Paruch Medicinal Chemistry C9115Drug metabolism
Roskoski, J. Pharmacol. Res. 2016, 107, 249.
• Metabolic breakdown of drugs by living organisms, usually through specialized enzymatic systems.
Functional outcomes of drug metabolism:
• Inactivation and accelerated elimination of drugs.
• Activation of prodrugs.
• Formation of active metabolites with similar or novel activity.
• Detoxification of toxic xenobiotics
• Toxification of non-toxic xenobiotics.
Drug
(liphophilic)
Enzyme Metabolite
(polar)
Excretion
• It may lead to the formation of inactive and non-toxic compounds, hence the term detoxification.
• Some studies have shown that some metabolites are not only active, but may be toxic.
Example:
Igarashi, K. Life Sci. 1995, 57, 2439.
Sampson, D. Bioorg. Med. Chem. Lett. 2014, 24, 4294.
Paruch Medicinal Chemistry C9115Drug metabolism
Site of drug metabolism:
1. Organ sites
• Liver : major site, most drug metabolizing enzymes.
- orally administered drugs first pass through the liver and thus are susceptible to firstpass
effect. This can lead to lower bioavailability.
- For example, Lidocaine is inactive when given orally due to the first-pass effect.
• Other sites : intestine, kidneys, lungs, skin, placenta, brain, adrenal glands.
2. Cellular sites: cytosol, mitochondria, lysosomes, smooth endoplasmic reticulum.
Paruch Medicinal Chemistry C9115Drug metabolism
Categories of drug metabolism reactions:
1. Phase I reactions (modification):
• oxidation, reduction, hydrolysis, cyclization, decyclization.
• which converts parent drug to more polar metabolite by introducing or unmasking a functional
groups, e.g.: –OH, -COOH, -NH2.
• usually involve enzymes like cytochrome P450 monooxygenase, flavin-containing monooxygenase,
alcohol dehydrogenase, aldehyde dehydrogenase, monoamine oxidase, peroxidases.
(a). Oxidation: may occur at several centres in drugs.
Oxidation at carbon centre: includes oxidation at aromatic ring, olefinic centre, and aliphatic groups
Paruch Medicinal Chemistry C9115Drug metabolism
Oxidation at carbon-heteroatom systems:
- involves reaction on C–N, C–S and C–O systems.
- The oxidation reactions on C–N systems comprise of N-dealkylation, oxidative deamination,
formation of N-oxide, or N-hydroxylation.
- The reactions in C–S systems may involve S-dealkylation, desulfuration and S-oxidation.
- O-dealkylation, oxidative dehalogenation and oxidative aromatization are other important
oxidation reactions at carbon centre.
Paruch Medicinal Chemistry C9115Drug metabolism
(b). Reduction:
• can take place in aliphatic and aromatic aldehydes and ketones.
• Drugs such as methadone (analgesic), chloral hydrate (sedative and hypnotic) and naltrexone
(management of alcohol dependence) undergo this metabolic process.
• The N-containing compounds having nitro, azo or N-oxide undergo this metabolic reaction. For
example: nitrazepam (hypnotic and anxiolytic) and prontosil (antibacterial antibiotic), which get
reduced to the corresponding amines.
• The halogen atom present in various drugs may undergo reduction via replacement by H-atom, e.g.,
halothane.
• usually involve enzymes like NADPH-cytochrome P450 reductase, reduced (ferrous) cytochrome
P450.
Paruch Medicinal Chemistry C9115Drug metabolism
(c). Hydrolysis:
• esters are administered as prodrugs, which on hydrolysis are converted to active forms, e.g.,
aspirin (analgesic, antipyretic).
• drug molecules containing amide functionality undergo slow hydrolysis as compared to esters.
• The reaction occurs in secondary and tertiary amides, and rarely in primary amides, e.g.,
procainamide (antiarrhythmic).
• usually involve enzymes like esterases, amidase, and epoxide hydrolase.
Paruch Medicinal Chemistry C9115Drug metabolism
2. Phase II reactions (conjugation): combination type reaction. e.g. A + B = AB.
- which attaches polar and ionizable endogenous groups to achieve complete solubility, e.g.:
sulfate, acetate, amino acid.
- sites on drugs where conjugation reactions occur include carboxy (-COOH), hydroxy (-OH),
amino (NH2), and thiol (-SH) groups.
- It can also lower or terminate biological activity.
- These reactions are catalyzed by a variety of transferase enzymes, such as uridine
diphosphate (UDP)-glucoronsyltransferases, sulfotransferases, glutathione transferases.
- In these reactions, a suitable moiety such as glucuronic acid, glutathione, sulphate, glycine,
etc., get conjugated to the metabolites of Phase I reaction.
- An example of glutathione conjugation as the Phase II metabolic reaction in the body.
Paruch Medicinal Chemistry C9115Drug metabolism
The conjugation reactions along with the enzymes involved and the examples of drugs
metabolized by the same pathway.
Paruch Medicinal Chemistry C9115Drug metabolism
3. Phase III reactions (further modification and excretion):
• further metabolism of xenobiotic conjugate after phase II reactions.
Paruch Medicinal Chemistry C9115Drug metabolism
Cytochrome P450 enzyme system (CYP450)
Meunier, B. Chem. Revi 2004, 104, 3947.
• Superfamily of enzymes
• contain a heme prosthetic group, where heme group is the iron-porphyrin unit.
• It oxidizes hydrophobic compounds to hydrophilic or more polar metabolites for subsequent
excretion.
• are membrane-bound proteins, present in the smooth endoplasmic reticulum of liver and other
tissues.
• CYPs catalyze the transfer of one atom of oxygen to a substrate producing an oxidised substrate
along with a molecule of water.
• Cytochromes P450 have been named on the basis of their cellular (cyto) location and
spectrophotometric characteristics (chrome).
• Reduced heme iron combines with CO, P450 enzymes absorb light at wavelengths near 450 nm
(Soret peak)
Paruch Medicinal Chemistry C9115Drug metabolism
• The detailed mechanism of metabolism by CYPs has been described in the catalytic cycle
Paruch Medicinal Chemistry C9115Drug metabolism
• There are more than 300 different CYP enzymes, which have been grouped into several families
and subfamilies based on the amino-acid sequence.
• Out of these, 18 CYP families have been identified in mammals, comprising majorly of families
CYP1, CYP2 and CYP3.
• Fraction of clinically used drugs (248 drugs) metabolized by P450 isoforms and factors influencing
variability.
Zanger, U. M. Pharmacol Thera 2013, 138, 103.
Paruch Medicinal Chemistry C9115Drug metabolism
P450 enzyme classification:
• In human around 30 CYP enzymes which are responsible for drug metabolism.
• however, that 90% of drug oxidation can be attributed to six main enzymes: CYP 1A2, 2C9, 2C19,
2D6, 2E1 and 3A4.
• CYP450 3A4 is involved in the metabolism of many different drugs (almost 50% of those on the
market).
• The most significant CYP isoenzymes in terms of quantity are CYP3A4 and CYP2D6.
• CYP450 2E1 is involved in the oxidation of small molecules such as ethanol and chlorzoxazone.
• In humans - Central role in phase I drug metabolism
- Significant problems in clinical pharmacology
- Drug interactions
Paruch Medicinal Chemistry C9115Drug metabolism
Biotransformations Leading to Toxic Metabolites: Chemical Aspect
• Differential metabolism of drugs, where one istoxic and the other non-toxic, representing
structure–toxicity relationship.
Paruch Medicinal Chemistry C9115Drug metabolism
Biotransformations Leading to Toxic Metabolites: Chemical Aspect
Paruch Medicinal Chemistry C9115Drug metabolism
Biotransformations Leading to Toxic Metabolites: Chemical Aspect
Paruch Medicinal Chemistry C9115Microsomal stability
Background Information
• Hepatic metabolic stability is a key parameter in drug discovery.
• It is essential to identify metabolic liabilities early in drug discovery so they can be addressed
during lead optimization.
• Both in vitro half-life (t1/2), intrinsic clearance (CLint) or compound hepatic clearance (CLH) are
utilized to express metabolic stability.
• Metabolic stability is typically first measured in vitro using liver microsomes and data from this
assay is used to guide structural modifications to improve stability or select the best compounds
for in vivo pharmacokinetic (PK) and efficacy testing.
• Liver microsomes are enriched with CYP450 enzymes, localized in the endoplasmic reticulum
membrane, which are responsible for the metabolism of the majority (70–80%) of clinically
approved drugs.
• The metabolic stability assays offer a method to calculate the rate of clearance of a test
compound over time in microsomal incubations, and these data are used to evaluate intrinsic
clearance.
Paruch Medicinal Chemistry C9115Microsomal stability
benefits of using liver microsomes for drug metabolism studies:
• The liver is the main organ of drug metabolism in the body.
• Subcellular fractions such as liver microsomes are useful in vitro models of hepatic clearance as
they contain many of the drug metabolising enzymes found in the liver.
• Microsomes are easy to prepare and can be stored for long periods of time.
• They are easily adaptable to high throughput screens which enable large numbers of compounds to
be screened rapidly and inexpensively.
Paruch Medicinal Chemistry C9115Microsomal stability
overview of microsomal stability assay:
• The microsomes are incubated with the test compound at 37°C in the presence of the co-factor,
NADPH, which initiates the reaction.
• The reaction is terminated by the addition of 90% acetonitrile-water containing internal standard.
• Following centrifugation, the supernatant is analysed on the LC-MS/MS.
• The disappearance of test compound is monitored at 5-different time points over a 40 minute time
period.
• An example of a typical depletion profile is shown in Figure below.
• The elimination constant (kel), half-life (t1/2) and intrinsic clearance (Clint) were determined in plot
of ln(AUC) versus time, using linear regression analysis.
Paruch Medicinal Chemistry C9115Microsomal stability
Interpretation of microsomal stability assay data:
• The test compounds can be classified in terms of their microsomal stability into low, medium and
high clearance groups.
• The intrinsic clearance classification bands for mouse, rat, and human species are calculated
according to the well stirred model equation:
where CLH is a hepatic clearance (mL/min/kg), CLH = E x QH
QH = liver blood flow (mL/min/kg)
E = extraction ratio, assumed at 0.3 for low clearance and at 0.7 for high clearance compounds
fu = fraction unbound in plasma, assumed at 1.
Table : classification bands typically used for categorising compounds into low, medium or high clearance.
Houston J.B. Biochemical Pharmacology 1994, 47, 1469; Barter Z.E. Current Drug Metabolism 2007, 8, 33;
Iwatsubo T. Journal of Pharmacology and Experimental Therapeutics 1997, 283, 462.
Paruch Medicinal Chemistry C9115Microsomal stability
Example of mouse microsomal stability for reference and test compound:
Compound ID
Incubation
time
(min)
Microsomal
proteins
(mg/ml)
Compound
concentration
(µM) % Remaining
% Remaining
without
cofactor R
kel ,
(min-1)
t1/2
min
Clint
(µl/min/
mg)
MU1656 40 0.415 2 41 112 0.997 0.023 29.8 56
Pinometostat 40 0.415 2 25 106 0.992 0.036 19.0 88
Paruch Medicinal Chemistry C9115
• Strategies to enhance metabolic stability
- Deactivation of aromatic rings to facilitate oxidation through substitution with strongly electronwithdrawing
groups (e.g. CF3, SO2NH2)
- Introduction of an N-t-butyl group to prevent N-dealkylation.
- Replacement of a labile ester linkage with an amide group.
- Constraining the molecule in a conformation that is unfavorable to the metabolic pathway, more
typically, protecting the labile moiety by steric shielding.
Microsomal stability
Paruch Medicinal Chemistry C9115Microsomal stability
Nassar, A. F. Drug Discovery Today 2004, 9, 1020.
Paruch Medicinal Chemistry C9115Microsomal stability
Nassar, A. F. Drug Discov Today 2004, 9, 1020.
Paruch Medicinal Chemistry C9115Microsomal stability
Nassar, A. F. Drug Discovery Today 2004, 9, 1020.
Paruch Medicinal Chemistry C9115
improvement of metabolic stability via incorporation of Fluorine
• the relatively small size of the fluorine atom (vander Waals radius of 1.47 A˚ ), comparable to
hydrogen (van der Waals radius of 1.20 A˚).
• the highly electron withdrawing property of fluorine.
• greater stability of the C–F bond compared to the C–H bond.
• greater lipophilicity of fluorine compared to hydrogen.
Microsomal stability
Insertion of fluorine atoms into molecules can
• Improved metabolic stability
• Altered physicochemical properties
• Increased binding affinity
Example : Ezetimibe (SCH 58235)
- approved in 2002 by the FDA to reduce
cholesterol levels in patients with
hypercholesterolaemia.
- developed from the compound SCH 48461, which
is susceptible to metabolic attack in four
primary sites via demethylation, hydroxylation,
and/or oxidation.
Paruch Medicinal Chemistry C9115
Fluorine substitution to extend the biological half life
• The prostanoids, short half-lives in vivo of less than 5 minutes.
• The platelet-aggregating agent, thromboxane A2 (TxA2) has an unusual oxetane acetal structure
that undergoes hydrolytic cleavage at pH 7.4 and has a half-life of around 30 seconds.
• By introducing fluorine into the oxetane ring (7,7-difluoro-TxA2) the rate of hydrolysis is 108fold
slower than TxA2.
Microsomal stability
• Prostacyclin an inhibitor of platelet aggregation contains an acid labile enol-ether group that is
responsible for its short half-life.
• By introducing a fluorine atom a to the enol-ether group, the electron density on the enol-ether
group is reduced via its inductive effects, which improves the stability of the molecule to acid
hydrolysis.
Paruch Medicinal Chemistry C9115Microsomal stability
improvement of metabolic stability via incorporation of Deuterium
• Selective replacement of hydrogen with deuterium leads to increased bond strength which in
turn increases the biological half-life and thus metabolic stability of the drug.
• Metabolic shunting resulting in reduced exposure to undesirable metabolites or increased
exposure to desired active metabolites.
• Reduced systemic clearance resulting in increased half-life.
• Decreased pre-systemic metabolism resulting in higher bioavailability of unmetabolized drug.
Harbeson, S. Med-Chem News 2014, 24, 8. Sukhninder, K. Glob J Pharma Sci. 2017, 1, 555566.
Paruch Medicinal Chemistry C9115Microsomal stability
1. CTP-499: A deuterated pentoxifylline (Trental)
• Pentoxifylline, an active metabolite of Trental, is an effective agent for the treatment of
nephropathy in Type-II diabetic patients.
• It is significantly metabolized to HDX 1.
• The deuterated version of 1, i.e., CTP-499 (2) has showed enhanced metabolic profile of the
compound.
• Phase II study showed a delay of end stage of renal failure, suggesting CTP-499 can be used
for type 2 diabetes and chronic kidney disease.
Sabounjian, L. Clin Pharmacol Drug Dev 2016, 5, 314.
Paruch Medicinal Chemistry C9115Microsomal stability
2. Fludalanine
• developed by Merck, one of the earliest deuterated drug candidates to enter the clinic.
• The combination of 22 with cycloserine provides a broadspectrum and potent antibacterial.
• The hydrogen analog is also an effective antibacterial, but preclinical studies reportedly
demonstrated that 23 was metabolized to form 3-fluorolactate 24 a toxin that caused brain
vacuolization, which is in equilibrium with 3-fluoropyruvate 23 during metabolism.
• The deuterated analog 22 showed reduced level of 3-fluorolactate 24 production in healthy
volunteers.
• However, higher levels of 3-fluorolactate 24 were observed in patients; therefore, studies on
fludalanine were discontinued at Phase IIb.
Kahan, F. Chem Engin News 2009, 87.
Paruch Medicinal Chemistry C9115Microsomal stability
3. CTP-347
• CTP-347, a selectively deuterated analogue of paroxetine a centrally acting SSRI (selective
serotonin reuptake inhibitor) for the treatment of major depressive disorder, panic disorder,
social anxiety disorder.
• Low doses of 26 have been reported to have good efficacy in treating hot flashes.
• However, 26 is not only metabolized by CYP2D6 but also potently inhibits its own metabolism
by irreversibly inactivating that enzyme.
• Its use, therefore, can be complicated in patients potentially benefiting from this agent due
to possible drug-drug interactions with other drugs metabolized by CYP2D6. In the case of
thioridazine, coadministration of paroxetine is contraindicated.
Uttamsingh V. J. Pharmacol. Exp. Ther 2015, 354, 43.
Paruch Medicinal Chemistry C9115Microsomal stability
4. BMT-052
• the development of second generation pan-genotypic inhibitor BMS-986139 halted due to
unexpected microcrystallization in multiple tissues at elevated doses in both rats and dogs in
investigational new drug (IND) toxicology studies.
• through iterative SAR studies and systematically incorporating deuterium into both the C5
and amide substituents, the promising preclinical compound 14 was identified.
• Compound 14 exhibited low clearance (Cl), a moderate volume of distribution (Vss), and good
oral bioavailability (F%) across the species.
• Compound 14 had an improved solubility and overcomes the micro crystallization problems
observed during the IND toxicology studies of BMS-986139.
Parcella, K. ACS Med. Chem. Lett. 2017, 8, 771.
Paruch Medicinal Chemistry C9115Pharmacokinetics
• defined as the study of the quantitative relationship between administered doses of a drug and
the observed plasma/blood or tissue concentrations.
• pharmacokinetics is concerned with the drug ADME - absorption, distribution, metabolism, and
excretion or elimination.
• When should they be determined? – early in the process
• Drug delivery routes
1. Enteral routes : delivers the compound into the body through the gastrointestinal (GI) tract.
- oral (PO), sublingual, rectal.
2. Parental routes : administration can be performed by injection.
- intramuscular (IM), subcutaneous (SC) and
intravenous (IV) etc.
Paruch Medicinal Chemistry C9115Pharmacokinetics
Absorption (will my drug get into the body??)
• To be in solution compound must be fully hydrated (surrounded by water).
• The greasier your compound, the less it likes to be hydrated.
• The more polar, the more aqueous soluble.
H
O H
H
O
H
H
O H
O
H
H
O H
H
O
H
N
N
CF3
S
O
O
N
H
O H
H
H
H
O H
H
H
O
H H
O
H
H
O
H
H
O
H
H
H
HH O
H
H O
H
H
H
O
H
H
O
H
Absorption pathways
• Most compounds absorbed by transcellular route.
• It is possible for molecules to be absorbed between the cells of the gut wall (paracellular
absorption), but this is relatively rare.
Paruch Medicinal Chemistry C9115Pharmacokinetics
Paruch Medicinal Chemistry C9115Pharmacokinetics
Pharmacokinetic parameters:
1. Volume of distribution (Vd)
2. Clearance
3. half-Life
4. Bioavailability
5. Bioequivalence (BE)
Paruch Medicinal Chemistry C9115Volume of distribution
• pharmacokinetic parameter representing an individual drug’s propensity to either remain in the
plasma or redistribute to other tissue compartments.
• Vd is a proportionality constant that relates the total amount of drug in the body to the plasma
concentration of the drug at a given time.
• Volume of Distribution (L) = Amount of drug in the body (mg) / Plasma concentration of drug
(mg/L).
• It is a major determinant of half-life and dosing frequency of a drug.
Smith, D. A. J. Med. Chem. 2015, 58, 5691.
• The volume of distribution values for small molecule drugs should be compared to the
physiological tissue volumes.
Physiological volumes of body fluids in
humans.
Paruch Medicinal Chemistry C9115Volume of distribution
• the most calculated parameter is volume of distribution at steady state (Vss).
• it represents the apparent distribution volume associated with the steady-state dosing paradigm
under which most drugs are developed.
• the volume of the central compartment (Vc) represents the apparent volume of distribution
immediately following an intravenous bolus dose.
• Vβ can be derived from the terminal elimination phase half-life.
• The volume of distribution values for small molecule drugs should be compared to the physiological
tissue volumes.
Smith, D. A. J. Med. Chem. 2015, 58, 5691.
Definition of the different volume of
distribution terms: Vc, Vss, and Vβ.
Classification of steady state volume
of distribution (Vss, L/kg).
species low moderate high very
high
all <0.6 0.6−5 5−100 >100
Paruch Medicinal Chemistry C9115Volume of distribution
• Vss affects half-life and duration of action. For compounds with equal daily dose, the compound
with lower Vss (shorter half-life) may need to be dosed more frequently (at lower individual doses)
to achieve a similar pharmacodynamic profile as the one with higher Vss.
• Large Vss does not indicate that drugs will reach the remote sites of action and be
pharmacologically active. Similarly, low Vss does not mean that the compound will not reach the
remote site of action.
• High Vss and high total tissue concentration do not necessarily translate to better activity for
disease targets in the tissues.
• Transporters, pH gradients, and electrochemical potential scan impact Vss, but the magnitude is
usually small. Binding typically plays a dominant role in determining Vss.
• Structure modification can be applied to increase/decrease Vss and duration of action while
maintaining clearance. Achieving this is not straightforward and requires well-directed strategies
and design.
• The major approaches to modulate Vss are introducing basic functional groups and increasing
lipophilicity in a way that does not increase unbound intrinsic clearance.
• Acidic compounds will need to have very low intrinsic clearance to have long duration of action
because their Vss values are typically small.
Smith, D. A. J. Med. Chem. 2015, 58, 5691.
Paruch Medicinal Chemistry C9115Clearance
• It quantitates the irreversible removal of a drug from the measured matrix (blood or plasma).
• clearance parameter (CL):
rate of elimination = CL × concentration
• Units: CL = volume/time.
• Clearance = rate at which a drug is removed from plasma (mg/min)
concentration of that drug in the plasma (mg/mL)
Smith, D. A. J. Med. Chem. 2019, 62, 2245.
• While CL is typically constant, the rate of drug
elimination is concentration dependent.
• measurement of the concentration of drug in plasma
over a time course, the area under the plasma
concentration versus time course (AUC) can be
calculated.
CL = dose/AUC
• The relationship between CLb and CLp is: CLb = CLp/Rb
where CLb = blood clearance
CLp = plasma clearance
Rb = blood-to-plasma ratio
iv route
Paruch Medicinal Chemistry C9115Clearance
Introduction:
• The route of drug administration is critical for clearance assessment.
• iv route : all of the administered dose reaches the systemic circulation (blood).
• po route: potential barriers, where the dose is applied to the gastrointestinal (gi) tract and has to
be absorbed and avoid first pass extraction by the gut wall and liver prior to reaching the
systemic circulation.
• The AUC following po is often lower than that following iv of the same dose due to incomplete
absorption, first-pass intestinal extraction, and/or hepatic extraction.
Smith, D. A. J. Med. Chem. 2019, 62, 2245.
• Clearance following po (CLpo) is most often higher
than CLs.
• The
• relationship between oral clearance and systemic
clearance relies on an understanding of oral
bioavailability (F):
CLpo = CLs/F
Paruch Medicinal Chemistry C9115Clearance
Importance of clearance in drug disposition:
(a) Effect of clearance on half-life:
• Together with volume of distribution (Vd), clearance governs the elimination rate (kel) of a drug,
and ultimately half-life (t1/2).
• approximation of half-life from Vd and CL:
CL ≅ Vd × kel
t1/2 ≅ ln 2 × Vd/CL
(b) Effect of clearance on oral bioavailability:
• During absorption, drugs are exposed with metabolizing enzymes in the GI tract.
• the liver can also extract a portion of the administered dose by metabolism and/or transport due
to the fact that drugs directly enter the liver via the hepatic portal vein before reaching systemic
circulation. This process is termed hepatic first-pass extraction.
• the hepatic extraction is a key component of both oral bioavailability and clearance.
• minimizing hepatic extraction by lowering clearance is a key component in optimizing oral
bioavailability.
F = Fa × Fg × (1 − Eh) where, F = fraction bioavailable, Fa = fraction absorbed
Eh = CL/Q Fg = fraction escaping gut wall extraction
Eh = hepatic extraction ratio, Q = hepatic blood flow
Smith, D. A. J. Med. Chem. 2019, 62, 2245.
Paruch Medicinal Chemistry C9115Clearance
(c) Effect of clearance on efficacious dose.:
• The importance of clearance in the relationship between steady state concentration and dose was
recognized early by Wagner in 1965.
• modulation of clearance has a direct effect on efficacious dose within a particular chemical series.
dose = Css,av × CL × τ/F
where Css,av = average concentration at steady state (related to potency and target concentration)
τ = dose interval.
Smith, D. A. J. Med. Chem. 2019, 62, 2245.
Paruch Medicinal Chemistry C9115half-Life
• Relevance of half-life (t1/2) in drug design:
• t1/2 is defined as the time required for the concentration of a drug (typically in blood or plasma) to
reduce to half of its initial value when the concentrations of the drug are in simple exponential
(log−linear) decline.
• A half-life of 12−48 h is generally ideal for once daily dosing of oral drugs.
• If the half-life is too short, it may require more frequent dosing in order to maintain desired
exposures and avoid unnecessarily high peak concentrations.
• When considering a design effort to optimize t1/2, there are several key questions that need to be
understood, namely:
- What is the optimal t1/2 to support once daily administration?
- How can t1/2 be modulated?
- What are the important considerations in measuring t1/2 during compound optimization?
Smith, D. A. J. Med. Chem. 2018, 61, 4273; Gunaydin, H. ACS Med. Chem. Lett. 2018, 9, 528.
first order absorption and
elimination rates.
Ka = first order rate constant of absorption.
Kel = first order rate constant of elimination.
Paruch Medicinal Chemistry C9115half-Life
• the characteristic exponential decline following iv bolus administrations.
Smith, D. A. J. Med. Chem. 2018, 61, 4273; Gunaydin, H. ACS Med. Chem. Lett. 2018, 9, 528.
• absorptive phase followed by monoexponential decline following oral administration.
• Drug is eliminated via another first order rate constant (kel) which determines the t1/2 of the drug
under conditions in which the rate of absorption is more rapid than the rate of elimination.
• kel can be estimated from the slope obtained after plotting the log10 of concentration versus time.
• The kel parameter determines the t1/2 of a drug according to :
t1/2 = ln(2)/kel
Paruch Medicinal Chemistry C9115half-Life
relation between half-life, volume of distribution and clearance:
t1/2 = ln(2)Vss/CL
• t1/2 will be proportional to volume and inversely proportional to clearance.
Smith, D. A. J. Med. Chem. 2018, 61, 4273; Gunaydin, H. ACS Med. Chem. Lett. 2018, 9, 528.
• How can t1/2 be modulated?
• reducing plasma CL (e.g., increase metabolic stability, reduce renal clearance, reduce hepatic
uptake by active transporters and biliary clearance) and/or second increasing Vss.
• modulation of plasma protein binding in the absence of changing CL or Vss will not affect t1/2.
• changing fraction unbound in plasma will often change CL and Vss in equal and opposite directions.
• it is also possible to improve the t1/2 by modifying the delivery of the compound (formulation)
depending upon aqueous solubility, high membrane permeability, and low efflux transport.
Paruch Medicinal Chemistry C9115half-Life
Smith, D. A. J. Med. Chem. 2018, 61, 4273; Gunaydin, H. ACS Med. Chem. Lett. 2018, 9, 528.
Matched molecular pairs (MMP):
• are pairs of molecules that differ by a single chemical transformation and are used to relate the
changes in measured activities to changes in chemical structures.
• Examples of MMPs that show the change in half-lives upon changing the lipophilicity of the
molecules.
Paruch Medicinal Chemistry C9115Bioavailability
• the fraction (percentage) of an administered drug that reaches the systemic circulation (blood).
• The route of administration and the dose of a drug have a significant impact on both the rate and
extent of bioavailability.
• to have a therapeutic effect, the active substance
- has to enter the body.
- needs to be available in the correct dose at the specific site (target site).
- needs to reach the target site within a certain time.
- needs to available there for a defined time.
• Tmax = time where the highest concentration
of the medicine is found in the blood.
• Cmax = maximum concentration of the medicine
found in the blood.
• When giving an injection directly into the bloodstream, i.e. an intravenous injection (IV), the
bioavailability is defined as 100%.
Paruch Medicinal Chemistry C9115Bioavailability
Oral bioavailability:
• Lower bioavailability can be the result of poor or no absorption from the stomach and the
intestines.
• When the active substance is absorbed, it reaches the hepatic portal vein first, and is
transported to the liver. This is the first time the active substance is metabolised in the liver,
referred to as the ‘first pass metabolism’.
• The non-metabolised part of the active substance, normally less than 100%, will reach systematic
circulation via the hepatic vein. The amount that actually reaches systemic circulation is referred
to as the ‘absolute bioavailability’.
Paruch Medicinal Chemistry C9115Bioequivalence
• the property wherein two drugs with identical active ingredients or two different dosage forms of
the same drug possess similar bioavailability and produce the same effect at the site of
physiological activity.
• If two drugs are bioequivalent, there is no clinically significant difference in their bioavailability.
• Bioequivalence implies that different drugs release their active ingredient in the equivalent dose,
rate of absorption, and quality.
A bioequivalency profile comparison of 150 mg
extended-release bupropion as produced by
Impax Laboratories for Teva and Biovail for
GlaxoSmithKline.
• bioequivalence studies are also performed for
brand name drugs in some situations such as:
- early and late clinical trial formulations.
- between the formulations used in clinical trials
and the product to be marketed for new
medicines.
- changes in formulation have occurred after a
brand name drug has been approved.
Paruch Medicinal Chemistry C9115Genotoxicity Testing
Why genotoxicity assessment of drugs?
• for safety assessment of drugs/products.
General purpose:
• detect compounds which induce genetic damage directly or indirectly by various mechanisms.
• positive compounds may induce cancer and/or heritable defects.
A standard genotoxicity testing includes:
• AMES test – a test for gene mutations in bacteria
• micronucleus test - chromosomal fragment in cytoplasma of erythrocytes or binuclear cells,
lymphocytes.
• Cytogenetic analysis of bone marrow cells of experimental animals or of human peripheral
lymphocytes - chromosomal breaks or rearrangements.
• Comet assay – single strand DNA breaks.
Paruch Medicinal Chemistry C9115Genotoxicity Testing
Ames test:
• Ames test is developed by Bruce N. Ames in 1970s to test for determining if the chemical is
mutagens.
• based on the principle of reverse mutation or back mutation. So, the test is also known as bacterial
reverse mutation assay.
• can detects suitable mutants in large population of bacteria with high sensitivity.
Test organism:
• Ames test uses several strains of bacteria (Salmonella, E.coli) that carry mutation.
e.g. - A particular strain of Salmonella Typhimurium carry mutation in gene that encodes histidine.
• So it is an auxotrophic mutant which loss the ability to synthesize histidine utilizing the
ingredients of culture media. Those strains are known as His- and require histidine in growth
media.
• Culturing His- salmonella is in a media containing certain chemicals, causes mutation in histidine
encoding gene, such that they regain the ability to synthesize histidine (His+).
• This is the reverse mutation. Such chemicals responsible to revert the mutation is actually a
mutagen. So, this Ames test is used to test mutagenic ability of varieties of chemicals.
Mortelmans, K. Mutation Research 2000, 455, 29.
Paruch Medicinal Chemistry C9115Genotoxicity Testing
General procedure:
• The bacteria are spread on an agar plate with small amount of histidine.
• This small amount of histidine in the growth medium allows the bacteria to grow for an initial time
and have the opportunity to mutate.
• When the histidine is depleted only bacteria that have mutated to gain the ability to produce its
own histidine will survive.
• The plate is incubated for 48 hours.
• The mutagenicity of a substance is proportional to the number of colonies observed.
Paruch Medicinal Chemistry C9115Genotoxicity Testing
Limitations:
• Salmonella typhimurium is a prokaryote, therefore it is not a perfect model for humans.
• Rat liver S9 fraction is used to mimic the mammalian metabolic conditions so that the mutagenic
potential of metabolites formed by a parent molecule in the hepatic system can be assessed;
however, there are differences in metabolism between humans and rats that can affect the
mutagenicity of the chemicals being tested.
• the use of human liver S9 fraction; its use was previously limited by its availability, but it is now
available commercially and therefore may be more feasible.
• Mutagens identified in the Ames test need not necessarily be carcinogenic, and further tests are
required for any potential carcinogen identified in the test.
- e.g. drugs that contain the nitrate moiety sometimes come back positive for Ames when they are
indeed safe. The nitrate compounds may generate nitric oxide, an important signal molecule that can
give a false positive.
Paruch Medicinal Chemistry C9115Genotoxicity Testing
Micronucleus test:
• micronucleus formation resulting from agents that cause chromosomal damage is a hallmark of
genotoxicity.
• to determine if a compound is genotoxic to cells in culture by evaluating the presence of
micronuclei.
• simple, reliable and reproducible.
• performed in erythrocytes generally, but currently its use has been extended to other tissues like
liver, lung, skin etc.
• A mutagenic effect of tested compound is presented by increased number of micronuclei in 1000
of cells in comparison with control.
• There are two major versions of this test, in vivo and in vitro.
Micronuclei Characteristics:
• micronuclei are morphologically identical to the main nuclei, but are
smaller than it.
• it is not linked or connected to the main nuclei.
• it may touch but do not overlap the main nuclei.
• it is non-retractile and they can therefore be readily distinguished
from staining particles.
Paruch Medicinal Chemistry C9115Genotoxicity Testing
In vitro micronucleus test:
• a recommended regulatory test for genotoxicity that is capable of detecting the micronuclei
formation resulting from clastogens or aneugens.
• Clastogens - agents that induce chromosomal breaks mainly through interaction with the DNA
• Aneugens - agents that induce chromosomal loss mainly through interference with the spindle
apparatus.
• Cultures are incubated with several concentrations of the test compound for three to four hours
in the presence and absence of metabolic activation (S9) and for 21 to 24 hours in the absence of
S9.
Paruch Medicinal Chemistry C9115Genotoxicity Testing
In vitro micronucleus test:
• Micronuclei frequency with cyclophosphamide treatment: Cyclophosphamide, an indirect clastogen,
induces an increase of micronuclei frequency only in the presence of metabolic activation (+S9
fraction).
• Example of automated high content analysis of bi-nucleated cells
Paruch Medicinal Chemistry C9115Genotoxicity Testing
In vivo micronucleus test: design
• Male and/or female rats or mice are treated with the test compound at three dose levels, usually
two or three times at 24-hour intervals.
• Approximately 24 hours after the last dose, bone marrow or peripheral blood is collected to
determine the frequency of micronucleated polychromatic erythrocytes (MN-PCEs) or
micronucleated reticulocytes (MN-RETs), respectively.
• A positive outcome is characterized by a statistically
significant, dose-dependent increase in MN-PCEs or
MN-RETs that exceeds historical control limits.
0h 24h 48h
Species: mouse/rat/….
Gender: 6 or 7 males in single gender
5 males/5 females
Samples: bone marrow/peripheral blood
Single dose/Multiple sampling