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Practical Application of Toxicology in Drug Development Course

Pharmacokinetics and

ADME

Gerry Kenna

Drug Safety Consultant ([email protected])

Pharmaceutical Director, Safer Medicines Trust ([email protected]; www.safermedicines.org)


Overview

• Principles of drug ADME (Absorption, Distribution,

Metabolism, Excretion)

• Pharmacokinetics and toxicokinetics

• Reactive metabolites and toxicity

• Membrane transporters and their Regulatory Guidance

• Metabolites in Safety Testing (MIST)

• Optimising ADME properties to design safer drugs

• Summary and Conclusions


ADME overview

Administered drug

Free drug

Bound drug

Interaction with target molecule(s)

Excretion

Biotransformation

Efficacy or toxicity

Systemic circulation

Metabolic organs e.g. liver Target organ(s)

Metabolites

Tissue reservoir

Free Bound


Drug absorption

Entry from site of administration into bloodstream

• Drug dissolution and membrane penetration is required

• Passive diffusion of uncharged nonpolar molecules through biomembranes

o pH dependent ionisation weak acid and base permeability and distribution (“ion trapping”)

HA ↔ A- + H+

BH+ ↔ A + H+

e.g. aspirin, pKa 3.5: predominantly uncharged in gastric juice (pH 3.5), ionisedin plasma (pH = 7.4)


Route of administration

• Oral absorption requires stability in gastric juice and suitable physchem props

o sublingual or rectal routes may be suitable alternatives

• If aqueous solubility is poor, parenteral administration may be suitable -intravenous, subcutaneous, intramuscular

o control of dissolution rate important (rapid vs. delayed)

• Other routes enable drug targeting to particular sites (pulmonary, topical, eye drops)

o e.g. bronchodilators and topical steroids for inflammatory airway diseases

• Formulation will influence stability and rate of dissolutiono e.g. controlled release preparations to delay drug dissolution,

enteric coating to prevent intestinal dissolution

Drug safety testing should be undertaken using the intended clinical dose route when this is feasible


Drug distribution• Affected by reversible and saturable drug binding to plasma proteins

o Many acidic drugs bind to albumin

o Basic drugs bind to α1-acid glycoprotein or β-globulin

• Cellular uptake required for tissue penetration, involving solute carriers

o Tissue accumulation may lead to cellular reservoirs

o e.g. OATP1B1 and hepatic statin accumulation

o As may tissue specific binding

o e.g. lipophilic drugs to fat; tetracycline antibiotics to bone

o Phys chem props may cause organellar accumulation

o e.g. lysosomal accumulation of basic drugs

� The blood brain barrier controls central nervous system access

� Lipid soluble, nonionized drugs enter foetal blood readily via placental transfer


Metabolism (biotransformation)

• Biotransformation often abolishes or minimises pharmacological activity – “metabolic clearance”

• Enhances polarity, aiding excretion

• May lead to toxicity

Compound (drug)

Metabolite

Conjugate

Phase 1

Phase 2

Oxidation, hydroxylation, dealkylation, deamination

Conjugation

Excretion


Phase I biotransformationImipramine

• Most commonly oxidative, although may be reductive

• Enzymatically catalysed - most often, but not exclusively, by cytochromes P450

• Most drugs have multiple metabolic fates

• Major biotransformation organ is the liver, but may occur elsewhere e.g. GI tract,

kidney

N

(H2C)3

NCH3 CH3

N

(H2C)3

NHCH3

N

(H2C)3

NCH3 CH3

OH

NH

Demethylation

Hydroxylation

Dealkylation


Cytochrome P450s

• Multi-enzyme membrane protein superfamily

• Reside primarily in the endoplasmic reticulum

• Some enzymes catalyze essential intermediary metabolic processes(e.g. steroid hormone and bile salt biosynthesis

• Others mediate metabolic clearance of drugs and other foreign compoundso Predominantly in the liver, in hepatocytes

o Multiple P450s metabolise most drugs –although some drugs are metabolised by individual enzymes

o Typically, different P450s catalyze formation of different metabolites

• Activities of individual enzymes may vary markedly in the human populationo Due to genetic polymorphisms, enzyme

induction and enzyme inhibition

Proportion of drugs metabolised by

different P450s


Cytochrome P450 polymorphisms

• Genetically inherited variation in DNA encoding CYP2D6 result in variable enzyme activities, e.g. debrisoquine hydroxylation

• Affects all major P450s

• Can result in marked population differences in drug kinetics and efficacy


Cytochrome P450 induction

• May arise via enhanced gene transcription, mRNA stabilisation, protein stabilisation

o Enhanced transcription mediated by activation of nuclear hormone receptors, especially PXR and CAR (also AhR, PPAR)

• P450 induction by drugs (e.g. CYP3A4 by rifampicin) may enhance metabolic clearance of the culprit drug (auto-induction), or of other drugs, and can influence toxicity


Cytochrome P450 inhibition

• Caused by many drugs and other xenobiotics (e.g. CYP3A4 by grapefruit juice)

• May lead to altered metabolic clearance and hence undesired drug-drug interactions

• Can occur via competitive or non-competitive mechanisms

• An important mechanism of time-dependent and non-competitive CYP inhibition is via formation of reactive intermediates, which covalently modify the enzyme active site, e.g. dihydralazine


Reactive intermediates

• Electrophilic, unstable metabolites

• Most often formed by P450s

• Interact with cellular macromolecules to cause:o Time dependent P450 inhibition

o Genetic toxicity (via DNA modification)

o Organ toxicity

o Immune activation

• Often react with reduced glutathione (GSH) to form glutathione conjugates, which can be detected

o Catalyzed by GSTs

• Or bind irreversibly to proteins to form protein adducts

DrugReactive

metabolite

Glutathione

conjugate


The glutathione cycle

Cellular glutathione synthesis is induced by electrophilic chemicals, which activate gene transcription via Keap1/Nrf2 dependent signalling

Respiratory Research 2002 3:26


Cytochrome P450 species differences

Hence it is important to understand whether drug metabolites formed in humans are also formed in animal species used in preclinical safety studies

Bars with different superscripts are significantly different (P<0.05 or less)


• Addition of a substituent group, adding polarity and aiding excretion

• Enzymatically mediated, often involves SULTs and/or UGTs o Sulfotransferases (SULTs) catalyze sulfation

o UDP-glucuronyltransferases (UGTs) catalyze glucuronidation

� SULTs and UGTs are gene superfamilies. Exhibit functional genetic polymorphisms & their expression is inducible by CAR, PXR etc.

• Also acetylation (NATs), methylation, amino acid conjugation etc.

Drug conjugation


Drug excretion• Polar compounds are eliminated more

efficiently than lipophilic compounds, therefore often requires metabolism

• Major routes are via kidney (urine) and liver (bile)

• Renal excretion may arise via:o Glomerular filtration

o Tubular secretion or reabsorption

o Passive diffusion

• Plasma protein binding limits glomerular filtration of small drugs e.g. penicillin

• Tubular secretion is transporter mediated and so can be inhibited, e.g. by probenecid

• Passive diffusion of weak acids and bases is influenced by pH or renal filtrate

• Biliary excretion requires active biliary transporter activity


Pharmacokinetics

“Pharmacokinetics may be simply defined as what the

body does to the drug,

as opposed to pharmacodynamics which may be defined

as what the drug does to the body”

Benet LZ (1984). "Pharmacokinetics: Basic Principles and Its Use as a Tool in

Drug Metabolism". In Horning MG, Mitchell J. Drug metabolism and drug toxicity.

New York: Raven Press. ISBN 0-89004-997-1.


Key pharmacokinetic parameters

• D = dose administered

• f = systemically available fraction (“bioavailability”)

• C = amount per volume of plasma or tissue (concentration)o Cmax = maximum plasma concentration

o Cmin = lowest concentration reached before next dose is administered

o Css = steady state concentration

• tmax = time to reach Cmax

• Vd = apparent volume in which drug is distributed

• CL = volume of plasma cleared of drug per unit time (“clearance”)

• t½ = time required for concentration to decrease to half Cmax

• ke = elimination rate constant

• AUC = area under plasma concentration/time curve


Single dose pharmacokinetics

http://www.medscape.org/viewarticle/416455_2


Multiple dose pharmacokinetics

• Accumulation of drug to a plateau level

• Plasma concentrations then fluctuate between a minimum and a

maximum value


Pharmacokinetic modelling

Two compartment model assumes distribution to and from rapidly and slowly perfused compartments


PBPK modelling

• Physiologically based pharmacokinetic (PBPK) modelling provides more accurate description and prediction of kinetics in animals and humans

• Mathematically rigorous, multi-compartment

• Requires significant expertise. Various proprietary models and commercial service providers.


Toxicokinetics

Mathematical description and modelling of the kinetics of compounds which cause adverse effects

• Utilises PK principles

• When relationship between dose and toxicity is well defined, provides useful prediction of toxicity outcome at specific doses / plasma exposure levels

• Underpins environmental risk assessments for environmental and dietary chemicals and consumer products (e.g. cosmetics)


Drug toxicokinetics

Quantifies dose-toxicity relationship in preclinical species used in safety studies, to aid human risk assessment

• Understanding of the

relationship between dose,

plasma exposure, exposure

at efficacy site and site of

toxicity is needed; plus

dynamics of efficacy vs.

toxicity responses

• Often confounded by

species variability in

ADME, PK and/or

mechanism of toxicity

• TK modelling of parent

compound does not

address toxicity caused by

metabolites


• Widely used over-the-counter analgesic

• Considered safe at doses up to 4 g per day

• Human overdose may cause severe / fatal liver injury

• Dose dependent hepatotoxicity also evident in animals (hamster,mouse>rat)

Reactive metabolite mediated

toxicity: Paracetamol


• Tropane alkaloid, dopamine reuptake inhibitoro Topical anaesthetic used in eye, nose and

throat surgery

o CNS stimulant and appetite suppressant - “…. a euphoric sense of happiness and increased energy” - hence recreational abuse

• Causes dose dependent liver injury in mice, but not ratso Requires repeated dosing

o Potentiated by enzyme induction (with phenobarbitone)

o Prevented by CYP inhibition

• Reported to cause very liver injury in humans, without overt dose dependence


toxicity: Cocaine



toxicity: Methapyrilene

• Histamine H1 receptor antagonist

• Anti-histamine / sleep aid

• Introduced ~ 1955, withdrawn 1980

• Carcinogen in rat (43-64 wk), not mouse

• Potent hepatotoxin at high doses (150 mg/day) in rat, but not mouse

o Due to thiophene bioactivation: J PharmacolExp Ther 326:657, 2008; Toxicol Appl Pharmacol 239:297, 2009.

• No reports of hepatotoxicity or carcinogenicity during 25 years of human clinical use (10-20mg/day dose)



toxicity: Halothane

• Volatile anaesthetic, introduced in the 1950s and widely used worldwide

• Causes rare but severe liver injury in humans

• P450 dependent bioactivation triggers potent hapten driven immune response

• Now largely replaced by alternative anaesthetics, which exhibit minimal bioactivation and cause even less frequent liver injury

Anesth Analg. 1995;81(6 Suppl):S51-66.


Membrane transporters

Transport-related~5-10%; >2000 proteins

Protein Foldingand degradation

13%Signal transduction

11%

Unclassified8%

Ribosomal proteins6%

Cytoskeletal5%

Intermediary metabolism

28%

DNA /RNA metabolism

18%

1. SLC transporters

2. ABC transporters (49)

3. Pumps

4. Channels

5. Aquaporins

• High prevalence, multiple types

• Drug permeation of cell plasma membranes is mediated by:o Facilitated solute

carriers (SLC)

o Active ATP-dependent (ABC) transporters

Transmembrane proteins which carry molecules across plasma and intracellular membranes


Passive diffusion Diffusion through a membrane without expending metabolic energy, driven by a concentration or electrochemical gradient

Active transport Transport against a concentration gradient, requiring metabolic energy

Primary active transport

Uses energy directly to mediate transport (e.g. ABC transporters)

Secondary active transport

Coupled transport, involving electrochemical potential (e.g. sodium/calcium exchanger, sodium-glucose co-transporter)

Solute carriers Transport solutes down their electrochemical gradients or via coupling to solutes which are transported down their gradients.Fifty two families, which include organic anion, cation and zwitterion transporters.

ABC transporter ATP binding cassette transporters. Seven subfamilies (ABCA –ABCG). Include P-glycoprotein (Pgp; MDR1, ABCB1), Multi-Drug Resistance Proteins (MRPs; ABCCs), Breast Cancer Resistance Protein (BCRP; ABCG2), and the Bile Salt Export Pump (BSEP: ABCB11).

Transporter terminology


• Polarized expressiono Solute carriers on sinusoidal domain (passive cell entry)

o ABC transporters on apical domain (active biliary efflux)

• Statins enter hepatocytes via OATP1B1o Inhibition causes drug-drug interactions (e.g. cyclosporin/statins)

• ABC transport from hepatocytes is essential for bile flowo Genetic defects in BSEP expression cause accumulation of toxic bile salt

in hepatocytes and severe cholestatic liver injury (PFIC2 )

• Many drugs inhibit BSEP activityo A key mechanism underlying human drug induced cholestatic liver injury

Hepatocyte plasma membrane

transporters


Other tissues

Membrane transporters in drug development. Nature Reviews Drug Discovery 2010;9, 215-236

Plasma membrane transporters

in other tissues


Membrane transporter mediated

toxicity of trichloroethylene

Toxicol Sci. 2006;91(2):313-31


Organ toxicity of GSH-derived

conjugates

• Nephrotoxicity (proximal tubule uptake)

o Halogenated hydrocarbons (TCE, HCBD etc)

o Polyphenols (hydroquinone etc.)

• Neurotoxicity (blood brain barrier uptake)

o Amphetamine derivatives (MDA, MDMA)

o 2-chloropropionic acid

Monks and Jones (2002) Current Drug Metab 3:425, 2002


Transporter mediated DDI

safety problems


Membrane transporter FDA guidance

• Focus on potential of transporter interactions to cause DDIs

• Initial in vitro evaluation, which may trigger follow on in vivo clinical studies

• All investigational drugs should be evaluated to determine whether they are substrates of P-gp or BCRPo When hepatic clearance is significant,

OATP1B1 and OATP1B3 transport should be assessed

o When active renal secretion is important, OAT1 and OAT3 transport should be assessed

• Possible inhibition of P-gp, BCRP, OATP1B1/OATP1B3, OAT1/OAT3 and OCT2 should also be evaluated

http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm292362.pdf


Membrane Transporter EMEA Guidance

• Highlights need to evaluate inhibition of P-g, OATP1B1, OATP1B3, OCT2, OAT1, OAT3 and BCRP.

• Possible inhibitory effects on OCT1, MATE1 and MATE2 should also be considered.

• Inhibition of BSEP should also preferably be investigatedo If in vitro studies indicate BSEP

inhibition, in vivo monitoring (including of serum bile salts) is recommended during drug development

• In vitro data on transporter inhibition should preferably be available before initiating Phase III

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/07/WC500129606.pdf


Metabolites in Safety Testing (MIST)http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm079266.pdf

• When and how to identify and characterize drug metabolites whose nonclinical toxicity needs to be evaluated directly in nonclinical safety studies

• Safety of drug metabolites may need to be determined directly where:o These are identified only in humanso Levels identified in humans are

disproportionately higher than in animal species used in nonclinical drug toxicology testing

• Applies to all small molecule non-biological drug products • Possible exception is some cancer

therapies, where risk-benefit is considered


• Large biologically active molecules, often produced via recombinant DNA technology

o Includes antibodies, cytokines, inflammatory proteins (e.g. interferons)and hormones (e.g. insulin)

• Disposition and clearance processes quite different from small molecules o Cell membranes penetration limited, does not occur via passive

diffusion or ABC transporters. May arise via endocytosis, phagocytosis or pinocytosis

o No biotransformation by “conventional” Phase I and Phase II enzymes and no inhibition of these enzymes - although modulated enzyme expression and activity may arise (e.g. interferons)

o Plasma biological t typically low (minutes, not hours) due to rapid clearance via binding to target macromolecules. May necessitate depot formulation or continuous infusion

o Major human safety issues in humans likely to be due to unexpected sustained efficacy (e.g. super-agonism) and/or immunogenicity

Biotherapeutics


Optimising ADME to design safer drugs

Target Selection

LeadGen

LeadOptim

Pre clinical Phase I Phase IIa Phase IIb

Phase III/Launch

In silico tools

In vitro high volume screens

Refinedin vivo

preclinical models Clinical Safety Evaluation

In vitro low volume complexassays

• Currently, ADME tools used in drug discovery guide design and selection of compounds with best possible PK properties and minimal DDI potential

• Can such tools also aid design and selection of safer drugs?


In vitro (microsomes, hepatocytes, supersomes)• Metabolite id• Chemical trapping (GSH, CN, methoxyamine)• Covalent binding to macromolecules • CYP MBI

ADME tools for assessing reactive

metabolite formation

In silico• Structural alerts• Computational tools

In vivo (preclinical species, occasionally humans)• Metabolite id• Covalent binding to macromolecules

� Useful awareness/avoidance tools

� But do the signals they provide correlate with drug safety risk?


� Covalent binding to human hepatocyte proteins in vitro “flagged” many high ADR concern drugs, when considered alongs ide oral drug dose


� Evaluation of in vitro CYP MBI and glutathione adduct formation “flagged” many licensed drugs which cause liver injury in humans, when considered alongside oral drug dose (100 mg daily dose cut-off)

� And also “flagged” 3 of 6 candidate drugs terminated due to liver injury in preclinical species

Chem Res Toxicol. 2012;

25:2067-82.


A Zone classification scheme, which combined:

� A Panel of Safety Screening assays (cell toxicity plus BSEP and Mrp2 inhibition)

� Covalent binding of radiolabelled drugs to human hepatocyte proteins, adjusted human daily dose and in vitro metabolic turnover

Chem Res Toxicol. 2012 ;

25:1616-32.


Systems Modeling

The DILI-sim Consortium (Watkins et al, Hamner Institutes USA http://www.dilisym.com/)

“ The long-range goal ….. will be to produce a model capable of predicting the

hepatotoxic response across a wide range of patient types and to use simulations to

propose candidate biomarker combinations and/or mechanistic links to hepatocellular

drug induced liver injury ”


Summary and conclusions (1)

• Understanding ADME properties of small molecule

drugs provides understanding of the biological

processes which determine kinetics and dynamics of

plasma and tissue exposure and elimination

o For many drugs, biotransformation results in loss of

pharmacological activity and enables safe elimination

o Some drugs are bioactivated by drug metabolising enzymes

to reactive intermediates which cause toxicity

o Minimisation or elimination of bioactivation during drug

discovery may aid design and selection of safer drugs



• Human safety assessment requires comparison

between drug kinetic parameters in preclinical species

used in toxicology studies and drug kinetics in humans

• Such studies should consider possible species

differences in ADME

o MIST Guidance from FDA has specified when safety testing

of metabolites is required



• Transporters play key roles in drug disposition and drug toxicity.

o Current FDA and EMEA Regulatory Guidances require evaluation of

uptake and efflux transporter interactions, to predict and avoid DDIs

o BSEP inhibition potential should be considered and evaluated if liver

injury signals are observed in animals or humans

o Proactive evaluation of BSEP inhibition potential during drug

discovery aids prediction and avoidance of drug induced liver injury

• Physiologically based Systems Modelling will enhance

understanding of exposure based differences in toxicological

responses between species and within the human population


Thanks for your attention

Any questions?

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