The Role of Drug Metabolism Studies in Optimizing Drug Candidates

Kenneth Santone, PhD

Bristol-Myers Squibb

Metabolism and Pharmacokinetics / Pharmaceutical Candidate Optimization

ALTERNATE TITLE:

Why All the Chemist's Wonderful Compounds Don't Become Drugs!

Our Focus

Unmet medical needFirst in classBest in classNeed for efficiency and

productivity enhancement

What are we faced with?

Industrialization of pharmaceutical research

– Unprecedented increase in identification of targets

– Corresponding increase in throughput of chemistry

– Blurring of traditional discovery-development interface Focus and emphasis on “developability”

(early go/no go decisions) Improve success rate Reduce development timeline

– Necessity for increasing efficiency and productivity

Drug Discovery Paradigm Shift

‘Old’ Modelof Drug Discovery

Validated Hits

Detailed Physicochemical,ADME & Tox Workup

Development Compound

Efficacy &Selectivity

Testing

Hits

Lead Candidates

Physicochemical, ADME & Tox Workup

Development Compound

Efficacy &Selectivity Testing

Design & Synthesis PAT

Screening &Predictions

Design & Synthesis

‘New’ Modelof Drug Discovery

More informed decision making

during Lead Optimization,

through quicker and earlier

evaluation of PAT attributes

The Hand-off from Drug Discovery to Development: The Top Ten Quotations We All Know and Love*

“The molecular weight? 850. Why? Is that a problem?”

“We’ll need eight different capsule strengths for Phase I.”

“The compound is very potent in the in vitro screen but does not work well in the animal efficacy model.”

“Now that you mention it, our solutions were a little cloudy.”

“The compound is highly insoluble but Pharmaceutical Development will fix the problem.”

“BMS-XXXXXX is a highly potent and selective inhibitor of (the target).In mouse models, the optimal dose was 200 mg/kg.”

“Toxicity?! It’s not the drug; must be a metabolite unique to that animal species.”

“Animal bioavailability ranged from 65% to <1%, depending on species.”

“Gee, we didn’t have any problems when we gave it in DMSO.”

“It’s a great compound, but it has formulation problems.”

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Partially adapted from R.A. Lipper *why great compounds don’t always become drugs

Chemistry

Biology

Activity

Metabolism & Pharmacokinetics

Optimized Compound

Safety Pharmaceutics

Critical Interfaces in Drug Discovery*

*Analytical Chemistry (Bioanalysis) involved in every one of these disciplines

Role of ADME* StudiesSelection of quality drug candidate for development

– Developability

– First-in-class vs. best-in-class

– Crisp go/no go decisions

Optimization of drug discovery and early development processes

– Multi-tiered approach for ADME studies

– Equal partnership with all functional areas

Lead Discovery BiologyChemistry PharmaceuticsDrug Safety Analytical R&DClinical Pharmacology Process Chemistry

Blurring of traditional discovery-development interface

* Absorption, Distribution, Metabolism, Excretion

Selection of Drug Candidates:Focus on Developability

Permeability

Transport

Metabolic stability

P-450 mediated drug interactions

PK/PD assessment

Distribution

Protein binding

Biopharmaceutics

Active/reactive/toxic metabolites

In vivoPK/bioavailabilityin animals

Prediction of PKand efficacious doses in humans

Tiered-Approach for ADME Studies

Hits to Lead

• In vitro Studies•Permeability•P450 inhibition•Metabolic Stability

•In silico predictions

•Objective•Develop SAR•Chemotype selection

Tiered-Approach for ADME StudiesLead Optimization In vitro Studies

•Permeability/transport•P450 inhibition•Metabolic Stability•Reaction phenotyping•Protein binding

•In vivo PK•Cassette dosing•Individual PK

•Tissue penetration•Early biotransformation

•Objective•Identify a lead compound•Feedback to chemistry/biology

Tiered-Approach for ADME StudiesLead Selection• Absolute bioavailability in pharmacology/toxicology models• Dose dependency in PK• Mechanism of absorption• Assess potential for DDI• Characterization of metabolites, routes of elimination• Assess formation of active metabolites• Interspecies differences in metabolism and in vitro-in vivo correlation• Extrapolation of ADME properties to man from in vitro and in vivo data• Determination of PK/PD relationships; help selection of doses for First in Human studies

•Objective•Characterize the lead compound•Identify risks/opportunities

How In Vitro Metabolic Stability Relates to Clearance?How In Vitro Metabolic Stability Relates to Clearance?

Intrinsic Clearance (CLi) = Vmax / Km = vo / Cu

TBC = CLhepatic + CLrenal + CLother

CLhepatic = CLmetabolism + CLbiliary

CLmetabolic = fB * CLintrinsic * Qh / fB * CLintrinsic + Qh

through rearrangement of the Michaelis-Menton eqn, assuming drug conc is < Km

Depletion or Half-Life Method: CLi = (0.693 * liver wt) / (in vitro t1/2 * amount of liver)

well stirred model of organ extraction

Tools to Predict Metabolic ClearanceTools to Predict Metabolic Clearance

In Vitro Systems

Liver microsomes

– high throughput and most common

– mostly oxidative (CYP & FMO)

S9 fraction

– high throughput

– Phase I & Phase II metabolism

Hepatocytes

– low throughput

– cell membrane/transporters

– intracellular concentration

– Phase I & Phase II metabolism

In Vivo Animal Clearance

In Silico

In Vitro - In Vivo Correlation

Metabolic Stability to Select Compounds with Potentially Longer Half-Life

Rate of Human Microsomal Metabolism

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Oxidation

Glucuronidation

Human Metabolic Stability: Microsome vs Hepatocyte

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Hepatocyte Metabolic Rate

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• Lead compound is primarily glucuronidated in humans• Human in vitro systems with combination of oxidation and glucuronidation employed for selection of back up

Major Reactions Involved in Drug Metabolism

OXIDATIVE REACTIONS (CYP, LM+NADPH)

•N-Dealkylation: erythromycin, morphine, caffeine

•O-Dealkylation: codeine, dextromethorphan

•Aliphatic Hydroxylation: tolbutamide, midazolam

•Aromatic Hydroxylation: phenytoin, amphetamine, warfarin

•N-Oxidation: chlorpheniramine, dapsone

•S-Oxidation: cimetidine, omeprazole

•Deamination: amphetamine

Major Reactions Involved in Drug Metabolism

HYDROLYSIS REACTIONS (Esterase, ?LM+NADPH)

•Ester Hydrolysis: aspirin, cocaine

•Amide Hydrolysis: lidocaine, procainamide

CONJUGATION REACTIONS (Phase II, hepatocytes)

•Glucuronidation: morphine, ibuprofen

•Sulfation: acetaminophen

•Acetylation: sulfonamides, isoniazid

Metabolic Stability SummaryMetabolic Stability Summary• Not all metabolism is hepatic.

• Incubation concentration < Km balanced with assay sensitivity.

• Need to correlate with in vivo model.

• Fast in vitro clearance generally implies fast in vivo clearance, the reverse need not be true.

• Confounding physical-chemical properties.

solubility, stability, purity, non-specific binding

• Real concentration at enzyme active site?

protein binding, cell penetration, non-specific binding

• In vitro systems generally underestimate CLi due to non-specific binding.

• Can the stability be too good? Yes, in certain situations.

• Many unknown factors to can contribute to a poor in vitro - in vivo correlation or poor estimation of human metabolic stability.

Nonetheless, in vitro methods are still the best method for predictions

Drug-Drug Interaction SummaryDrug-Drug Interaction Summary• Major drug interactions are caused by either inhibition or induction of

drug metabolizing enzymes.

• Semi-quantitative predictions of drug interactions many unknown factors human ADME properties in vivo

• Models provide numbers that must be placed in context with multiple factors: therapeutic area therapeutic index, route of administration market competition

• Animal models are not predictive of human interaction potential ???

• Static nature of in vitro systems compared to the dynamic in vivo system

• Mixtures of interaction mechanisms from the same compound are extremely difficult to predict: reversible + irreversible inhibition inhibition + induction

Assessment of Active Metabolites

Issue• Similar metabolism and in vitro activity profile but different in vivo

activity profile• Apparent PK/PD disconnect

Solution• Rapid in vitro metabolism and biological activity assays

Compound Met Ratea Cmaxb (M)

AUC(0-8)b (M.h)

IC50 (nM)

Efficaceous Dose

(mole/kg) BMS-X 0.64 8.5 35 19 1.4 BMS-Y 0.58 13.2 64.4 19 >60

a: metabolism rate in nmol/min.mg protein in rat liver microsomes b: rat oral exposure studies at 0.1 mmol/kg

Assessment of Active Metabolites

Structural identification of active metabolites• MS/MS indicated presence of monohydroxylation• NMR showed site of hydroxylation

Subsequent steps• Monohydroxylated metabolite synthesized• Activity and PK properties confirmed

In vitro Activity of Liver MicrosomalProduct in Cell Based Assay (IC50 (nM))

CompoundParent 0 min

incubation30 min

incubation

% parentremaining

BMS-X 19 12 19 <1BMS-Y 19 60 490 20

Assessment of Reactive Metabolites

•A number of functional (chemical) elements have been associated with problems in drug discovery leading to toxicity

Metabolic activation to reactive intermediates

Interference with metabolic processes

•Clinical manifestations include (preclinical measure)

Cellular (hepatic) necrosis (animal toxicity)

Idiosyncratic toxicity (glutathione adducts, protein covalent binding, immunogenic response)

Drug-drug interactions (mechanism-dependent CYP inhibition)

Examples of Reactive Metabolites

Furans

Furan substructure is associated with toxicity (eg. aflatoxin) and with CYP inhibition (eg. bergamottin)

O O

O

O

O

O

OO

OCH3

CYP3A4(epoxidation)

Aflatoxin B

O O

O

O

OH

OH

6',7'-dihydroxybergamottin

CYP3A4(epoxidation)

Examples of Reactive MetabolitesThiophenes

Thiophene substructure has been associated with several types of toxicity (predominately hepatotoxicity). Other thiophene containing drugs: ticlopidine, clopidigrel, raloxifene.

S S

O

SO

O

O

HO

Cl Cl

SO

N

OO

H2N

ClTienilic acid Tenidap

CYP2C9

S

O

Nu

Examples of Reactive MetabolitesAnilines, Nitroaromatics

Anilines are associated with a number of types of toxicity (eg. methemoglobinemia, skin rashes, etc.). Nitroaromatics are primarily activated by initial reduction, often in the gut, followed by N-oxidation.

Anilines of polycyclic aromatic systems are often potent mutagens and carcinogens (eg., naphthylamine, aminofluorene) through conjugation of the hydroxylamine and subsequent loss of the conjugate to leave a nitrenium ion.

NO2 NH2 HNOH

SNH

O O

NO

H2N

SO O

H2N NH2

Sulfamethoxazole Dapsone

NO

Examples of Reactive MetabolitesAmines, alkylamines

The metabolism of amines or alkylamines is generally related to time-dependent inhibition of CYP enzymes, with the nitroso species forming a tight complex with the heme iron, known as a MI complex. Other compounds that undergo this type of transformation and inhibit CYPs are TAO, erythromycin and verapamil

N N

O

N

S

O

N

O

O

O

Diltiazem

Examples of Reactive MetabolitesQuinone, Quinoid

Quinone-like compounds can exert their effects through direct alkylation of nucleophiles or through redox cycling between their oxidized and reduced forms

O

X

O

X

HN

OH

O

HO

OO

NHS

O

O

Acetaminophen Troglitazone

X = O, N, C

Examples of Reactive Metabolites

Acetylenes

Acetylenes have been found to be time-dependent inhibitors of CYP enzymes.

O

OH

O

Gestodene

OH

O

Mifepristone (RU 486)

N

Examples of Reactive MetabolitesAcyl glucuronidation formation

Acyl glucuronides have been implicated in both direct hepatic damage and idiosyncratic toxicities

O

OH

O

OGlucAmidori rearrangement, then reaction with nucleophiles

Direct reaction with nucleophiles

N

O

OH

O

Cl

N

O

OH

O

Zomipirac Tolmentin

Challenges and Opportunities HTS screens for prediction of permeability, metabolic stability, metabolic

reactivity and DDI– How are we using these data?– Retrospective analysis on return of investment– The numbers in gray zone!– Secondary assays for better predictability

Application of animal PK/bioavailability data for lead optimization– Adequacy of permeability and metabolic stability data– Animals vs. humans: quantitative and qualitative differences in ADME

properties

Informed decision based on drug metabolism and pharmacokinetic data– Low bioavailability vs. oral efficacy– Role of metabolite(s), reactivity of metabolite(s)– Protein binding– In vitro- in vivo correlation in animals and extrapolation to humans

Issue of enzyme induction in humans– In-vitro models and predictability– False and real alarm from in-vivo animal data

Challenges and Opportunities

Use of biomarkers– In-vivo biology, animals vs. humans– Development and validation of assays– Transfer from preclinical to clinical laboratories– Biomarkers = Surrogate marker = Efficacy/Toxicity– A balancing act of emerging science

The feedback loops– To and from chemistry– To and from biology– To and from drug safety– To and from pharmaceutics– To and from clinical pharmacology

Volume of data– Conversion of information into knowledge– Timing and availability

A Focused Application of ADME Studies

• Active involvement earlier in the Discovery Process

• Timely guidance to Chemistry to select chemotypes with desirable ADME properties

• Maximize informed decision making during Lead Optimization

• Improved ability to predict human metabolism and pharmacokinetics

• Stronger partnerships with Drug Discovery and all areas of Pharmaceutical Development

To ensure that no development candidate fails in the clinic due to anunforeseen metabolic or

pharmacokinetic property

Our Mission

Acknowledgements

And finally ….

David Rodrigues and Griff Humphreys

Saeho Chong, Punit Marathe, Wen Chyi Shyu and Mike Sinz