Therapeutic peptides
© doc. PharmDr. Oldřich Farsa, PhD., 2012
Classification of therapeutic peptides
1. Hormones
1.1 Liberins a statins („releasing“&“inhibiting“)
1.2.Soma(to)tropin
1.3 Oxytocin, vasopressin and their analogues
1.4 Insulines, glucagon and GLP-1 analogues
1.5 Calcitonin
2. Blood factors of erythropoietine type
3. Colony stimulating factors
4. Non-specific antibodies
One- and three-letter symbols of L-α-amino acid rests
One-letter Three-letter
A Ala alanine
B Asx asparaginic acid or asparagine
C Cys cysteine
D Asp asparaginic acid
E Glu glutamic acid
F Phe phenylalanine
G Gly glycine
H His histidine
I Ile isoleucine
K Lys lysine
L Leu leucine
M Met methionine
N Asn asparagine
P Pro proline
Q Gln glutamine
R Arg arginine
S Ser serine
T Thr threonine
U Sec selenocysteine
V Val valine
W Trp tryptofane
X Xaa unknown or „other“ amino acid
Y Tyr thyrosine
Z Glx glutamic acid or glutamine (or compounds such
as 4-carboxyglutamic acid 5-oxoproline)
1. Hormones
1.1 Liberins and statins („releasing“ & „inhibiting“)
Gonadorelin (GnRH = LHRH) and its analogues
●hormone of hypothalamus
●stimulates releasing of folicules stimulating hormone (FSH) and luteinizing hormone (LH)
from pituitary gland; GnRH receptors also in various non-reproductive tissues
gonadorelin acetate Gonadorelini acetas EP
buserelin Buserelinum EP
goserelin Goserelinum EP
Gonadorelin agonists
Gonadorelin and its analogues
Agonists
leuprorelin (syn. leuprolide) Leuprorelinum EP
Eligard ®
●
longer-term application lowers testosterone levels ⇒ treatment of prostate cancer
⇒ treatment of sexual deviations
OH
OH
OH
O
OH
O
NN
H
O
O
O N
H
O
NH2
NH
O
NH
NH NH2
CH3
CH3
N
H
N
N
H
N
H
N
H
N
H
N
H
N
H
O
O
OH
O
O
O
O
OH
N
H
O
OH
OHO
OH
OH
Gonadorelin and its analogues
Agonists
Short- and long term action of gonadorelin agonists
●long term action leads to receptors internalisation and stopping of the effect (due to
decreasing LH and FSH levels and thus also levels of sexual hormones)
Gonadorelin analogues
Gonadorelin antagonists
. CH3
COOH
. CH3
COOH
N
H O
N
H
Cl
N
H
O
N
H
OH
N
H
O
OH
N
H
O
N
H
O
CH3
CH3
N
H O
N
O
N
H
O
CH3
NH2
N
O
CH3
N
NH N
H
CH3
CH3
N
N
H
N
H
CH3
CH3
OO
ganirelix
N
H O
N
H
O
Cl
N
H
O
N
H O
OH
N
H O
OH
N
H
O
NH
N
H
O
CH3
CH3
N
H
NH2
NH
N
H
O
N
O
N
H
O
CH3
NH2
O NH2
N
O
CH3
cetrorelix
Gonadorelin and its analogues
●preparation: chemical synthesis
●usage: assisted reproduction, treatment of prostate cancer, sexual deviation ...
●
advantages of analogues: significantly higher stability ⇒ longer elimination half-time ⇒
⇒ possibility of application in markedly longer intervals; a single injection of an agonist can
replace a continuous infusion of gonadorelin
Structure – activity relationships (SAR)
●replacement of Gly in position 6 with a more bulky amino acid leads to stability increase
●the sequence of the first three amino acids is needed for receptor binding and is kept in
agonists
● antagonists have Trp in position 3 replaced with an non-physiologic amino acid, they bind
to GnRH and avoid its action on receptors
Corticotropin and its analogues
Corticotropin = Adrenocorticotrophic hormone (ACTH); an anterior pituitary hormone that stimulates the
adrenal cortex and
its production of both gluco- and mineralocorticoids and growth of adrenal glands
●polypeptide of 39 amino acids; N-terminal 24 identical in all species
●N-terminal 24 AA are responsible for biologic activity; C-terminal 15 AA for immunospecificity
corticotropin
tetracosactide
syn. cosyntropin [USAN]
Tetracosactidum EP
Synacten®
SynVL
●compound used as a standard
for determination of
tetracosactide by mass
spectrometry
Usage of corticotropin and tetracosactide
●diagnosis of adrenal glands function
●substitution treatment in lack of glucocorticoids
●substitution of depot administration of glucocorticoids in a long-term treatment
N
N
N
N
H
N
N
HOH
N
H
N
H
N
H
N
H
N
H
NH
OH
O
NH2
O
OH
O
S
CH3
O
OHO
O
O
N
H
N
H
N
H
O
NH
NH2 NH
O
O
ONH
O
NH2
O
NH
CH3
CH3
O
NH
N
H
O
N
H
N
H
N
H
NH2
O
NH2
O
NH
NH2 NH
O
NH
NH2
NH
O
O
N
H
N
H
N
H
CH3 CH3
O
O
NH2
CH3CH3
O
NH
OH
O
OOH
tetracosactide
●used since 1961
●prepared by synthesis
●misused for doping in sport
Protirelin – synthetic thyreotropin-releasing hormone (TRH)
●a hormone sythetized in paraventricular nucleus of hypothalamus, stimulating release of
thyreotropin and prolactin from the anterior pituitary gland
●also neurotransmitter in CNS, takes part in food intake regulation, control of energy
metabolism etc.
N
O
NH2
O
N
H
N
O NH
N
H
O
protirelin
5-oxoprolyl-histidyl-prolinamide
Protirelinum EP
●structure elucidated 1969, used approx. 1976 – 1991, then abandoned
●administered p.o.
●used as cognitive functions enhacer for treatment of post-traumatic conditions in injuries
of brain and spinal cord and of neurodegeneration diseases (Alzheimer, Parkinson,
motoric neuronal disease etc.)
Metabolism of TRH and its regulation
Somatostatin
●cyclic tetradecapeptide formed namely in hypothalamus, but also in peripheral nervous
●system, the gut, and other organs
●inhibits pituitary growth hormone (somatotropin) release and probably also release of TRH,
prolactin,insulin and glucagon
●has impact to functions of kidneys, pancreas and GIT
●also acts as neurotransmitter in CNS („neuropeptide“)
NH
N
H
NH
O
O
N
H
N
H
O
O
N
H
N
H
O
CH3
OH
NH2
O
N
H
N
H
O
O
NH2
O
N
H
N
H
O
CH3OH
O
N
H
S
NH2
N
H
O
OH
S
O
N
H
OH O
O
CH3
NH2
somatostatin
Somatostatinum EP
Somatostatin Eumedica®
inf.
●prepared by synthesis
●treatment of acromegaly
1.2 Soma(to)tropin
= growth hormone (GH)
●peptide consisted of 191 AA secreted from anterior pituitary gland
●stimilates mitosis, growth and differentiation of cells of some tissues
●influences expression of genes and metabolism
●sequence of AA known since 1972, nucleotide sequence of the encoding gene since 1977
somatropin
Somatropinum EP
●human, prepared by recombinant technology, used since 1985
●substitution treatment of natural GH deficiency
Genotropin ® , Humatrope ® , Nutropinaq ® , Omnitrope ® ...
tertiary structure of porcine GH
1 MATGSRTSLL LAFGLLCLPW LQEGSAFPTI PLSRLFDNAM LRAHRLHQLA FDTYQEFEEA YIPKEQKYSF LQNPQTSLCF SESIPTPSNR EETQQKSNLE 100
101 LLRISLLLIQ SWLEPVQFLR SVFANSLVYG ASDSNVYDLL KDLEEGIQTL MGRLEDGSPR TGQIFKQTYS KFDTNSHNDD ALLKNYGLLY CFRKDMDKVE 200
201 TFLRIVQCRS VEGSCGF
Primary structure of human somatropin
Śomatropin (GH) analogues
pegvisomant
●analogue – antagonist of human GH, in which 9 AA are changed; which enables it to block
binding of native GH to its receptor by means of preventing receptor dimerisation
●pegylation is performed on 4 – 5 sites randomly selected from Phe1
and various 8 Lys residues
●prepared by the recombinant technology followed by a controlled reaction with oxiran
(polyadition) which results to covalent binding of 4 – 5 polyoxoethylene chains of Mr
∼ 500
●pegylation lowers antigenicity and prolongs the biologic half-time
●using: treatment of acromegaly
1.3 Oxytocin, vasopressins and their analogues
Vasotocin
= fylogenetic precursor of oxytocine and vasopressins in organisms lower than mammals
●prepared by synthesis
●used for triggering of the birth and enhancing of uterine contractions
Oxytocinum EP; Oxytocin Ferring-Léčiva ® inj. sol.
Oxytocin
● a cyclic nonapeptide released from the posterior pituitary gland (neurohypophysis)
●acts on smooth muscle cells, such as causing uterine contractions and milk ejection
N
O
N
H
O
NH
S
O
S
NHO
NH
N
H
O
NH2
O
NH2
NH2
O
N
H
N
H
O
NH2
NH
O
NH
NH2
O
O
CH3
CH3
OH
H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH2
Vasopressin(s)
=antidiuretic hormone(s) (ADH)
●octapeptides released from the neurohypophysis of all vertebrates (precursor synthetized in
hypothalamus)
●control body water content (regulation of kidneys, lungs etc.)
●potential neurotransmitters
●semi-synthetic derivatives used predominantly
N
O
N
H
O
NH
S
O
S
NHO
NH
N
H
O
NH2
O
NH2
NH2
O
N
H
N
H
O
NH2
NH
O
NH
NH2
O
O
OH
N
O
N
H
O
NH
S
O
S
NHO
NH
O
NH2
O
NH2
NH2
O
NH2
N
H
N
H
O
O
NH
NH2
O
O
OH
arginine-vasopressin
argipressin
●predominant form of mammalian ADH
lysine-vasopressin
lypressin
●Suidae family only
●treatment of diabetes insipidus and low blood pressure
Vasopressin analogues
Desmopressin
Desmopressinum EP
●cyclic pseudononapeptide
●prepared by synthesis
●antidiuretic (enuresis nocturna, ...)
Vaspressin analogues
Felypressin
Felypressinum EP
●vasoconstrictor with reduced antidiuretic activity
T
T
Terlipressin
NH2Pro
PheGly TyrCys
LysGln
GlyNH
2 Gly
Asn Cys Gly
H-Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2
●vasoconstrictor, treatment of variceal bleeding, circulation and septic shock
Glypressin ®
inj., Remestyp ®
inj.
1.4 Insulines, glucagon and GLP-1 analogues
Insuline
●
Secreted mostly by β-cells of Langerhans islets of pancreas
●Enables utilisation of glucose by cells of body
●First isolated by Banting and Best from dog´s pancreas in 1921
Human insuline
TP
C
COOHF
NS
H
Q N
ES C Y
Y
C
T
A
I
V TVL LG
E SV QI Y CC
KN L R GENH
2
L
F G
G L
FV
E
Q
C
H YL
NH
2
COOH
chain B (30 AA)
chain A (21 AA)
●formed from its precursor proinsuline consisted of 110 AA
10 20 30 40 50 60
MALWMRLLPL LALLALWGPD PAAAFVNQHL CGSHLVEALY LVCGERGFFY TPKTRREAED
70 80 90 100 110
LQVGQVELGG GPGAGSLQPL ALEGSLQKRG IVEQCCTSIC SLYQLENYCN
1-24 signal sequence; 25-54 chain B; 57-87 peptide C; 90-110 chain A
●today produced by recombinant technology, or by partial synthesis from
the porcine one
Insulinum humanum PhEur
●syn. humuline
Bovine (cow´s) insuline
Insulinum bovinum PhEur
●isolation from beef pancreases
Porcine (swine) insuline
Insulinum porcinum PhEur
Insuline analogues
human
aspart
Insulinum aspartum
PhEur
Novorapid ®
●recombinant technology
insulin-lispro
Insulinum lisprum PhEur
●recombinant
Humalog ®, Liprolog ®
insulin-detemir
●
chain B has only 29 AA, tetradecanoyl (myristoyl) attached to LysB29
●recombinant-semi synthetic
Levemir ®
insulin-glargin
Gly21A
-L-Arg30B
-L-Arg31B
-insulin
Lantus®
, Optisulin ®
●
insulin of 1st
choice in diabetes of 2nd
type when oral antidiabetics are not satisfactory
●long T½
, typically administered 1x daily s.c. before sleeping
insulin-glulisin
Apidra ®
Summary of the used insuline analogues
Glucagone
●peptid consisted of 29 AA from pancreas supporting cleavage of liver glycogene and increasing
glycaemia
●causes relaxation of smooth gastric muscules similarly to cholinergics
Glucagonum PhEur
●isolated from porcine or bovine pancreases
Glucagonum humanum PhEur
●produced by recombinant technology; AA sequence is identical
●usage: treament of serious hypoglycaemia, X-ray GIT diagnostic etc.
GLP-1 analogues
GLP-1: Glucagon-like peptide 1 = an intestinal hormone, which together with glucosedependent
insulinotropic polypeptide(GIP)* potentiates insulin secretion induced by food
●potetiates all steps of insulin biosynthesis; has positive impact to function and surviving of
β-cells
●decreases redundant glucose production in liver, slows down stomach emptying leading to
postprandial hypoglycaemia, its central effect leads to appetite decrease (⇒ body weight
loss), probably also positive effects to cardiovascular system
●disadvantages of GLP-1 as a drug: necessity of administration in a continual infusion,
extremely short biological half-time T1/2
= 2 – 3 min (fast decomposition by peptidases) ⇒
need of more stable analogues
*Both are known also as incretins.
GLP-1 analogues
liraglutide
Victoza ® inj. sol.
γ-L-glutamoyl(N-α-hexadecanoyl)-Lys26
, Arg34
-GLP-1(7–37)
●sequence of amino acid rests shares 97 % identity with the fragment 7-37
of the native GLP-1
●strong binding to serum albumin, mutual association of molecules, does not
come under glomerular filtration ⇒ T1/2
= 12.5 hours after s.c. injection
●
improves functions of both α and β cells
Calcitonin
●released from thyroidal C-cells ( = parafolicular cells – Baber 1876), in lower
vertebrates from ultimobranchial bodies, originated from 5th
branchial fissure
●peptide from 32 amino acid residues (salmon's – Onchorhyncus kisutch; human has
139 AA)
●receptors on osteoclasts (also in kidneys and brain)
●
↓ excretion of Ca2+
from the bone (⇒ ↓ calcaemia)
●
↓ osteoclasts formation
●used together with Ca2+
for treatment of osteoporosis
Calcitonin
N
N
N
H
N
H O
NH2
O
O
NH
N
H
N
H
N
H
N
H
S
S
N
H
NH2
O
O O
O
OH
O
OH
O
O
NH2
OH
N
H
N
HO
O
N
H
O
N
H
O
NH2
O N
H NH
O
OH
ONH
NH2
O
O
NH
O
N
H
O
N
H
N
H O
N
NH
O
NH2
O
N
H
OH
N
H
O
OH
O
O
N
H
O
NH
NH
NH2
O N
H
NH
OH
N
H
N
H
N
H
NH2
O
O
OH
O
O
OH
N
H
N
HO
O
OH
O
ONH2
OH
Calcitoninum salmonis EP = calcitonin salmon (synthetic; AA sequence
coresponds with salmon hormon)
Miacalcic®
inj., nasal; Osteodon®
; Tonocalcin®
2. Blood factors of erythropoetine type
erythropoietin
= glycosylated protein from 165 AA
Erythropoietini solutio concentrata EP
= a solution containing a group of closely related glycoproteins, which are not to distinguish
from the natural human erythopoietin (urine erythropoietin) from the point of view of 165
amino acids sequence and their average profile of glycosylation
●naturally released fromkidneys of adults and in liver of foetus
●stimulates stemcells of bone marrow to proliferation and differentiation
●produced in vitro in rodent cell lines by a method based on the recombinant DNA
technology
●treatment of haematopoietic disorders, misused for doping
Mr
about 30 600
3. Colony stimulating factors
molgramostim
= a factor stimulating granulocytes and macropfages coloies released from various kinds
of blood cells
●not glycosylated
●stimulates differentiation and proliferation of leukocyte pluripotent stem cells into matured
granulocytes and macrophages
●production by a recombinant technology using bacteria as host cells
● treatment of leukopenia in cancer chemotherapy or HIV infections
Filgrastim and pegfilgrastim
Filgrastim = human granulocytes colony-stimulating factor (G-CSF); glycosylated, 174 AA
Sequence of filgrastim precursor
●
treatment of neutropenia in cancer chemotheapy and in AIDS
Pegfilgrastim has covalently attached PEG chain of Mr
cca 20 000 on N-terminus
●longer elimination half-time
●recombinant and semi-synthetic production
4. Non-specific antibodies - interferons
interferon α2
Interferoni alfa-2 solutio concentrata EP
X1 = Lys α2a
X1 = Argα2b
●antiviral activity during viral RNA and protein syntheses
●antiproliferation activity
●produced by a recombinant technology on bacteria
Pegylated interferons α
● peginterferon α2a
(Pegasys ® ) - on some Lys residues attached N2
, N6
-dicarboxyLys
esterified with PEG-monomethylether of Mr about 20 000
– substitution is stable, free interferon is not released
– peginterferon α2a
interacts directly with receptors on surface of the infected
cell
– lowered activity (only 7 % of free interferon α2a
) is counterbalanced by
much longer half-time
– treatment of hepatitis B and C combined with ribavirin
● peginterferon α2b
(Pegintron ® ) - only one PEG chain of Mr
about 12 000 attached
via urethane linker to a His, most frequently to His34
– urethane moiety is labile, free interferon α2b
is released into the circulation
and directly interacts with receptors
– treatment of hepatitic C
Pegylated interferons α
Differences in their substitutions
n
n
O
O
CH3
O
O
CH3
O
NH
NH
NH
O
O
NH
O
n
O
O
CH3
NH
N
N
O
O
His34
Lys
α2a
α2b
interferon β
= a glycosylated peptide consisted of 166 AA
●produced by fibroblasts in response to stimulation by a living or inactivated virus or doublestrained
RNA
●treatment of multiple sclerosis
Variants of interferon β
● β1a
(Avonex ® , Betaferon ® , Rebif ® )
– Mr
cca 20 000
– prepared by a recombinant technology on Chinese hamster ovary cell lines
– preparations are not equally active probably due to different glycosylation
– recommended i.m. application once weekly
● injected s.c. is much more painfull than β1b
● β1b
(Extavia ® )
– Cys17
changed to Ser
– recombinant technology on E. coli
– s.c. application every other day
interferon γ1b
●released by human T-lymfocytes in response to viral infections and other agents
●imunomodulatory effects
●non-covalent dimer of 2 identicas monomers consisted of 141 AA
Sequence of the monomer:
●production by recombinant technology on bacteria
●supporting treatment of idiopatic lung fibrosis; only increases the hope of patients live to see
lungs transplantation