invitrogen) Intratumoral inj. 1.2nmolEvery3daysInhibitionof tumor growth NotassessedHeetal.,2012 LoVomiR-K-rasRTSXSCID-C.B- 17/IcrHsd- Prkdcscid mice (females) PlasmidDNA encodingmiRNA specifictoK-ras Intratumoral inj.followedby percutaneous electroporation 50µgSingledoseTransient suppression oftumor growthfor6 days, increased necrosis Nosideeffects observed Vidicetal., 2010 MC38(murine colon adenocarcinoma) miR-27aRTSXNormal C57B/l6 mice miR-27aprecursor (GenePharma) withLipofectamine 2000(Invitrogen) Intratumoral inj. 6.26µgEvery3days for3times Inhibitionof tumor growth, reductionof tumorsizes andweight NotassessedBaoetal., 2014 HCT-116miR-133aRTSXBALB/c nudemice (females) miR-133a preincubatedwith Lipofectamine 2000(Invitrogen) Intratumoral inj. 0.3nmolEvery3days for4times Reduced tumor growthrate NotassessedDongetal., 2013 (Continued) Frontiers in Pharmacology | www.frontiersin.org 13 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers TABLE5|Continued Xenografted cellline MiRNA(s)of interest Therapeutic strategy Animal model Animal strain (gender) Deliverysystem andchemical modifications Routeof administration DosageFrequencyof administration ResultsToxicity, undesired effects Reference HT29let-7 IT+ immunotherapy SX Athymic nudemice (females) Anti-Fasactivating mAbcloneCH11 (Millipore Corporate) Intratumoral inj. 20µg Threetimes (days4,6, 8) Reductionin tumorsize, increased sensitivityof tumorcells to Fas-related apoptosis NotassessedGengetal., 2011 let-7inhibitor (GMR-miRTM microRNA inhibitor, GenePharma) 20µg DLD1 (KRASG13D) miR-4689RTSXNudemice (females) Carbonateapatite nanoparticles conjugatedwith mature hsa-miRNA(Gene Design) Tail-veininj.40µgThreetimesa weekfor8 times Inhibitionof tumor growth Nomortalities orbodyweight loss,no significant differencesin blood chemistry tests,except fortheslight increasein BUN, histological damagenot observed (brain,heart, lung,liver, kidney,spleen, smallintestine, andcolon) Hirakietal., 2015 SW620miR-429RTSXNudemice (males) MaturemiRNAIntratumoral inj. 1µgSingledoseInhibitionof tumor growth, reductionof tumorweight NotassessedSunY.etal., 2014 (Continued) Frontiers in Pharmacology | www.frontiersin.org 14 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers TABLE5|Continued Xenografted cellline MiRNA(s)of interest Therapeutic strategy Animal model Animal strain (gender) Deliverysystem andchemical modifications Routeof administration DosageFrequencyof administration ResultsToxicity, undesired effects Reference SW620miR-27bRTSXNOD/SCID mice (females) Cholesterol- conjugatedmimics (GenePharma) Intratumoral inj. 1OD/mouseTwiceaweek for5weeks Inhibitionof angiogenesis, severetumor necrosis, one xenograft disappeared completely(a scab remained) Twomicefrom negative controlgroup diedafter 4-week treatment, causeofdeath wasnot determined Yeetal.,2013 FIGURE 4 | Pie chart of miRNA therapeutic strategy in the selected studies. FIGURE 5 | Pie chart of animal models and xenograft methods in the selected studies. animal models, and also from human volunteers. Potential adverse effects could be provoked by hybridization-dependent or independent mechanisms, and could be linked with specific sequence motifs or length of an oligonucleotide. It means that some of the findings from antisense therapy in general are quite relevant for extrapolation to miRNA-based therapy. Immunostimulation was described for phosphorothioate antisense oligonucleotides. It is a sequence-dependent, Frontiers in Pharmacology | www.frontiersin.org 15 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers FIGURE 6 | Pie chart of routes of administration of miRNA-based therapy in the selected studies. hybridization-independent process, which leads to the reversible activation of various immune cells (e.g., NK cells, B lymphocytes, mononuclear cells) and increased production of cytokines such as IL-6, IL-12 and interferon γ (Levin, 1999; Henry et al., 2002). The main responsible sequence motif is CpG (p stands for phosphodiester bond) or CG palindromic sequences naturally occurring mostly in bacterial genome (Krieg et al., 1995). While unmethylated, this motif is recognized by TLR receptors on immune cells and activates them. The effect is also exerted by oligonucleotides with both phosphorothioate, and phosphodiester bonds in the structure. In rodents, which are more sensitive than primates to this effect, splenomegaly, lymphoid hyperplasia, and multiple organ mononuclear infiltrates were described (Levin, 1999). Another severe adverse effect relating with immunity is an activation of complement cascade. It prevails over TLR-mediated immune stimulation in primates and its mechanism is probably hybridization/sequence-independent originating from physicochemical properties (polyanionic character) of oligonucleotides. In the study of Henry et al., after reaching a threshold plasma concentration after i.v. infusion, macaques suffered from emesis, ataxia, and facial edema. Hemodynamic changes (fluctuation of blood pressure and tachycardia), changes in blood count (neutropenia followed by neutrophilia), and increase of cytokines mentioned above were described (Henry et al., 2002). When maintaining plasma concentration below the threshold, symptoms were mild or not present, which is in accordance with the results of phase I clinical study with antisense oligonucleotide against intercellular adhesion molecule-1 (ICAM-1) (Glover et al., 1997). Similar hybridization/sequence-independent mechanism leads also to the influencing of blood coagulation cascade observed in rodents, primates and human (Glover et al., 1997; Henry et al., 1997). Negative charge of oligonucleotides could inhibit intrinsic tenase complex (consisting of factor IXa and VIIIa, which activate factor X), and thus leads to the reversible prolonging of blood clotting and to the increase of activated partial thromboplastin time (aPTT) (Sheehan and Lan, 1998; Levin, 1999). After administration of relatively high doses of antisense oligonucleotides (above 100 mg/kg in rodents), histological or laboratory signs of hepatotoxicity and renal toxicity were present in experimental animals. Mostly, immune-mediated cellular infiltrations in liver, multi-focal liver necrosis, and proximal tubules infiltrations were found in rodents. Posology studies indicate that lower doses (below 3 mg/kg) do not cause liver and kidney pathologies in monkey and human (Levin, 1999). Different mechanism could potentially lead to hepatotoxicity, which was proven in rodents (Grimm et al., 2006). By introducing oligonucleotides into the cell, enzymes and other proteins that physiologically deal with these molecules could be saturated, and thus processing of other endogenous RNAs sharing these pathways could be diminished (Bader et al., 2010). This effect was described on mice treated with shRNA (short hairpin RNA) expression vectors. Mice suffered from multifocal liver necrosis followed by ascites, edema, increase of bilirubin and liver enzymes, and decrease of plasma proteins and body weight. Several mice died within 1 month. There were no signs of blood count changes, or increases in cytokine productions. ShRNAs compete of Dicer cleavage and exportin-5 stabilization in cytoplasm with endogenous pre-miRNAs, therefore mature liver miRNAs were found decreased and shRNAs precursors increased in mice with symptoms of hepatotoxicity. As Grimm et al. studied almost 50 distinct shRNAs, they assume that the effect was not sequence related (Grimm et al., 2006). Introducing of miRNA-precursors into the cell could produce the same effect, but the data concerning safety of miRNA-based therapies are limited. Again, proper posology studies are needed. Another possible mechanism of toxicity is hybridizationdependent. But the toxicity arisen from both binding to the desired mRNAs, and off-target binding is hypothesized to be rare (Bader et al., 2010; van Rooij et al., 2012). MiRNA-mimics and precursors are suggested to be generally better tolerated than antisense therapy (Bader et al., 2010). One miRNA could regulate number of genes, frequently functionally linked in a specific pathway. Targeting more genes in one or more pathologically deregulated pathway could be beneficial. The potential for targeting other genes in different pathways still remains, but influencing of target or off-target genes with impact on cell viability should be revealed during accurate in vitro testing. MiRNA-BASED THERAPEUTICS IN CLINICAL TRIALS Certain chemical modifications of oligonucleotides structure and also several delivery systems for miRNAs have already Frontiers in Pharmacology | www.frontiersin.org 16 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers entered clinical phase of drug development. There are no reports of clinical trials of miRNA-based therapies in gastrointestinal malignancies on which we have focused in this review— colorectal, pancreatic, gallbladder and gastric cancer. Two experimental miRNA-based therapies are now listed on ClinicalTrials.gov. MiR-34a mimics in an amphoteric liposomal formulation administered i.v. are tested in the phase I in patients with primary liver cancer and advanced or metastatic lung and kidney cancer, melanoma, multiple myeloma and lymphoma (NCT01829971, Adams et al., 2015). MiR-16 mimic is evaluated in the treatment of malignant pleural mesothelioma also in the phase I (NCT02369198). The therapeutic system used is termed TargomiR and it is based on specific nanoscale delivery system—nonliving bacterial minicells (EnGeneIC Delivery Vehicle, EnGeneIC, New York, USA), and targeted to cancer cells by an anti-EGFR antibody, since EGFR is known to be overexpressed by certain types of cancer. Kao et al. even published some of the preliminary results achieved in the cohort of six patients with malignant pleural mesothelioma describing significant radiologic and metabolic responses indicated by PET-scan (Kao et al., 2015; Quinn et al., 2015). In non-cancer diseases, the first miRNA-based drug in clinical settings was miravirsen (LNA miR-122 inhibitor) tested as a hepatitis C treatment. The drug entered clinical trials phase II (NCT02031133, NCT02508090, NCT02452814), but van den Ree has recently referred that the development of miravirsen had been ceased. A more potent miR-122 inhibitor conjugated with N-acetylgalactosamine entered phase II (RG-101, 2016; van der Ree et al., 2016). Other delivery systems, used in the selected animal studies as carriers for miRNA-based therapeutics, are evaluated in clinical trials for non-miRNA treatment. Future results from these trials may serve also for the development of miRNA-based therapies, as we may obtain e.g., the information about the potential toxicity, or pharmacokinetic aspects of a specific delivery system regardless of its cargo. Lentiviral vectors are mostly used to transfect cells that are subsequently injected into the patient, e.g., in the treatment of lymphoma (NCT02337985). Adenoviral vectors are evaluated in various solid cancers and are usually administered locally, intraperitoneally, or even intratumorally. They are tested in urinary bladder cancer (NCT00003167), ovarian (NCT00964756), breast (NCT01703754), prostate (NCT01931046), or pancreatic carcinoma (NCT02705196). Adeno-associated viruses are tested in non-cancer diseases to deliver genes for the experimental treatment of hemophilia B (coagulation factor IX; NCT01620801), lipoprotein lipase deficiency (NCT00891306), Pompe disease (α-glucosidase; NCT00976352), or genetic retinopathies (NCT01482195). In gastric cancer, AAV is used to transfect patient’s dendritic cells, which are later mixed with his T lymphocytes to produce specific cytotoxic T lymphocytes injected i.v. back to the patient (NCT02496273). IONPs are investigated in various applications in biomedicine, above all in diagnostics and tissue imaging (e.g., NCT00147238, NCT01895829). Ferumoxyde, superparamagnetic iron oxide, has already been used in clinical practice in the United States for the treatment iron-deficiency anemia in patients with chronic kidney disease. Finally, PEI particles for delivering gene therapy are utilized in the clinical trials phase I and II of pancreatic ductal adenocarcinoma (NCT01274455), hepatocellular carcinoma (NCT00825474) and urinary bladder carcinoma (NCT00595088, NCT01274455, NCT00393809). FUTURE PERSPECTIVES In addition to animal models and techniques described in this review, there are also novel and promising approaches to target miRNAs under development. Very intriguing strategy present small-molecule inhibitors that target specific miRNAs (SMIRs, e.g., diazobenzene inhibiting miR-21) that usually interfere with miRNA biogenesis and maturation (Wen et al., 2015). SMIRs constitutes a reasonable and evidence based strategy with strong potential and chance for success. The progress of screening techniques and computational stimulation may address bright future in this field. CRISPR/Cas 9 technology is another emerging technique to be used in miRNA targeting therapy. For instance, construction of sequence specific CRISPR/Cas9 based miRNA inhibitor was reported to downregulate miR-17-92 cluster and miR- 21, two canonical oncogenic miRNAs in cancer (Ho et al., 2015; Narayanan et al., 2016). Since single miRNA has the potential of regulating thousand genes, long non-coding RNA (lncRNA) that is capable of binding multiple miRNAs could consequently impact the expression of thousands of genes. In light of this potentially fundamental biological role, all the lncRNAs that act as endogenous miRNA sponges presents another promising strategy to target miRNAs in cancer. Finally, it can also be envisioned that blocking production, transportation and release of exosome miRNAs may have beneficial effects in controlling cancer development, and this may be achieved by targeting other non-cancerous cells such as the inflammatory cells in the cancer microenvironment. CONCLUSIONS MiRNA-based therapies as a new class of targeted therapy are heading toward from bench to the bedside. It is now generally accepted and many times proved that influencing pathologically changed intracellular levels of miRNAs change oncogenic phenotype of cancer cells in vitro and in vivo. However, as there is no ideal animal model of a human pathology, the translational potential of most studies is somehow limited. In the studies selected for this review, change of a specific miRNA was followed by significant diminishing of tumor size or volume in vivo. The subcutaneous tumor model used in the bulk of the studies clearly do not respond with microenvironment of the normal tumor cells, and also the necessary immunodeficiency of experimental animals do not correspond with immune status of an average Frontiers in Pharmacology | www.frontiersin.org 17 September 2016 | Volume 7 | Article 329 Merhautova et al. MiRNA Therapy in Gastrointestinal Cancers oncology patient, nevertheless, the results of animal studies are promising. Serious obstacles still lie in the way to the clinical practice. The main issue is efficient delivery of miRNA-mimics, precursors, expression vectors, or inhibitors. Other important difficulty is an assessment of a proper dose sufficient for anticipated intracellular effects, but lacking or possessing acceptable adverse effects relating to immunostimulation, blood coagulation, or toxicities that account for the specific delivery systems. We also see the importance of nonrodent models in the development of new drugs, as shown on the immunostimulation triggered by oligonucleotides which is significantly different in nature in rodents and primates. Several miRNAs and delivery system are now tested in clinical trials. Most of them are in phase I or II. Together with more information obtained from preclinical experiments, the results could move us forward on the way to a new approach in targeted therapy—drugs that aim on epigenetic mechanisms of pathophysiological processes. AUTHOR CONTRIBUTIONS JM did the literature search and wrote the text, JM and OS made the figures, RD and OS advised on the concept of the review and lately rearranged, corrected and critically revised the text. FUNDING This work was supported by the grant GA16-18257S of The Grant Agency of Czech Republic, by the internal Masaryk University Faculty of Medicine grants MUNI/A/1284/2015 and MUNI/11/InGA09/2014, by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601) and by the Czech Ministry of Health under the project MZ CR – RVO (MOU, 00209805). ACKNOWLEDGMENTS The authors gratefully thank for the funding. We apologized to the many authors whose work could not be quoted due to lack of space. REFERENCES Adams, B. 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(2015). miR-29c suppresses pancreatic cancer liver metastasis in an orthotopic implantation model in nude mice and affects survival in pancreatic cancer patients. Carcinogenesis 36, 676–684. doi: 10.1093/carcin/bgv027 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2016 Merhautova, Demlova and Slaby. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Pharmacology | www.frontiersin.org 21 September 2016 | Volume 7 | Article 329 Research Article miR-155 and miR-484 Are Associated with Time to Progression in Metastatic Renal Cell Carcinoma Treated with Sunitinib Jana Merhautova,1 Renata Hezova,2,3 Alexandr Poprach,3 Alena Kovarikova,2 Lenka Radova,2 Marek Svoboda,2,3 Rostislav Vyzula,3 Regina Demlova,1 and Ondrej Slaby2,3 1 Department of Pharmacology, Faculty of Medicine, Masaryk University, 625 00 Brno, Czech Republic 2 Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic 3 Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic Correspondence should be addressed to Ondrej Slaby; on.slaby@gmail.com Received 3 March 2015; Accepted 17 April 2015 Academic Editor: Paul L. Crispen Copyright © 2015 Jana Merhautova et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Sunitinib is a tyrosine kinase inhibitor used in the treatment of metastatic renal cell carcinoma. The main difficulty related to the treatment is the development of drug resistance followed by rapid progression of the disease. We analyzed tumor tissue of sunitinib treated patients in order to find miRNAs associated with therapeutic response. Methods. A total of 79 patients with metastatic renal cell carcinoma were included in our study. miRNA profiling in tumor tissue samples was performed by TaqMan Low Density Arrays and a group of selected miRNAs (miR-155, miR-374-5p, miR-324-3p, miR-484, miR-302c, and miR-888) was further validated by qRT-PCR. Normalized data were subjected to ROC and Kaplan-Meier analysis. Results. We reported decreased tissue levels of miR-155 and miR-484 as significantly associated with increased time to progression (miR-155: median TTP 5.8 versus 12.8 months, miR-484: median TTP 5.8 versus 8.9 months). Conclusion. miR-155 and miR-484 are potentially connected with sunitinib resistance and failure of the therapy. miR-155 is a known oncogene with direct influence on neovascularization. Biological role of miR-484 has to be clarified. Stratification of patients based on miRNA analysis would allow more personalized approach in therapy of metastatic renal cell carcinoma. 1. Introduction Targeted therapy with tyrosine kinase inhibitors (TKIs) is used in the first line of metastatic renal cell carcinoma (mRCC) treatment. TKIs inhibit multiple receptor tyrosine kinases needed for the activation of intracellular signaling pathways controlling cell proliferation, survival, or angiogenesis. Almost all treated patients will eventually develop secondary resistance to TKIs [1]. Other therapeutic alternatives, such as TKIs pazopanib or sorafenib, mTOR inhibitor temsirolimus, VEGFR antibody bevacizumab, cytokine therapy with interferon-𝛼, or clinical trials [2], could be provided, if there would be a possibility to distinguish individuals with and without benefit from sunitinib therapy. Emerging evidence suggests that microRNAs (miRNAs) could be suitable biomarkers with diagnostic, prognostic, and predictive potential [3–6]. These small (18–25 nt) noncoding RNAs are posttranscriptional regulators of gene expression. miRNAs affect most cellular processes and the dysregulation of their network has been linked to various malignant diseases including RCC [7]. miRNAs as biomarkers could be measured in tissues and body fluids and are relatively resistant to decay. The aim of our study was to find tissue miRNAs associated with the time to progression of mRCC in patients treated with sunitinib. To have an effective tool for distinguish patients according to the expected therapy outcome would contribute to more personalized mRCC therapy. 2. Materials and Methods 2.1. Study Design, Patients, and Tissue Samples. The study protocol was approved by the local ethical committee and written informed consent was obtained from all patients. Metastatic RCC patients included in the study were from Hindawi Publishing Corporation BioMed Research International Volume 2015,Article ID 941980, 5 pages http://dx.doi.org/10.1155/2015/941980 2 BioMed Research International Table 1: Clinicopathological characteristics of patients. Screening cohort Validation cohort Responders 𝑁 = 8 Nonresponders 𝑁 = 8 Responders 𝑁 = 44 Nonresponders 𝑁 = 19 Gender Male 6 (75%) 8 (100%) 34 (77.3%) 11 (57.9%) Female 2 (15%) 0 (0%) 10 (22.7%) 8 (42.1%) Age Median 64 64 66 66 Range 40–80 53–73 41–84 45–84 Histology Papillary carcinoma 1 (12.5%) 1 (12.5%) 3 (6.8%) 3 (5.8%) Clear cell carcinoma 7 (87.5%) 7 (87.5%) 41 (93.2%) 16 (84.2%) Grade 1 0 (0%) 0 (0%) 6 (13.6%) 0 (0%) 2 2 (25%) 3 (37.5%) 11 (25%) 5 (26.4%) 3 5 (62.5%) 3 (37.5%) 13 (29.5%) 7 (36.8%) 4 1 (12.5%) 2 (25%) 5 (11.4%) 7 (36.8%) Unknown 0 (0%) 0 (0%) 9 (20.5%) 0 (0%) Response to sunitinib according to RECIST criteria Complete response 0 (0%) 0 (0%) 1 (2.3%) 0 (0%) Partial response 6 (75%) 0 (0%) 19 (43.2%) 0 (0%) Stable disease 2 (25%) 0 (0%) 24 (54.5%) 0 (0%) Progressive disease 0 (0%) 8 (100%) 0 (0%) 19 (100%) South Moravian region of Czech Republic with uniform exposure to the environmental factors. Hereditary cases of RCC were excluded from the study. Two cohorts of patients with mRCC treated with sunitinib in a standard regimen were set up retrospectively. The screening group included 16 patients from Masaryk Memorial Cancer Institute, Brno, Czech Republic (MMCI). Response to the treatment was assessed according to RECIST criteria after 9 months and patients were divided into two groups: (a) responders to the treatment (complete, or partial response, and stable disease) and (b) nonresponders with rapid progression. A group of candidate miRNAs was chosen and the expression was analyzed by qRT-PCR in the validation cohort of 63 mRCC patients from MMCI. Clinicopathological characteristics of both cohorts are summarized in Table 1. 2.2. Tissue Samples and RNA Isolation. Tumor tissue was provided as formalin-fixed paraffin embedded (FFPE) samples. Total RNA enriched with small RNA was isolated using mirVana miRNA Isolation Kit (Ambion, Austin, USA). Concentration and purity of the isolated RNA were determined spectrophotometrically using Nanodrop ND-1000 (Thermo Scientific, Rockford, USA). 2.3. Microarray Profiling. miRNAs profiling was conducted using TaqMan Low Density Array (TLDA) technology. Megaplex miRNA RT primers set (pools A and B, version 3.0, Applied Biosystems, Foster City, USA) and TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) were used for reverse transcription. Reactions were carried out according to the manufacturer’s protocol. 667 miRNAs were simultaneously quantified using ABI 7900 HT Instrument (Applied Biosystems). 2.4. RT-PCR Quantification. Gene-specific primers were used in reverse transcription according to the TaqMan MicroRNA Assay protocol (Applied Biosystems). qRT-PCR was performed on ABI 7500 HT Instrument (Applied Biosystems) using the Applied Biosystems 7500 Sequence Detection System. TaqMan (NoUmpErase UNG) Universal PCR Master Mix and specific primer and probe mix (Applied Biosystems) for each miRNA were used. PCR reactions were run in duplicates, and average threshold cycles and SD values were calculated. 2.5. Data Normalization and Statistical Analysis. Expression data from TLDA profiling were normalized using miR-625∗ , which was uniformly expressed in all samples from screening cohort. Normalized miRNA expression data were evaluated using Bioconductor Limma differential expression analysis. 𝑃 value lower than 0.01 was selected according to the potential of identified miRNAs to accurately discriminate responders and nonresponders in consequent HCL analysis. In validation phase of the study, average expression levels of miRNAs in RT-PCR quantification were normalized using miR-1233 as a reference gene. miR-1233 was selected according to our previous experience with normalization of renal cell carcinoma FFPE samples. Normalized expression data were BioMed Research International 3 A5 A7 A4 A57 A66 A3 A2 A1 A16 A14 A55 A22 A11 A33 A9 A10 hsa-miR-636-002088 hsa-miR-483-5p-002338 hsa-miR-214-002306 hsa-miR-888-002212 hsa-miR-302c-000533 hsa-miR-484-001821 hsa-miR-196b-002215 hsa-miR-29c-000587 hsa-miR-30d-4373059 hsa-miR-204-000508 hsa-miR-324-3p-002161 hsa-miR-21-000397 hsa-miR-454-002323 mmu-miR-374-5p-001319 hsa-miR-150-000473 hsa-miR-155-002623 hsa-miR-30a-4373061 hsa-miR-30e-4395334 hsa-miR-30a∗ -4373062 hsa-miR-30e∗ -4373057 Figure 1: Hierarchical clustergram of miRNAs differentially expressed in sunitinib responding and nonresponding patients. Cluster analysis groups samples and miRNAs according to the expression similarity. miRNAs are in rows and samples in columns. Upregulated miRNAs are marked as red and downregulated miRNAs as green. Blue color indicates responders, yellow color indicates nonresponders. 𝑃 < 0.01. evaluated by ROC analysis (MedCalc 14.12.0) and KaplanMeier analysis followed by log-rank test (GraphPad Prism 5.03). 𝑃 values lower than 0.05 were considered statistically significant. 3. Results 3.1. Microarray Profiling Revealed 19 Differentially Expressed miRNAs between the Responders and Nonresponders Group. High-throughput miRNA analysis of tumor tissue of 16 patients treated with sunitinib belonging to either responding (𝑁 = 8) or nonresponding (𝑁 = 8) group was performed. Limma analysis of normalized expression data identified 19 miRNAs differentially expressed (Figure 1). Six miRNAs (miR-155, miR-374-5p, miR-324-3p, miR-484, miR-302c, and miR-888) were chosen as candidates for the verification using qRT-PCR (𝑃 value < 0.01, CT < 35). 3.2. Association between miR-155 and miR-484 Expression and Time to Progression in mRCC Treated with Sunitinib. The results obtained from the screening cohort were verified on the independent cohort (𝑁 = 63) by qRT-PCR. Normalized data were analyzed by ROC analysis and patients were separated into two groups according to the calculated criterion. Kaplan-Meier analysis revealed that lower level of miR-155 is associated with increased time to progression in patients on sunitinib treatment (Table 2 and Figure 2(a), median TTP 5.8 versus 12.8 months). Similar result was obtained for miR-484 (Table 2 and Figure 2(b), median TTP 5.8 versus 8.9 months). Kaplan-Meier plots of other miRNAs did not reach statistical significance, although some of them indicate potentially interesting trends (data not shown). 4. Discussion Our findings suggest a link between two miRNAs (miR-155 and miR-484) and disease progression in mRCC patients treated with sunitinib. Tyrosine kinase inhibitors inhibit tyrosine kinase domains of growth factor receptors, albeit their main activity is promoted by the inhibition of VEGF receptor cascade, which leads to the decrease in blood tumor perfusion and to the inhibition of neovascularization. Tumors of TKI treatment-refractory patients are able to escape from the VEGFR blockade [1]. miR-155 is a potent oncomiR upregulated in diverse types of cancer including renal cancer [8, 9], which is in accordance with our findings. The role of miR-155 in angiogenesis is well described. Positive feedback loop between VEGF and miR-155 exists, and miR-155 decreases the expression of VHL tumor suppressor, a protein with ubiquitin ligase activity sequestrating, for example, hypoxia-induced 4 BioMed Research International Table 2: Validation of miR-155 and miR-484 on the independent cohort (𝑁 = 63) and their correlation with TTP (months). Number of patients (𝑁 = 63) Median TTP (months) Log-rank 𝑃 HR 95% CI miR-155 Low, <0.2381 42 12.8 0.0092 2.412 1.243–4.680 High, ≥0.2381 21 5.8 miR-484 Low, <1.4408 52 8.9 0.0296 2.623 1.100–6.254 High, ≥1.4408 11 5.8 miR-155 0 20 40 60 0 20 40 60 80 100 High level Low level TTP (months) Survival(%) (a) miR-484 0 20 40 60 0 20 40 60 80 100 High level Low level TTP (months) Survival(%) (b) Figure 2: Kaplan-Meier survival curves estimating TTP in sunitinib treated mRCC patients (𝑁 = 63) according to miR-155 ((a); 𝑃 value < 0.01) and miR-484 ((b); 𝑃 value < 0.05) tumor tissue expression levels. Patients with low expression of the relevant miRNA are illustrated by dashed line. factors (HIFs). Higher levels of HIFs promote expression of genes involved in angiogenesis, proliferation, and other aspects of the tumorigenesis, even in the condition of VEGFR blockade [10, 11]. Our data imply that patients with higher tissue expression of miR-155 have decreased time to progression on sunitinib treatment and thus limited benefit from the therapy. However, we have detected a discrepancy between the results obtained from the screening and independent cohort. TLDA screening indicated that the nonresponders from the screening group have lower expression of miR-155 than the responders. Opposite result was achieved by qRT-PCR in the independent cohort (data not shown). We suppose that a bias might occur due to a small number of the specimens analyzed by TLDA, which is also significant limitation of our study. The expression of miR-484 in mRCC patients treated with sunitinib has already been noticed. Prior et al. described high tumor tissue levels of miR-484 as significantly associated with decreased TTP and overall survival [12]. Our findings are in agreement with this study. Research in ovarian cancer proved that miR-484 is excreted from tumor cells as a paracrine regulator of tumor microenvironment [13] and it is also measurable in plasma [14, 15]. Therefore, it was found decreased in the tumor tissue [13] and increased in plasma [16]. However, adrenocortical cancer is typical with high tissue expression of miR-484 [17]. The role of this miRNA is probably diverse and depends on the tumor type and miRNA localization. Up to date, there are no reports of possible targets of miR-484 in renal cell carcinoma. Its paracrine function was described in ovarian cancer, where miR-484 targets VEGF B in tumor cells and VEGFR2 in adjacent endothelial cells [13]. Increased levels of miR-484 attenuate the intrinsic apoptotic pathway rising from mitochondria in anoxia, which was unveiled in experiments with myocardial infarction [18]. 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Atypical antipsychotics substantially differ in their propensity to induce metabolic alterations. Aripiprazole is considered to represent an antipsychotic drug with low risk of metabolic syndrome development. The aim of this study was to evaluate metabolic phenotype of neurodevelopmental polyI:C rat model and assess metabolic effects of chronic aripiprazole treatment with regard to complex neuroendocrine regulations of energy homeostasis. Polyinosinic:polycytidylic acid (polyI:C) was administered subcutaneously at a dose of 8 mg/kg in 10 ml on gestational day 15 to female Wistar rats. For this study 20 polyI:C and 20 control adult male offspring were used, randomly divided into 2 groups per 10 animals for chronic aripiprazole treatment and vehicle. Aripiprazole (5 mg/kg, dissolved tablets, ABILIFY® ) was administered once daily via oral gavage for a month. Altered lipid profile in polyI:C model was observed and a trend towards different dynamics of weight gain in polyI:C rats was noted in the absence of significant antipsychotic treatment effect. PolyI:C model was not associated with changes in other parameters i.e. adipokines, gastrointestinal hormones and cytokines levels. Aripiprazole did not influence body weight but it induced alterations in neurohumoral regulations. Leptin and GLP-1 serum levels were significantly reduced, while ghrelin level was elevated. Furthermore aripiprazole decreased serum levels of pro-inflammatory cytokines. Our data indicate dysregulation of adipokines and gastrointestinal hormones present after chronic treatment with aripiprazole which is considered metabolically neutral in the polyI:C model of schizophrenia. © 2017 Elsevier Ltd. All rights reserved. 1. Introduction Atypical antipsychotics, drugs with indisputable benefits in the treatment of a wide spectrum of psychiatric disorders, substantially differ in their propensity to induce metabolic alterations including weight gain, dyslipidemia, impaired glucose tolerance or insulin resistance, yet the underlying pathophysiological mechanisms are complex and have not been fully elucidated (Henderson et al., 2015). Aripiprazole (ARI) is considered a metabolically neutral antipsychotic agent with low risk of metabolic syndrome development (Kasteng et al., 2011; Nasrallah et al., 2016). Switching to antipsychotics characterized by lower propensity to induce metabolic dysregulation represents one of the recommended strategies to reduce cardio-metabolic risk in patients experiencing metabolic alterations during antipsychotic treatment (American Diabetes Association et al., 2004). This approach is supported by clinical data as switching from olanzapine to ARI led to weight reduction and a decrease in cholesterol and triglyceride serum levels (Newcomer et al., 2008; Stroup et al., 2011; Takeuchi et al., 2010). However, ARI was also reported to induce significant weight gain (Malla et al., 2016). In addition, there is evidence that antipsychotic-naïve first episode schizophrenia patients are more prone to metabolic abnormalities (Enez Darcin et al., 2015). Therefore, it seems that * Corresponding author. Department of Pharmacology, Faculty of Medicine, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic. E-mail address: jkucer@med.muni.cz (J. Ruda-Kucerova). Contents lists available at ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm http://dx.doi.org/10.1016/j.neuropharm.2017.06.003 0028-3908/© 2017 Elsevier Ltd. All rights reserved. Neuropharmacology 123 (2017) 148e158 schizophrenia per se is linked to higher incidence of metabolic syndrome development (Kritharides et al., 2016; Kucerova et al., 2015; Malan-Müller et al., 2016) and as the common underlying pathophysiology of schizophrenia and metabolic syndrome distorted inflammatory pathways have been suggested (Leonard et al., 2012). Pro-inflammatory cytokines have been implicated in etiology of neuropsychiatric disorders, while the low-grade pro-inflammatory state is associated with obesity, diabetes mellitus and cardiovascular morbidity (Aguilar-Valles et al., 2015; Reisinger et al., 2015). The neurodevelopmental theory of schizophrenia postulates the association between the pre- and perinatal environmental factors such as prenatal infection, subsequent maternal immune activation and later risk of schizophrenia (Canetta and Brown, 2012; Ratnayake and Hill, 2016). This provides a concept for chronic neurodevelopmental animal models of neuropsychiatric disorders such as in utero exposure to a viral mimetic agent polyinosinic:polycytidylic acid (polyI:C) (Meyer and Feldon, 2012; Reisinger et al., 2015). Neurodevelopmental models of schizophrenia have several advantages over other types of preclinical models as the condition of the animals is chronic and reflects several aspects of schizophrenia-related symptomatology and pathophysiology (Micale et al., 2013). The polyI:C model is widely recognized and considered suitable for basic and translational schizophrenia research (Meyer and Feldon, 2012; Ratnayake and Hill, 2016). Similarly as in human it could be assumed that schizophreniclike phenotype in rodent models may involve intrinsic vulnerability to metabolic disturbances and susceptibility to metabolic alterations induced by antipsychotic medication further proving the validity and translational potential of the models (Kucerova et al., 2015). So far, preclinical research has extensively addressed metabolic effects of atypical antipsychotics (AAP) (Boyda et al., 2010) but despite the high clinical relevance, there is a lack of studies assessing the relation between metabolic abnormalities and schizophrenia-like phenotype in rodents per se. Moreover, to the best of our knowledge, metabolic abnormalities induced by antipsychotic treatment have not been evaluated in rodent schizophrenia-like models yet. Furthermore, metabolic alterations after chronic ARI exposure in healthy rodents have been investigated less intensely compared to other antipsychotic agents (Boyda et al., 2010). Therefore, the need to focus on evaluating metabolic dysregulation also in antipsychotics carrying lower metabolic risks has been expressed (Ersland et al., 2015) and this approach may also contribute to better understanding of the underlying mechanisms. In rodent experiments, metabolic abnormalities induced by antipsychotics, specifically weight gain, glucose metabolism dysregulation and altered lipid profile have been described (Boyda et al., 2010). Nevertheless, the frequently inconsistent findings do not allow definite conclusions on the potential pathophysiological mechanisms likely due to methodological heterogeneity. More recently, the attention in this field has been drawn to complex energy homeostasis regulation and the role of adipokines, gastrointestinal hormones and other modulators, yet few preclinical studies have assessed these putative alterations with regard to antipsychotic treatment (Horska et al., 2016, 2017; Skrede et al., 2012; Zhang et al., 2013). The aim of this study is to evaluate metabolic phenotype of polyI:C rat model and assess metabolic effects of chronic ARI treatment with regard to complex neuroendocrine regulations of energy homeostasis. Thus, in this study we analyzed apart from basic serum biochemical parameters a spectrum of gastrointestinal hormones, adipokines and markers of inflammation. This includes leptin, ghrelin, glucagon-like peptide 1 (GLP-1), glucagon, fibroblast growth factor 21 (FGF-21) and pro-inflammatory cytokines e interleukin 1 and 6 (IL-1, IL-6) and tumor necrosis factor a (TNF-a). These hormones are involved in energy homeostasis regulations and associated with obesity and insulin resistance (Blüher and Mantzoros, 2015; Quarta et al., 2016). The role of cytokines in energy homeostasis is well-described (Fontana et al., 2007; Glund and Krook, 2008) and it is highly relevant in a model based on a prenatal pro-inflammatory insult. We hypothesized that the polyI:C model may possess intrinsic metabolic derangements as a part of the schizophrenia-like phenotype and ARI treatment may induce dysregulations in the metabolic parameters to a higher extent in the polyI:C animals even in the absence of significant changes in body weight. This study was designed to assess potential metabolic disturbances present in the polyI:C model per se, the effect of ARI on metabolic variables in normal rats and the possible interaction between the polyI:C model and ARI treatment. 2. Material and methods 2.1. Animals Adult male and female Wistar rats were purchased from the Masaryk University breeding facility (Brno, Czech Republic) and time-mated. Polyinosinic:polycytidylic acid (polyI:C) was administered at a dose of 8 mg/kg in 10 ml subcutaneously on a gestational day (GD) 15 to 11 rats, while vehicle (saline) was injected to 10 control rats. The average surviving litter size was n ¼ 10.5 in both control and polyI:C treated mothers. The average proportion of male and female offspring was 52% of males and 48% of females. No cross-fostering was used, the mothers were regularly weighed and no differences were observed between control and polyI:C treated mothers. The offspring were weaned on a postnatal day (PND) 22 and group-housed. For this study 20 polyI:C and 20 control male offspring weighing 250e350 g were used. The polyI:C and control rats were randomly divided into 2 groups per 10 animals for chronic (28 days) ARI treatment or vehicle control. The treatment was initiated when the animals were 11 weeks old. All animals were pair-housed in standard polycarbonate housing cages. Environmental conditions during the whole study were constant: relative humidity 50e60%, temperature 23 C ± 1 C, normal 12-h light-dark cycle (6 a.m.e6 p.m. light). Standard rodent chow and water were available ad libitum. All procedures were performed in accordance with EU Directive no. 2010/63/EU and approved by the Animal Care Committee of the Faculty of Medicine, Masaryk University, Czech Republic and Czech Governmental Animal Care Committee, in compliance with Czech Animal Protection Act No. 246/1992. 2.2. Drugs and treatments PolyI:C was purchased from Sigma-Aldrich spol. s.r.o., Prague, Czech Republic as a sodium salt and dissolved in saline to obtain a concentration of 8 mg/kg in 10 ml. Subcutaneous administration of 8 mg/kg on gestational day 15 was chosen according to validated protocol using a Wistar strain of rats (Missault et al., 2014). This protocol was validated but the complex schizophrenia-like phenotype was not assessed in this study due to concerns about non-standard energy expenditure and stress related to behavioural testing which may bias the metabolic data. However, this is a limitation of the study. Aripiprazole (ARI) was obtained as ready-made preparation for human use in tablets (ABILIFY® non-coated tablets, 15 mg, Otsuka Pharmaceutical Europe Ltd., GB). The solution was prepared by dissolving the tablets to obtain ARI dose of 5 mg/kg in 1 ml. The K. Horska et al. / Neuropharmacology 123 (2017) 148e158 149 solvent vehicle (VEH) was purified water. All solutions were freshly prepared on the day of administration and both, ARI and water were administered once daily via oral gavage. The ARI dose was chosen based on our pilot experiments, when higher than 5 mg/kg doses administered orally produced apparent sedation. Importantly, ARI has a distinct pharmacokinetics and consequently pharmacodynamic actions in rats as it is extensively metabolized and, compared to human, different metabolites with the dissimilar pharmacological profile are formed in the rat (Wood et al., 2006). However, a relevant in vivo D2 receptor occupancy was reported already after 2.5 mg/kg given subcutaneously in rats (Wadenberg, 2007). 2.3. Body weight recording and analysis Body weight (BW) was recorded daily in all animals and cumulative weight gain was calculated by subtracting the BW recorded the day before ARI treatment commencement from the subsequent values obtained in the following 28 days. 2.4. Treatment groups and sample collection The rats were sacrificed immediately after completing the chronic treatment schedule (28 days) by decapitation under isoflurane inhalation anesthesia. The blood was collected and centrifuged to obtain serum. The serum samples were frozen in À70 C before being used to assay the basic biochemical parameters and hormonal levels. 2.5. Biochemical assays Basic biochemical parameters were determined spectrophotometrically (Dimension Xpand Plus® , kits Siemens® ). The markers assayed were: total cholesterol, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), triglycerides (TAG), alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Adipokines, hormones and cytokines: leptin, ghrelin, glucagon like peptide-1 (GLP-1), glucagon, interleukins IL-1a and IL-6, tumor necrosis factor-a (TNF) and fibroblast growth factor-21 (FGF-21), were assessed using Bio-Plex® Multiplex System (immunoassay kits Bio-Rad® ) and by immunochemical method (ELISA) using commercial sets (BioVendor® ). 2.6. Statistical data analysis Primary data were summarized using arithmetic mean and standard deviation (±SD). The cumulative body weight gain data were analyzed by repeated measures ANOVA with the factors of ARI treatment and polyI:C model while the day was the repeated factor, Tukey post-test was used when significant differences were detected. Basal and final BW, total BW gain, biochemical and adipokine parameters were evaluated by two-way ANOVA, factors: polyI:C model and ARI treatment, the dataset was distributed normally (Kolmogorov-Smirnov test of normality). The Pearson's correlation was calculated for identification of interrelationships among leptin, ghrelin, GLP-1 and glucagon. All analyses were calculated using Statistica 12 (StatSoft, USA). A value p < 0.05 was recognized as the boundary of statistical significance in all applied tests. 3. Results Body weight data are depicted in Fig. 1. There was found no significant difference between the groups both before and after chronic ARI treatment (two-way ANOVA, n.s.). However, in the total body weight (BW) gain a weak trend towards lower gain in the polyI:C model was identified (two-way ANOVA, the main effect of the model, F(1,32) ¼ 3.161, p ¼ 0.085). To assess possibly different dynamics of the weight gain, daily cumulative BW gain was calculated. Repeated measures ANOVA revealed a significant day*polyI:C model interaction (F(27,864) ¼ 1.7988, p ¼ 0.0078) suggesting slower BW gain in the polyI:C prenatally challenged rats but Tukey post-hoc test did not reveal any specific day of a significant difference. Fig. 2 shows the serum lipid profile. Two-way ANOVA identified a significant effect of the polyI:C model in total cholesterol levels: F(1,35) ¼ 4.953, p ¼ 0.033, Tukey post-hoc test: p ¼ 0.033. Total cholesterol was elevated in polyI:C rats and similar data were recorded in LDL levels: significant effect of the polyI:C model, F(1,35) ¼ 5.968, p ¼ 0.020, Tukey post-hoc test, p ¼ 0.017 and HDL levels: significant effect of the polyI:C model, F(1,35) ¼ 4.368, p ¼ 0.044, Tukey post-hoc test, p ¼ 0.043. There were no significant differences in TAG levels. Furthermore, in order to evaluate possible interrelationships among the lipid profile variables, atherogenic index (AI) and atherogenic index of plasma (AIP), were calculated as total cholesterol/HDL and log (TAG/HDL), respectively. Despite we found a significant influence of the polyI:C model on cholesterol parameters, two-way ANOVA analysis of AI revealed a significant effect of ARI treatment: F(1,32) ¼ 17.670, p ¼ 0.0002, Tukey post-hoc test, p ¼ 0.0004 but no influence of the polyI:C model. There was no difference in the AIP measure. To provide a complete overview of data, mean values of all lipid parameters ±SD are shown in Table 1. Markers of hepatic functions (ALT and AST) were not elevated after chronic ARI treatment (data not shown). Levels of adipokines, cytokines and hormones in all groups are depicted in Fig. 3. Two-way ANOVA did not detect a significant effect of the polyI:C model in any of the markers. Interestingly, the interaction between the two factors, i.e. polyI:C model and ARI treatment, was not found to be significant in any marker either. However, there were numerous changes induced by the chronic ARI treatment. Specifically, leptin level was decreased after ARI treatment in both polyI:C and control rats: two-way ANOVA: F(1,29) ¼ 5.761, p ¼ 0.023, Tukey post-hoc test, p ¼ 0.022. Similarly, two-way ANOVA showed a decrease of GLP-1 in both ARI groups: F(1,31) ¼ 11.890, p ¼ 0.002, Tukey post-hoc test, p ¼ 0.002. However, ghrelin showed an opposite trend and was elevated after ARI treatment disregarding the model, two-way ANOVA: F(1,29) ¼ 26.041, p ¼ 0.00002, Tukey post-hoc test, p ¼ 0.0002 and glucagon was not found to be different in any group probably due to high variability in the data. Importantly, significant correlations were identified between GLP-1 and leptin/glucagon levels, see Table 2. All assayed markers of inflammation were suppressed by ARI administration (two-way ANOVA: significant effect of treatment) while polyI:C model per se did not lead to systemic inflammation (two-way ANOVA: no effect of the model). The specific results of statistical analysis follow. IL-1a: two-way ANOVA, significant effect of treatment F(1,30) ¼ 16.570, p ¼ 0.0003, Tukey post-hoc test: p ¼ 0.0004. IL-6: two-way ANOVA, significant effect of treatment F(1,30) ¼ 6.924, p ¼ 0.013, Tukey post-hoc test: p ¼ 0.012. TNF-a: two-way ANOVA, significant effect of treatment F(1,30) ¼ 4.601, p ¼ 0.040, Tukey post-hoc test: p ¼ 0.027. Lastly, levels of FGF-21 did not differ among the experimental groups. 4. Discussion To the best of our knowledge, this is the first study focused on comprehensive metabolic profile characterization of K. Horska et al. / Neuropharmacology 123 (2017) 148e158150 schizophrenia-like phenotype in rodents. Below we discuss the observed metabolic alterations following chronic ARI treatment in the polyI:C neurodevelopmental model in rats taking into account the value of polyI:C model for the study of the metabolic disturbances related to the schizophrenia-like phenotype and effect of ARI. 4.1. Body weight and lipid profile In our study, a trend towards different dynamics of weight gain in polyI:C rats was observed, showing non-significantly lower body weight gains compared to healthy animals in the absence of the antipsychotic treatment effect. Further polyI:C model was associated with alterations in lipid profile. ARI treatment did not induce significant weight gain during the course of the experiment. Only few animal studies have evaluated metabolic effects of ARI with inconsistent findings. Specifically, a study by Han et al. (2008) found no effect of chronic ARI (2.25 mg/kg/day) treatment on body weight or fat deposits in female Sprague-Dawley rats (Han et al., 2008). Interestingly, body weight gain was reported after oneweek treatment of ARI (at a similar oral dose as in our study) in female Wistar but not in female Sprague-Dawley rats (Kalinichev et al., 2005). This may indicate that Wistar rat strain is well suited for modeling ARI-induced metabolic disturbances. In male Sprague-Dawley rats 10-week ARI treatment at a similar dose as in our experiment did not lead to significant changes in body weight (De Santis et al., 2014). Of note, for AAP-induced weight gain modeling female rats are considered more susceptible than male rats, whereas other metabolic derangements, e.g. increased adiposity have been reported in male animals (Boyda et al., 2010). In this regard, lipogenic activation in the liver after chronic olanzapine exposure and adverse lipid spectrum were reported in male rats without concomitant weight gain (Ferno et al., 2015). Since we focused primary on possible metabolic derangements not secondary to weight gain, in our study we also used male rats. Moreover, the polyI:C model is well validated in male offspring while in females the phenotype seems to be less affected (Reisinger et al., 2015; Zhang et al., 2012). Interestingly, after chronic ARI exposure unchanged triglyceride but decreased cholesterol parameters were found in female rats, in contrast to weight-independently elevated triglycerides in olanzapine-treated rats (Skrede et al., 2012). In line with these findings, our data show no alterations in triglyceride level. Contrary to the aforementioned findings, in our experiment we noted no Fig. 1. Body weight analysis. The bar graphs indicate mean þ/- SD levels of: basal body weight (BW), i.e. BW one day before commencing the aripiprazole treatment; BW at the end of the study, i.e. after 28 days of the treatment; and total BW gain over the course of the study. Two-way ANOVA did not detect any significant difference among the groups besides a trend to lower body weight gain in the polyI:C model (p ¼ 0.085, the factor is indicated in brackets). Daily cumulative BW gain did not show any difference either (repeated measures ANOVA, n.s.). K. Horska et al. / Neuropharmacology 123 (2017) 148e158 151 changes in cholesterol serum levels associated with ARI treatment. Since another study found no alteration in body weight or lipid parameters in male rats after chronic ARI treatment (Cai et al., 2015), the inconsistency might be related to the gender specific metabolic effects of AAP. However, in our experiment we observed that ARI treatment led to significantly elevated atherogenic index in both healthy and polyI:C prenatally exposed animals. The risk of AAP-induced dyslipidemia observed in clinic varies among specific agents (American Diabetes Association et al., 2004; Newcomer et al., 2008; Stroup et al., 2011; Takeuchi et al., 2015) and several mechanisms have been suggested including direct weightindependent effects (Cai et al., 2015; Henderson et al., 2015; Yan et al., 2013). It has been demonstrated that AAP exert a direct action on lipid and cholesterol metabolism in primary rat hepatocytes cultures. While olanzapine exposure induced de novo lipogenesis with a significant increase in triglycerides, cholesterol and Fig. 2. Serum lipid profile. The bar graphs indicate mean ± SD levels of total cholesterol (CHOL), HDL, LDL, triacylglycerides (TAG), atherogenic index (AI) and atherogenic index of plasma (AIP). Two-way ANOVA identified a significant effect of the polyI:C model which increased total cholesterol, HDL and LDL while ARI treatment did not show any influence. ARI was shown to have a significant increasing effect on AI while the model did not influence this parameter. All significant differences are marked by the dotted lines which pool the groups depending on the factor for which two-way ANOVA revealed a significant effect (the factor is indicated in brackets). The lipid parameters are reported as mmol/l. Table 1 Serum lipid levels. The table indicates mean ± SD levels of total cholesterol (CHOL), HDL, LDL, triacylglycerides (TAG), atherogenic index (AI) and atherogenic index of plasma (AIP). The grey background color indicates the statistically significant factor (either polyI:C model or ARI treatment). For details see the Results. model treatment CHOL HDL LDL TAG AI AIP CTR VEH 1.64 ± 0.29 1.69 ± 0.27 0.16 ± 0.05 1.21 ± 0.27 0.97 ± 0.02 À0.06 ± 0.12 ARI 1.67 ± 0.33 1.66 ± 0.27 0.13 ± 0.04 1.08 ± 0.04 1.03 ± 0.04 À0.21 ± 0.16 Polyl:C VEH 1.78 ± 0.24 1.79 ± 0.21 0.18 ± 0.04 1.32 ± 0.30 0.99 ± 0.03 À0.14 ± 0.13 ARI 2.00 ± 0.36 1.94 ± 0.30 0.19 ± 0.20 1.18 ± 0.20 1.03 ± 0.04 À0.26 ± 0.09 K. Horska et al. / Neuropharmacology 123 (2017) 148e158152 phospholipids, ARI decreased synthesis of cholesterol and had no effect on triglycerides (Lauressergues et al., 2010). Preclinical in vivo experiments appear to be consistent with these findings, since comparable olanzapine and risperidone effects on the triglyceride serum level were convincingly demonstrated in rodents (Cai et al., 2015; Horska et al., 2016, 2017; Skrede et al., 2012). Further, an altered lipid profile in polyI:C model was revealed, since virtually all cholesterol parameters were significantly Fig. 3. Adipokines, hormones and cytokines. The graphs indicate mean ± SD serum levels of hormones, cytokines and adipokines. Two-way ANOVA identified a significant effect of the ARI treatment only, the model did not show an influence. Ghrelin was increased by the ARI treatment in both groups of polyI:C rats and their control counterparts. An opposite effect is visible in leptin, GLP-1, IL-1a, IL-6 and TNF-a where the levels were decreased by the ARI treatment. All significant differences are marked by the dotted lines which pool the groups depending on the factor for which two-way ANOVA revealed a significant effect (the factor is indicated in brackets). All parameters are reported as pg/ml. K. Horska et al. / Neuropharmacology 123 (2017) 148e158 153 increased in the absence of the effect of ARI treatment. The pathophysiological basis of the observed lipid metabolism dysregulation could be linked to maternal immune activation following polyI:C exposure. The maternal pro-inflammatory state with altered interleukin production might have led to phenotype covering hyperlipidemia in the offspring, even though we observed no concomitant increases in cytokine levels in our study. Consequently, an altered lipid profile might represent a trait of the phenotype of polyI:C neurodevelopmental schizophrenia-like model. The shared genetic link between schizophrenia and cardiovascular risk factors, specifically dyslipidemia, has been unraveled (Andreassen et al., 2013) and the increasing evidence of the genetic overlap between schizophrenia and lipid homeostasis has been recently reviewed (Steen et al., 2016). The polymorphism in lipid biosynthesis related genes has been also suggested as a factor influencing inter-individual variability to AAP-induced metabolic syndrome (Yang et al., 2016). The genetic association between schizophrenia and cardiovascular risk factors is strong; however, the pathophysiology involves environmental influences and interacting inflammatory, hormonal, endothelial and other mechanisms (Dieset et al., 2016). 4.2. Cytokines Importantly, in our study the polyI:C prenatal exposure was not related to the apparent pro-inflammatory state, since no alterations were observed in IL-1, Il-6 and TNF-a serum levels. The association between the immune system and schizophrenia is strong (Ole, 2017). A low-grade inflammatory state is consistently reported in the acute phase of schizophrenia, some cytokines are considered as trait markers (Miller et al., 2011). However, results of our study indicate that this clinical finding is not reflected in the polyI:C model. The long-term antipsychotic treatment modulates the immune system in schizophrenia and attenuates the pro-inflammatory signaling (Leonard et al., 2012; Meyer et al., 2011; Miller et al., 2011). This reported also a clinical trial after switching the patients from a variety of antipsychotics to ARI after the first month of therapy (Sobis et al., 2015). In our study, the same effect of ARI treatment was observed, all of the assayed pro-inflammatory cytokines were significantly decreased. Nevertheless, it is in contrast to findings of our previous studies with depot risperidone and olanzapine in healthy rats; the treatment did not consistently affect these parameters (Horska et al., 2016, 2017). However, another study reported increased adiposity and low-grade inflammatory state after chronic olanzapine, with increased TNF-a expression in adipose tissue while plasma levels of TNF-a and IL-1 were not affected (Victoriano et al., 2010). Also in human adipocytes proinflammatory and lipogenic gene expression was induced by AAP including ARI (Sarvari et al., 2014). Taken together, it seems that anti-inflammatory effects of long-term AAP administration are not convincingly demonstrated in rodents with the exception of our study with ARI. Noteworthy, cytokine data presented in all of the above mentioned preclinical studies exhibit an extremely high variability and an experiment with a substantially higher number of subjects may provide conclusive results. Moreover, the cytokine-mediated model of antipsychoticinduced weight gain has been recently considered (Fonseka et al., 2016). Yet, the immuneeneuroendocrine interactions are more intricate as the regulatory effects of cytokines on adipokines e.g. leptin action have been described (Trujillo et al., 2004), while leptin is involved in immune-system modulation (Procaccini et al., 2017) and metabolic regulations discussed in the following section. 4.3. Adipokines and gastrointestinal hormones This study, as far as we know, is the first one focused on metabolic regulations and analysis of adipokine and hormone levels in polyI:C or another neurodevelopmental model of schizophrenia. The polyI:C induced phenotype was not characterized by significantly altered adipokine and gastrointestinal hormone serum level profile, while the effect of ARI treatment was revealed. ARI significantly reduced leptin and GLP-1 serum levels, while ghrelin level was elevated. Moreover, based on the correlation analysis, both polyI:C model and ARI treatment disrupted the interrelationship in hormone/adipokine regulations. 4.3.1. Leptin In clinical settings, increased leptin is consistently reported in patients with schizophrenia and this finding was recently interpreted as not exclusively associated with antipsychotic treatment, although it was concluded that atypical antipsychotics may possess an additional risk (Stubbs et al., 2016). However, also ARI elevated leptin serum level in medication-naïve first-episode psychosis patients after the first three months of treatment (Perez-Iglesias et al., 2014). Data from preclinical studies on comprehensive evaluation of antipsychotic-induced alterations in metabolically relevant hormones are limited. The majority of the studies focused on metabolic adverse effects induced by olanzapine as antipsychotic carrying high metabolic risks and particularly leptin, more recently ghrelin received attention (Skrede et al., 2012; Zhang et al., 2013). In our previous experiment, depot olanzapine treatment increased leptin level and even caused pronounced early leptin dysregulation in the absence of increased adiposity. We proposed that possibly due to methodological issues the long-term olanzapine effect on leptin level in other rodent studies was reported inconsistently either as elevated or unchanged (Horska et al., 2016). Chronic ARI treatment (6 mg/kg/day orally for two weeks) did not affect leptin level in female Sprague-Dawley rats (Skrede et al., 2012). Since we used a similar oral dose the possible interfering factors could be the strain and sex of the rats and length of treatment. The effect of chronic ARI in clinical setting resulting in increased leptin level concomitant to weight gain (Perez-Iglesias et al., 2014) is in stark contrast to our observation of reduced leptin in the absence of body weight changes in rats. A possible explanation may lie in the non-adequate translational validity of the animal model, an intervening variable playing a role could be the disparate ARI pharmacokinetic/pharmacodynamic profile in rats, compared to Table 2 Correlation analysis. The table indicates results of Pearson's correlation calculated for identification of interrelationships among leptin, ghrelin, GLP-1 and glucagon (r, correlation coefficient; #p ¼ 0.05, ##p ¼ 0.01, significant results are marked bold). model treatment GLP-1 vs. leptin GLP-1 vs. glucagon control vehicle r ¼ 0.733 p ¼ 0.038 # r ¼ 0.779 p ¼ 0.023 # aripiprazole r ¼ 0.256 p ¼ 0.624 n.s. r ¼ 0.948 p ¼ 0.004 ## Polyl:C vehicle r ¼ À0.427 p ¼ 0.251 n.s. r ¼ 0.438 p ¼ 0.238 n.s. aripiprazole r ¼ 0.668 p ¼ À0.200 n.s. r ¼ À0.387 p ¼ 0.391 n.s. K. Horska et al. / Neuropharmacology 123 (2017) 148e158154 that in humans (Wood et al., 2006), and D2 occupancy-functional relationship (Natesan et al., 2006). In this regard, one mechanism potentially involved could be represented by adipose tissue-specific regulation of leptin via dopamine D2 receptors (Cuevas et al., 2014). It is, however, reasonable to presume that more complex regulations are implicated. Moreover, leptin modulates dopaminergic system in both humans and rodents (Panariello et al., 2012). Nevertheless, based on our findings, the altered serum leptin level indicates direct effects of ARI on neurohormonal regulation independent of body weight changes. In this respect, our data may support the hypothesis that antipsychotics contribute to the development of leptin resistance, and this may establish a metabolic status resulting in weight gain later in subsequent treatment period (Panariello et al., 2012). In our study, adiposity which is associated with elevated leptin level both in rodents (Velasque et al., 2001) and humans (Considine et al., 1996) was not assessed and this is relevant to note as one of the limitations of the study. However, even a ten-week long 2.25 mg/kg/day ARI treatment was not found to affect adiposity in male Sprague-Dawley rats (De Santis et al., 2014). The intricate interplay of leptin with other hormones/adipokines assessed in this study is of importance. 4.3.2. Ghrelin Our results show elevated ghrelin serum level after ARI treatment. Although in our previous studies of similar length of treatment, olanzapine did not affect ghrelin level, risperidone led to an increase in parallel to leptin (Horska et al., 2016, 2017). Normally, leptin dose-dependently suppresses ghrelin secretion (Kamegai et al., 2004) and leptin and ghrelin interaction was also described in both animal and clinical studies in the context of antipsychotic treatment (Hegedus et al., 2015; Sentissi et al., 2008). Thus ARIinduced elevation of ghrelin in our study could be viewed as associated with reduced leptin level; however, the correlation analysis did not confirm this relationship, so it does not support this hypothesis. Tri-phasic effect of AAP on ghrelin serum level has been recently proposed in a clinical setting and also in preclinical experiments with initial elevation, followed by secondary feed-back reduction and final re-increase (Zhang et al., 2013). This phenomenon may have been detected in our earlier study with depot risperidone (Horska et al., 2017). The role of ghrelin signaling system in antipsychotic-induced weight gain has been recently highlighted and considered as a potential target for pharmacological strategies in metabolic adverse effects of antipsychotics (Zhang et al., 2013) and our data support this notion. Furthermore, there is an existing interplay between ghrelin and dopamine signaling (Muller et al., 2015) and ghrelin antagonism was found to suppress morphineinduced dopamine release in rat brain (Sustkova-Fiserova et al., 2014) which may be implicated in the potential underlying mechanisms. 4.3.3. Glucagon-like peptide-1 (GLP-1) The interactions of GLP-1 with leptin and ghrelin have been recently reviewed, though the understanding of the specific mechanism is incomplete (Ronveaux et al., 2015). Ghrelin enhances glucose-stimulated GLP-1 secretion (Gagnon et al., 2015) while leptin modulates GLP-1 and as proposed the leptin resistant state may lead to attenuation of GLP-1 mediated signaling (Lim and Brubaker, 2006). On the other hand, a long-acting GLP-1 agonist liraglutide, was shown to regulate leptin signaling (Kanoski et al., 2015) and there is substantial evidence that GLP-1 and also FGF- 21 act as leptin-sensitizers increasing leptin responsiveness. This constitutes a new strategy to overcome leptin resistant state associated with obesity (Quarta et al., 2016). In the present study, GLP-1 serum level was reduced following ARI treatment, though in other studies this effect was not consistently observed after chronic risperidone or olanzapine treatment (Hegedus et al., 2015; Horska et al., 2016, 2017). In our study the interaction between GLP-1 and leptin was also revealed by a correlation analysis in untreated healthy animals which disappeared after ARI treatment. Interestingly this relationship was not at all present in the polyI:C model. Furthermore, GLP-1 level was reported to be acutely reduced 1 h after drug administration during clozapine and quetiapine chronic treatment in rats (Smith et al., 2009). Interestingly, in healthy human subjects short-term (9 days) olanzapine administration led to dysregulation of postprandial GLP-1, but not leptin or ghrelin, whereas no similar effect was seen with ARI (Teff et al., 2013). Analogously as for obesity treatment, GLP-1 analogs were proposed to represent a potentially beneficial strategy in AAP-induced weight gain (Ebdrup et al., 2012). In animal experiments, chronic administration of liraglutide, reversed olanzapine-induced weight gain, accompanied with dyslipidemia (Lykkegaard et al., 2008; Sharma et al., 2015). However, very recent data from the first (randomized, placebo-controlled, double-blinded) clinical trial in obese schizophrenia patients treated with antipsychotics showed that 3 months treatment with exenatide (another FDA-approved long-acting GLP-1 analog) had no effects on metabolic parameters including body weight and serum lipid profile (Ishøy et al., 2017). However, the same treatment was seen to result in weight loss in non-psychiatric obese populations, thus the authors pointed also to the psychiatric condition per se or the effects of antipsychotic treatment as possible factors playing a role (Ishøy et al., 2017). This hypothesis is in line with the results of our correlation analysis showing loss of interrelationship between GLP- 1, glucagon and leptin in the polyI:C model. 4.3.4. Glucagon Normally, GLP-1 and leptin, both inhibit glucagon release (Lee et al., 2016). In this study despite reduced GLP-1 and leptin serum level, no significant glucagon serum level increase was detected. Correspondingly, we observed no alterations in glucagon serum level after olanzapine (Horska et al., 2016) or risperidone treatment (Horska et al., 2017). However, in this study the relationship was seen between GLP-1 and glucagon in healthy animals irrespective of ARI treatment, whereas this was not detected in any of polyI:C treated subgroups. These results indicate altered hormonal/adipokine regulation in the polyI:C model. 4.3.5. Fibroblast growth factor-21 (FGF-21) FGF-21 levels are known to be elevated in obesity and metabolic syndrome (Zhang et al., 2008) and also in first-episode schizophrenia patients (Qing et al., 2015). Neither polyI:C model nor ARI treatment in this study had no effect on FGF-21 serum level, similarly no effects of olanzapine or risperidone were observed in our previous studies (Horska et al., 2016, 2017). As far as we know, no other preclinical studies focused on the putative role of FGF-21 in antipsychotic-induced metabolic effects. Preclinical data showed that FGF-21 restored leptin responsiveness in diet-induced obesity (Müller et al., 2012). Similarly, the treatment with exogenous FGF21 analog in obese patients with type 2 diabetes significantly improved dyslipidemia, led to less atherogenic profile, further beneficial effects on body weight, fasting insulin and adiponectin were noted (Gaich et al., 2013). Whether this effect would be observed in psychiatric patients or even could be translated to preclinical experiments using neurodevelopmental schizophrenialike models remains to be clarified. 5. Conclusions The neurodevelopmental polyI:C model of schizophrenia-like K. Horska et al. / Neuropharmacology 123 (2017) 148e158 155 phenotype in rats exhibited altered lipid profile which suggests its translational value. In addition, the model was not shown to be more susceptible to ARI-induced metabolic effects. However, this does not exclude that it may possess higher sensitivity to metabolic disturbances and susceptibility to metabolic alterations induced by antipsychotic medication carrying higher metabolic risks. Moreover, despite ARI is considered metabolically neutral, it induced dysregulation of adipokines and gastrointestinal hormones reducing leptin and GLP-1 and increasing ghrelin levels in both control and polyI:C rats. Importantly, metabolic derangements were not developed consequently to body weight changes. Finally, ARI treatment did not affect the polyI:C induced changes in any parameter. The incomplete understanding of complex hormonal regulations in adverse metabolic alterations induced by AAP requires further investigation. Conflict of interest disclosure All authors declare no conflict of interest. Michal Karpisek works in the company Biovendor-Laboratorni medicina, which manufactures the ELISA kits used in this study. Acknowledgements This study was performed at Masaryk university as part of the project „Experimental and translational pharmacological research and development” number MUNI/A/1063/2016 with the support of the Specific University Research Grant, as provided by the Ministry of Education, Youth and Sports of the Czech Republic in the year 2017. 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Horska et al. / Neuropharmacology 123 (2017) 148e158158 Psychoneuroendocrinology 73 (2016) 177–185 Contents lists available at ScienceDirect Psychoneuroendocrinology journal homepage: www.elsevier.com/locate/psyneuen Olanzapine-depot administration induces time-dependent changes in adipose tissue endocrine function in rats Katerina Horskaa , Jana Ruda-Kucerovab,∗ , Zuzana Babinskab , Michal Karpisekc,a , Regina Demlovab , Radka Opatrilovad , Pavel Suchya , Hana Kotolovaa a Department of Pharmacology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic b Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic c R&D Department, Biovendor – Laboratorni Medicina, Brno, Czech Republic d Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic a r t i c l e i n f o Article history: Received 5 May 2016 Received in revised form 23 June 2016 Accepted 26 July 2016 Keywords: Adipokine Adipose tissue Dyslipidemia Leptin Olanzapine Sprague-Dawley rats a b s t r a c t Objective: Metabolic adverse effects of atypical antipsychotics (AAP) contribute significantly to increased risk of cardiovascular morbidity and mortality in patients suffering from schizophrenia. Extensive preclinical research has addressed this issue over the past years, though mechanisms underlying these adverse effects of AAP are still not understood completely. Recently, attention is drawn towards the role of adipose tissue metabolism and neurohormonal regulations. Methods: The aim of this study was to evaluate the time-dependent effects of olanzapine depot administration at clinically relevant dosing on the regulation of energy homeostasis, glucose and lipid metabolism, gastrointestinal and adipose tissue-derived hormones involved in energy balance regulations in female Sprague-Dawley rats. The study lasted 8 weeks and the markers were assayed at day 8, 15, 29, 43 and 57. Results: The results indicate that in the absence of hyperphagia, olanzapine chronic exposure induced weight gain from the beginning of the study. In the later time-point, increased adiposity was also observed. In the initial phase of the study, lipid profile was altered by an early increase in triglyceride level and highly elevated leptin level was observed. Clear bi-phasic time-dependent effect of olanzapine on leptin serum concentration was demonstrated. Olanzapine treatment did not lead to changes in serum levels of ghrelin, FGF-21 and pro-inflammatory markers IL-1a, IL-6 and TNF-␣ at any time-point of the study. Conclusion: This study provides data suggesting early alteration in adipose tissue endocrine function as a factor involved in mechanisms underlying metabolic adverse effects of antipsychotics. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction The benefits of atypical antipsychotics (AAP) in the treatment of schizophrenia and other disorders not limited to psychotic spectrum are indisputable in general. One of the great advantages of AAP in the treatment of psychotic disorders is their low propensity to induce extrapyramidal symptoms, though the neurological side effects are ‘replaced’ by adverse metabolic effects (Nasrallah, 2008; Newcomer, 2007). These metabolic alterations, including weight gain, dyslipidemia, increased adiposity, glucose intolerance and insulin resistance which correspond to the cluster of metabolic ∗ Corresponding author at: Department of Pharmacology, Faculty of Medicine, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic. E-mail address: jkucer@med.muni.cz (J. Ruda-Kucerova). syndrome (MetS) symptoms further increase the risks for development of obesity, type-2 diabetes, cardiovascular morbidity, and the overall mortality of patients with schizophrenia (De Hert et al., 2012; Leucht et al., 2007). The overall rate of MetS was 32.5% in patients with schizophrenia and related disorders according to a recent meta-analysis (Mitchell et al., 2013). The propensity to induce metabolic alterations substantially differs among the antipsychotic drugs. This can be partially explained by their unique receptor binding profiles (Nasrallah, 2008). The highest potential to induce weight gain, increased adiposity, dyslipidemia and to impair glucose tolerance has been reported in the context of treatment with multi-acting receptor targeted agents (MARTA) antipsychotics, especially olanzapine and clozapine. Olanzapine seems to be the drug most associated with the symptoms of metabolic syndrome (De Hert et al., 2012; Leucht et al., 2013; Mitchell et al., 2013). The exact pharmacological http://dx.doi.org/10.1016/j.psyneuen.2016.07.218 0306-4530/© 2016 Elsevier Ltd. All rights reserved. 178 K. Horska et al. / Psychoneuroendocrinology 73 (2016) 177–185 molecular mechanisms underlying metabolic adverse effects of AAP are poorly understood; however, there is increasing evidence suggesting metabolic dysregulations as an antecedent factor of obesity development (Correll et al., 2010; De Hert et al., 2012). Extensive preclinical research has addressed this issue over the past years. Nevertheless, findings from preclinical studies are frequently inconsistent due to methodological issues such as use of different animal strains and gender, drug dose, route of administration and duration of treatment (Boyda et al., 2010; van der Zwaal et al., 2014). Above all, one of the important challenges has been represented by possibly inadequate dosing of AAP in animal studies not corresponding to the clinical condition, which arises from incomparable pharmacokinetic profiles of AAP in rodents and humans, because rodents have a much shorter half-life of AAP (Kapur et al., 2003). Recently the availability of AAP in the form of long-acting injections has enabled researchers to optimize the dosing regimens of AAP in preclinical studies, ensuring stable drug exposure. In humans there is evidence supporting the assumption that adverse metabolic effects of AAP may be associated to serum concentration, since olanzapine and clozapine induced metabolic abnormalities appear to show concentration-dependent relationship (Simon et al., 2009). This was clearly observed with regard to weight gain in patients receiving different doses of long-acting injectable olanzapine (Kane et al., 2010). Despite the lack of definite evidence of gender-specific effects of AAP on weight changes or other metabolic parameters in humans (Correll et al., 2010; Mitchell et al., 2013), female rats were shown to be more suitable for modeling AAP-induced weight gain than males (Albaugh et al., 2006; Boyda et al., 2010; Davey et al., 2012; van der Zwaal et al., 2014). Furthermore, the AAPs with the least liability to metabolic dysregulation in humans, such as aripiprazole or ziprasidone (Leucht et al., 2013), were also associated with weight gain in rodent experiments (Boyda et al., 2010; Skrede et al., 2012; van der Zwaal et al., 2014). Lately, with regard to metabolic alterations induced by AAP, attention has been paid to dysregulation of adipose tissue metabolism and its endocrine/paracrine function in human studies and animal experiments (Potvin et al., 2015; Skrede et al., 2012; Zhang et al., 2013). Particularly, the research has been focused on the potential effects of AAP on adipokines, as well as proteins released by adipose tissue and neurohormonal regulations. The intricacy of interplay of modulators of food intake and energy balance and gastrointestinal hormones in general is also being investigated intensively (Perry and Wang, 2012) but these variables were incompletely explored in context of AAP-induced metabolic adverse effects in animal models. Furthermore, specific biochemical marker which enables the identification of patients at a high risk of AAP-induced MetS has not yet been proposed, and the underlying molecular mechanisms of AAP-induced metabolic effects remain to be elucidated. Therefore, the aim of this study was to evaluate the timedependent effects of olanzapine depot administration on the regulation of energy homeostasis, specifically feeding behavior, lipid profile, alterations of adipose tissue endocrine/paracrine functions and hormonal regulations in order to elucidate their interrelationships in the mechanisms of AAP-induced metabolic alterations. Apart from basic biochemical analysis (lipid spectrum, glucose serum level), serum levels of leptin, ghrelin, glucagonlike peptide-1 (GLP-1) and glucagon, fibroblast growth factor-21 (FGF-21) were assessed to describe alterations in adipose tissue endocrine functions and in neurohumoral regulation. Spectrum of adipokines and hormones was selected for the analysis, in order to explore their possible interrelationships and roles in the mechanisms of AAP-induced metabolic alterations, since eg. leptin and ghrelin are suggested to have opposite an/orexigenic effects in appetite and energy homeostasis regulation (Klok et al., 2007; Muller et al., 2015). These two factors have been studied most extensively with inconsistent findings in both preclinical and human studies (Potvin et al., 2015; Skrede et al., 2012; Zhang et al., 2013). However, the regulations are more complex, thus GLP-1 and adipokine FGF-21 were included as there is evidence confirming physiological role of GLP-1 in the complex regulation of appetite (Ronveaux et al., 2015), and FGF-21 is known regulator of glucose and lipid homeostasis, which possesses functions of endocrine hormones (Kharitonenkov, 2009). We also assessed pro-inflammatory cytokines (interleukin 1a and 6, tumor necrosis factor-␣), which are known to be elevated in MetS (Kucerova et al., 2015). 2. Material and methods 2.1. Animals Forty female 8 weeks old albino Sprague-Dawley rats weighing 200–225 g at the beginning of the study were purchased from Charles River (Germany) and housed individually in standard housing cages. Environmental conditions during the whole study were constant: relative humidity 50–60%, temperature 23 ◦C ± 1 ◦C, normal 12-h light-dark cycle (6 a.m.–6 p.m. light). Standard rodent chow and water were available ad libitum. All experiments were conducted in accordance with all relevant laws and regulations of animal care and welfare. The experimental protocol was approved by the Animal Care Committee of the Masaryk University, Faculty of Medicine, Czech Republic, and carried out under the European Community guidelines for the use of experimental animals. 2.2. Drugs and treatments Olanzapine (OLA) was administered in a depot formulation for human use (ZypAdhera®) by an intramuscular injection at dose 100 mg/kg every 14 days in the evening hours (administration on day 1, 15, 29 and 43). The solvent vehicle was injected to the control group. The food was removed from the cages and all rats were subjected to overnight fasting in order to prevent weight gain differences induced by sedation in the OLA-treated group as we observed in our previous experiments (unpublished data) and was also already recently validated (Skrede et al., 2014). 2.3. Food consumption and body weight recording Body weight (BW) and food consumption were recorded daily in all animals. Feeders in all cages were filled with 50 g of the rodent chow, the consumption was recorded after 24 h and then filled to 50 g again. On the days of drug administration when the chow was removed overnight the food consumption data were not recorded (day 1, 15, 29 and 43). 2.4. Treatment groups and sample collection Rats were randomly assigned to 2 treatment groups: vehicle (VEH) treated (n = 17) and olanzapine (OLA) treated (n = 23). Two subgroups of animals, vehicle (n = 7) and olanzapine-treated rats (n = 8), were sacrificed by decapitation under short isoflurane anesthesia to collect blood (for serum), liver and visceral fat tissue 8 days after the first administration. The dissection was performed after decapitation by wide laparotomy, the liver was excised and weighted and abdominal fat tissue was collected and weighed. Other two subgroups of rats, vehicle (n = 10) and olanzapinetreated (n = 15), were kept until the day 57 (8 weeks of treatment) and then sacrificed and dissected in the same manner. Furthermore, 1.5–2 ml of blood for serum was collected under short isoflurane anesthesia every 2 weeks (day 1, 15, 29 and 43) by retro-orbital puncture and the same amount of liquid was supplied by the K. Horska et al. / Psychoneuroendocrinology 73 (2016) 177–185 179 intraperitoneal injection of saline. The serum samples were used to assay the biochemical parameters – lipid profile (total cholesterol, high-density cholesterol – HDL, low-density cholesterol – LDL and triglycerides – TAG), glucose serum level, adipokine and hormone levels – leptin, ghrelin, GLP-1 (glucagon-like peptide-1), FGF-21 (fibroblast growth factor-21), interleukins IL-1␣, IL-6, TNF␣ (tumor-necrosis factor-alpha). 2.5. Biochemical assays Basic biochemical parameters were determined spectrophotometrically – lipid profile, glucose serum level (Dimension Xpand Plus®, kits Siemens®). Adipokines, hormones and cytokines were assessed using Bio-Plex® Multiplex System (immunoassay kits BioRad®) and by immunochemical method (ELISA) using commercial sets (BioVendor®). 2.6. Statistical data analysis Primary data were summarized using arithmetic mean and standard error of the mean estimate (SEM). The cumulative weight gain, cumulative food intake and feed efficiency data were analyzed by repeated measures ANOVA with the factor of OLA treatment and Dunnet post-hoc test. Organ weights (liver and adipose tissue) were compared at two time-points (day 8 and day 57) by 2 way ANOVA (factors: time-point and treatment) with Tukey post-hoc test. Biochemical parameters, adipokine, hormone and cytokine serum levels at day 8 were evaluated by t-test when the dataset was distributed normally (Kolmogorov-Smirnov test of normality) or Mann-Whitney U test when the normality test was significant (this was the case of interleukins and TNF-␣). For the longitudinally monitored groups repeated measures ANOVA with the factor of OLA treatment and Dunnet post-hoc test was used in normal data and Kruskal-Wallis test in interleukins and TNF-␣ results. The analyses were calculated using Statistica 12 (StatSoft, USA). A value p < 0.05 was recognized as the boundary of statistical significance in all applied tests. 3. Results 3.1. Food consumption and body weight recording Fig. 1 summarizes the body weight (BW) and standard rodent chow data. Repeated measures ANOVA revealed a highly significant increase in the mean daily cumulative weight gain of the OLA-treated group starting on the day 6 (Fig. 1A) except the last week of the study. Cumulative chow intake (assessed daily) is presented on days 8, 15, 29, 43 and 55 in both longitudinal groups and has shown no difference (Fig. 1B). Despite equal food intake, OLA treatment led to increase in body weight. Therefore feed efficiency was calculated as BW in grams/cumulative chow intake in grams at the same time-points (days 8, 15, 29, 43 and 55). Repeated measures ANOVA revealed a significant increase of the feed efficiency in the OLA-treated rats already at the beginning of the study (day 8). This effect was smaller yet significant until day 43, and no difference was found at the end of the study after 8 weeks of chronic treatment (Fig. 1C). Note that in the first time-point (8 days) data were pooled from the animals sacrificed at day 8 and the groups with the longitudinal assessment. Liver and adipose tissue mass were recorded at the time of sacrifice only (day 8 and 57). As expected, Fig. 2 indicates that 2 way ANOVA (factors: time-point and treatment) with Tukey post-test has revealed significant differences only in the adipose tissue mass. These changes were dependent on age in both treatment groups (significant increase when the time-points were compared) and more importantly on drug treatment at day 57 (an increase of adiposity in the OLA-treated group). The weight of liver did not change after chronic olanzapine exposure, which indicates that olanzapine chronic treatment did not lead to apparent metabolic or structural liver tissue changes. 3.2. Blood lipid and glycemic profiles Lipid profiles were evaluated independently in the groups sacrificed on day 8 and the longitudinal design. Fig. 3 depicts the total cholesterol, HDL, LDL and TAG data on the day 8 analyzed by ttest. There was a significant decrease in LDL and increase in TAG of the OLA-treated group. Fig. 4 reports the same variables in the longitudinal assessment. In longitudinal design, repeated measures ANOVA did not reveal any significant changes in lipid spectrum. The glycemic profiles did not differ significantly between the groups at any time-point (data not shown). 3.3. Cytokine and hormonal levels Analogously, the adipokine and hormone data were analyzed separately for the day 8 and the longitudinal study. Leptin, ghrelin, glucagon, GLP-1 and FGF-21 data were analyzed by parametric t-test, while IL-1a, IL-6 and TNF-␣ were evaluated using MannWhitney U test. As shown in Fig. 5, at this early time-point the only alteration in the parameters evaluated was a highly significant increase of leptin level in the OLA-treated group. Fig. 6 summarizes the longitudinal data on the same variables analyzed by repeated measures ANOVA (or Kruskal-Wallis test for IL-1a, IL-6 and TNF-␣ data). Interestingly, the leptin dysregulation was found significant again only in the last time-point of the study after 8 weeks of chronic treatment (day 57). Furthermore, decreases of glucagon and GLP-1 serum levels were found on day 29 in the OLA-treated group. Despite some apparent trends the Kruskal-Wallis test for IL-1a, IL-6 and TNF-␣ data did not detect significant differences between the groups. 4. Discussion This study reports comprehensive time-dependent data on basic biochemical profile, neurohumoral regulation and endocrine function of adipose tissue during chronic olanzapine exposure under conditions corresponding to clinical settings. The specific findings will be discussed in sections. 4.1. AAP-induced changes in feeding behavior and body weight As expected we recorded a significant increase in body weight increment after olanzapine treatment during the whole study. In contrast to findings from other studies, hyperphagia expressed as increased food intake concomitant to weight gain was not observed in this experiment. Hyperphagia has been considered as the key behavioral factor mediating weight gain (Davoodi et al., 2009; Weston-Green et al., 2011), however increases in adipose tissue mass were noted in male rats even without concomitant hyperphagia and weight gain (Girault et al., 2014; Minet-Ringuet et al., 2007; van der Zwaal et al., 2014). As described earlier (Skrede et al., 2014; Weston-Green et al., 2011), we calculated feed efficiency and noted markedly increased values in the olanzapine treatment group, starting from the first time-point (day 8) but not present at the end of the study (day 55). Our results demonstrate significantly increased visceral adipose tissue deposits after 8 weeks of chronic olanzapine treatment. Increased fat deposition, adipocyte size enlargement and alteration of adipocyte metabolism after chronic treatment with olanzapine have been characterized, indicating primary effects of AAP on adipose tissue (Minet-Ringuet 180 K. Horska et al. / Psychoneuroendocrinology 73 (2016) 177–185 Fig 1. body weight and food intake. Fig. 1A shows daily means ± SEM of cumulative body weight gain in VEH and OLA-treated animals. Till the day 8 the data are pooled with the early sacrificed groups. Repeated measures ANOVA, main effect of treatment p = 0.026, Dunnet post-test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Fig. 1B depicts cumulative chow intake at days 1, 15, 29 and 43, repeated measures ANOVA, n.s. Fig. 1C reports feed efficiency calculated as BW gain (g)/cumulative food intake (g). Repeated measures ANOVA, main effect of treatment p = 0.01, Dunnet post-test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). The last assessment of feeding behavior was on day 55 while animals were sacrificed on day 57. Fig. 2. liver and adipose tissue mass. ANOVA (factors: time-point and treatment) has detected no differences in the liver mass. Adipose tissue mass has shown the effect of both treatment (p = 0.015) and timepoint (p ≤ 0.001). Tukey post-test has revealed a significant increase in both groups at day 57 as compared to day 8 (symbol: *p ≤ 0.05, **p ≤ 0.01) and an OLA-induced increase of fat mass on the day 57 (symbol: $p ≤ 0.05). White bars: control rats, hatched bars: OLA treated rats. Fig. 3. blood lipid profile at day 8. The graphs indicate the levels of total cholesterol (CHOL), HDL, LDL and TAG on the day 8 analyzed by t-test. T-test identified a significant decrease in LDL and increase of TAG in the OLA-treated group (*p ≤ 0.05, **p ≤ 0.01). K. Horska et al. / Psychoneuroendocrinology 73 (2016) 177–185 181 Fig. 4. blood lipid profile at days 15, 29, 43 and 57. Repeated measures ANOVA did not reveal any significant changes in total cholesterol (CHOL), HDL, LDL or TAG levels. Fig. 5. blood adipokine, hormonal and cytokine levels at day 8. Parametric t-test detected a highly significant increase of leptin level in the OLA-treated group. There were no other significant results. Data on the IL-1a, IL-6 and TNF-␣ have shown high variability but did not reveal any elevated pro-inflammatory markers. et al., 2007). Furthermore, early morphological change of the subcutaneous adipose tissue has been described, characterized by an increase in the proliferation of undifferentiated adipocytes independent of weight gain but showing time- and dose-dependent relationship (Tan et al., 2010). Evidence and our data both support the conclusion that changes in body composition with increased adiposity are largely independent of changes in body weight or increased food intake. However, another confounding factor may be different energy expenditure in control and olanzapine treated rats. OLA is known to cause sedation in both open-field test (Zhang et al., 2014) and home-cage locomotor profile (van der Zwaal et al., 2010, 2012). Noteworthy, it seems that chronic treatment alleviates the effect on locomotor activity (Albaugh et al., 2011). 4.2. AAP-induced changes in lipid profile In the initial phase on the day 8 this study found lipid profile alterations characterized by an increase in triglycerides and lowered LDL-cholesterol plasma level. At later time-points, these changes did not persist. 182K.Horskaetal./Psychoneuroendocrinology73(2016)177–185 Fig. 6. adipokine, hormonal and cytokine levels at days 15, 29, 43 and 57. Repeated measures ANOVA revealed significant changes increase of leptin levels induced by OLA treatment at day 57 only, Tukey post-test (*p ≤ 0.05). Other changes detected comprise decreases of glucagon and GLP-1 on the day 29 in the OLA-treated group, Tukey post-test (*p ≤ 0.05). K. Horska et al. / Psychoneuroendocrinology 73 (2016) 177–185 183 Lipid profile has been characterized only in some of the earlier rodent studies mostly no increases in triglyceride and cholesterol plasma levels have been reported (Albaugh et al., 2006; Davoodi et al., 2009). Yet there are recent data demonstrating that AAP elevate plasma triglyceride levels after chronic administration in animals (Zugno et al., 2012). In female rats, increased triglyceride level has been reported as independent to weight gain after olanzapine chronic treatment (Skrede et al., 2012). Moreover, acute administration of olanzapine resulted in altered lipid parameters with increased serum free fatty acids, triglycerides, and reduced cholesterol levels followed by accumulation of lipids in the liver at the later time frame. Thus, direct dysregulation of lipid metabolism has been described prior to weight gain development (Jassim et al., 2012). Furthermore, evidence from in vitro experiments in three different human cell lines cultures points to direct effects of several AAP on the regulation of lipid metabolism. Inhibition of cholesterol biosynthesis was shown at several different steps and in contrast increased synthesis of triglycerides was observed. The elevation of triglycerides was suggested to be the consequence of compensatory mechanisms to inhibited cholesterol synthesis involving upregulation of gene expression (Canfran-Duque et al., 2013; Skrede et al., 2013). The potential mechanisms of AAP induced hypertriglyceridemia have been reviewed recently, highlighting direct effects of AAP on triglyceride metabolism as well as indirect actions on central nervous system pathways regulating energy homeostasis. Indirect effects of AAP on triglyceride metabolism may be mediated by obesity associated insulin resistance leading to lipogenesis stimulated by hyperinsulinemia. Regarding direct mechanisms, it has been consistently reported that AAP upregulate expression of sterol regulatory element binding proteins (SREBPs) transcription factors and related target genes involved in de novo lipid synthesis. Furthermore it was suggested that AAP may regulate activity or plasma level of lipoprotein lipase, thus may impair triglyceride catabolism (Yan et al., 2013). Other direct effect of AAP on adipose tissue was recently proposed with regard to expression of functional dopamine receptors in human adipocytes and potential regulatory role of dopamine in adipose tissue functions, e.g. leptin inhibition (Borcherding et al., 2011; Cuevas et al., 2014). Therefore, our data on lipid profile in the initial phase of the study correspond to the preclinical findings focused on the direct AAP effects on lipid metabolism. These alterations might be associated to dysregulation of adipokines’ secretion as discussed later. 4.3. AAP-induced changes in glycemic control markers Chronic olanzapine treatment did not demonstrate any effect on glucose level in this experiment, while decrease in GLP1 and glucagon levels was found on day 29. GLP1 is a potent insulinotropic intestinal hormone and its secretion is decreased in obesity (Anini and Brubaker, 2003). Dysregulation in glucose metabolism has been inconsistently documented in rodent models (Albaugh et al., 2006; Boyda et al., 2010; Girault et al., 2014). Direct and acute effects of AAP on glucose homeostasis and olanzapine-induced insulin resistance in the absence of weight changes have been convincingly demonstrated (Boyda et al., 2010; Jassim et al., 2012; Kovacs et al., 2015). Also chronic olanzapine treatment resulting in unaltered glucose plasma level was reported, though it was accompanied by hyperinsulinemia and caused desensitization to its acute effects on glucose metabolism (Girault et al., 2014). Therefore, it can be assumed that AAP-induced glycemic dysregulation and insulin resistance may involve mechanisms independent of either weight gain or adipos- ity. Our findings may support the hypothesis of GLP-1 dysregulation induced by chronic olanzapine exposure, yet on day 8 no alteration in GLP-1 serum level was observed. At this timepoint, high inter-individual variability in olanzapine-treated group should be noticed. Data from later time-points showed significantly decreased GLP-1 only on day 29. Similarly, decreased glucagon serum level was found on day 29 with no other significant alterations in the course of the study. Based on our data, olanzapine effects on the interrelationships of glucose homeostasis markers cannot be elucidated; however, the associations of GLP-1 with leptin and ghrelin are discussed below. 4.4. AAP-induced changes in adipokine/hormone levels In the initial phase of treatment (day 8), a pronounced increase in leptin and differences in lipid spectrum were found without concomitant increase in abdominal adiposity. At later time-points, a significant weight gain and increase in visceral adipose tissue mass were demonstrated together with increased leptin level (day 57). Interestingly, no alterations in leptin level were found at the intermediate time-points. Leptin is an anorexigenic hormone, however in obesity its levels are elevated; therefore, the concept of leptin resistance has been established (Klok et al., 2007). Leptin transport across hematoencephalic barrier is impaired by triglycerides, which implies hypertriglyceridemia as an important cause of leptin resistance (Banks et al., 2004). An alternative concept called “leptininsufficiency” postulates that hematoencephalic barrier limits the availability of leptin in response to its high concentration (Kalra, 2008). Consistently, this study revealed concomitant hypertriglyceridemia and hyperleptinemia as soon as on day 8. Human data consistently demonstrate increased leptin levels in patients treated with antipsychotics (Potvin et al., 2015; Sentissi et al., 2008). However, a recent meta-analysis has revealed that leptin levels increased in schizophrenia patients, may not be associated solely with antipsychotic medication (Stubbs et al., 2016). Moreover, it was suggested that peak in leptin concentration during initial phase of clozapine treatment predicts lower weight gain in later treatment period (Monteleone et al., 2002). The findings with regard to the effect of AAP on leptin plasma levels in rodent models appear to be controversial, showing no alteration (Davey et al., 2012; Kovacs et al., 2015; Skrede et al., 2012; Zugno et al., 2012), or significant increase after chronic treatment concomitant to increased adiposity (Albaugh et al., 2006; Girault et al., 2014; Hegedus et al., 2015). Possible explanation of this inconsistency lies in different methodological approaches, especially dosing scheme and duration of treatment. Currently, the attention has been paid to ghrelin, the gastrointestinal orexigenic hormone with central and peripheral actions, which include regulation of appetite, body weight and adiposity, lipid metabolism and glucose homeostasis (Muller et al., 2015). Similar to leptin, ghrelin is involved in regulation of broad central nervous system functions and appears to be closely linked to psychiatric disorders (Wittekind and Kluge, 2015). Moreover, dimerization of ghrelin receptors with wide spectrum of other receptors including dopaminergic and consequent modifications in signaling have been observed, e.g. oligomerization with ghrelin receptor is essential in anorexigenic actions mediated via D2 receptors (Wellman and Abizaid, 2015). Ghrelin levels in clinical studies seemed to be reduced during first several weeks of treatment with AAP and increased in long-term treatment. Also the interaction between leptin and ghrelin levels has been described as the increase in leptin and decrease in ghrelin levels both in preclinical and clinical settings (Hegedus et al., 2015; Sentissi et al., 2008). Later, tri-phasic effect of AAP on ghrelin levels has been postulated with initial acute elevation, peak in the first week of treatment, followed by a secondary decrease and re-increase as a long-term effect and a similar trend has been proposed regarding data from preclinical studies (Zhang et al., 2013). Though, dysregulation of ghrelin 184 K. Horska et al. / Psychoneuroendocrinology 73 (2016) 177–185 secretion was not demonstrated throughout the chronic olanzapine treatment in this experiment. Leptin and ghrelin modulate the anorexigenic effects of GLP-1 nevertheless the exact mechanisms of these interactions are not fully known (Ronveaux et al., 2015). Leptin was shown to stimulate in a dose-dependent manner secretion of GLP-1, a potent insulinotropic intestinal hormone. Consistent with the concept of leptin resistance, GLP-1 is decreased in obesity (Anini and Brubaker, 2003). From this perspective, the significant increase in leptin levels in this study leads to the assumption of reduced serum GLP-1 concentration. However, this was not confirmed concomitantly to elevated leptin concentration neither in the initial phase, nor after 57 days of chronic exposure. Decrease in GLP-1 level was observed in the intermediate time-point of the study; nonetheless, it might indicate possible interaction and time-dependent changes in GLP-1 secretion during chronic olanzapine treatment. Also our results do not indicate any changes in FGF-21 serum level, despite elevated serum FGF-21 levels reported in obesity which was independently associated to adverse lipid profile (i.e. increased levels of TAG), visceral adipose tissue deposits and metabolic syndrome in humans (Zhang et al., 2008). To the best of our knowledge, alterations in FGF-21 secretion were not addressed in preclinical or clinical studies focused on AAP-induced adverse metabolic effects. This parameter could also show time-dependent changes. However, there are insufficient data to explain its putative interactions and role in the complex mechanisms of AAP-induced metabolic dysregulation. 4.5. AAP-induced changes in pro-inflammatory cytokine levels In our study, no significant alteration in IL-1a, IL-6 and TNF-␣ secretion was observed at any time-point. In contrast to our results, previous report indicates that increased adiposity and low-grade inflammatory state follows chronic olanzapine treatment in rats, with increased TNF-␣ expression in adipose tissue and slightly elevated plasma levels of TNF-␣ and IL-1 (Victoriano et al., 2010). Though the immunomodulatory effects of AAP may contribute to the pro-inflammatory state, which is associated to schizophrenia per se (Kucerova et al., 2015), the long-term AAP treatment may enhance anti-inflammatory cytokine signaling (Meyer et al., 2011). The current evidence in this matter remains inconclusive in clinical studies, and there is a lack of data in preclinical research. 5. Conclusions The key finding of this study was concomitant with hyperleptinemia and altered lipid profile characterized by hypertriglyceridemia and lowered LDL levels in the initial phase of olanzapine treatment. The early dysregulation of leptin occurred prior to significant changes in adiposity. Since leptin decreases triglyceride levels and hypertriglyceridemia inhibits leptin transport across hematoencephalic barrier resulting in leptin resistance, this feedback loop represents a mechanism of olanzapine induced adverse metabolic effect of interest. Leptin serum level was also increased after 8 weeks of olanzapine treatment, thus our data showed clear bi-phasic time-dependent effect of olanzapine on leptin serum concentration. Hyperphagia was not observed in this study, but increased feed efficiency was noted in the course of the study, resulting in weight gain and increased visceral fat deposits after 8 weeks of olanzapine exposure. Therefore, it can be assumed that altered adipose tissue endocrine function contributes to mechanisms underlying metabolic adverse effects of antipsychotics. However, a better understanding of interrelationships in neurohumoral regulation and the mechanisms involved is essential for identification of suitable biomarkers as predictors of metabolic adverse effects of AAP, and ultimately for the development of new antipsychotics with a lower propensity to induce adverse metabolic effects or adjuvant treatment strategies. Conflict of interest disclosure All authors declare no conflict of interest. Michal Karpisek works in the company Biovendor-Laboratorni medicina, which provided ELISA kits for laboratory measurements. Role of the funding source This study was supported by the project of Internal Grant Agency (IGA) VFU Brno (IGA VFU 312/2016/FaF) with co-financing of the project “Experimental pharmacological development in neuropsychopharmacology and oncology” number MUNI/A/1284/2015 with the support of the Specific University Research Grant at Masaryk University, as provided by the Ministry of Education, Youth and Sports of the Czech Republic in the year 2016 and funds from the Faculty of Medicine MU to junior researcher Jana Ruda-Kucerova. Authors’ contributions Katerina Horska developed the original idea, designed the study, contributed to the statistical analysis and wrote a substantial part of the introduction, methods, results and discussion sections of the manuscript. Jana Ruda-Kucerova managed the experimental work, collected biological samples and the data and processed them for analysis, performed the statistical analysis and wrote a substantial part of the introduction, methods, results and discussion sections of the manuscript. Zuzana Babinska was responsible for the practical experimental work and contributed to writing the methods and results sections of the manuscript. Michal Karpisek was responsible for ELISA assays and contributed to the final version of the manuscript. Regina Demlova contributed to the final version of the manuscript. Radka Opatrilova contributed to the final version of the manuscript. Pavel Suchy contributed to the final version of the manuscript. Hana Kotolova developed the original idea, contributed to experimental work, cross-checked the materials and methods section of the manuscript and contributed to the final version of the manuscript. 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