Eliminating Problems in Drug Development – Understanding the Role of

Drug Metabolism

Andrew G. Taylor, Ph.D. Manager, Technical Support for Services

SEKISUI XenoTech

Abstract: This talk will discuss processes for the metabolism of a drug, referred to as ADME (Absorption, Distribution, Metabolism, Excretion), and the safety testing to provide requisite information to move a drug through IND and into clinical trials. Prior to marketing a new drug and actually prior to administering a candidate drug to humans in clinical trials, it is important to ensure that drug will not only be effective in the therapeutic manner prescribed, but also be safe and not bring any undo harm to patients. How a drug is metabolized is one of the most important aspects to understand. If you are involved in the development of potential new therapeutics, this discussion will help you gain necessary knowledge to inform your drug development decisions.

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• What is ADME?• Example Case Studies of Metabolism

Based DDIs• Regulatory Requirements and in

vitro Studies to Predict Metabolism Based DDIs

• Areas of concern: Proper design & interpretation

• Timing of in vitro ADME studies

Outline

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What is ADME?

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What is ADME and why is it important?

It is a scientific field of study examining how compounds interact with the human body.

Used to assess and predict potential clinical safety concerns of new medications/treatments prior to

clinical trials.

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Compounds are evaluated for ADME properties:

• Absorption – Drug Transporters, passive diffusion

• Distribution – Drug Transporters, passive diffusion

• Metabolism – Drug Metabolizing Enzymes (CYP450s,

UGTs, etc.)

• Excretion – Drug Metabolizing Enzymes and Drug

Transporters

Retrieved from https://aidsinfo.nih.gov/understanding-hiv-aids/glossary/213/drug-drug-interaction

ADME & DDI

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Absorption (Oral Drugs)• Orally administered drugs are delivered

to the gastrointestinal (GI) tract• Compound has to be able to get into

the bloodstream • Biological barriers keep foreign

substances out of cells• E.g., skin, cell membranes

• Affected by numerous characteristics• e.g., Solubility, dissolution, ionization,

particle size, molecular size etc.

Retrieved from http://cephalicvein.com/2016/05/gi-tract/

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Oral vs. Intravenous Administration

Oral drugs traverse the gut and liver to get into the bloodstream

Intravenous drugs go directly to the bloodstream

There is no absorption phase

Ahmad et al., Eur. J. Pharmaceut. And Biopharmaceut. 2017, 112: 234-248.

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Distribution• Drug moves to site of action – via

bloodstream• Biological considerations

• Vascularity – where the bloodvessels go

• Transport – passive or active• Blood/placental barriers• Plasma binding proteins

Retrieved from http://7mlkbodysystems.weebly.com/circulatory-system.html

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Metabolism• The biochemical structural modification of a drug• Often changes the chemical structure of a lipophilic compound to

make it more hydrophilic (water-soluble) for elimination• Often by DMEs in the liver; sometimes intestines, kidney etc.

Retrieved from https://pharmaxchange.info/2014/09/phase-i-metabolism-oxidative-reactions-oxidation-of-aliphatic-and-alicyclic-compounds/ibuprofen/

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Many Enzyme Families Involved in Drug Metabolism• CYPs: Oxidation of steroids, fatty acids and many xenobiotocs

• UGTs and SULTs: Nucleophilic groups (aliphatic and aromatic hydroxyls, carboxylic acids,

amines, hydroxylamines, thiols, etc.)

• AO & XO: Heterocycles with low electron density (e.g., pteridines, quinolones, etc.)

• FMO: Soft nucleophiles (sulfur or nitrogen heteroatoms)

• CESs: Ester moieties (often for prodrugs)

• CBRs and AKRs: Ketone and quinone moieties

• MAOs: Amines (often problematic PK and other liabilities)

• Hydrolases: Often exploited for prodrug activation (e.g., esters or amides)

Foti & Dalvie. DMD. 2016; 44(8):1229-45

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Excretion

• Irreversible loss of drug from system

• Drug-related material (drug and metabolites) has to be cleared from the organism

• Commonly occurs through• Kidneys (renal) - urine • Liver (biliary) - bile/feces• Lungs (respiratory) - breath• Other – sweat, tears, saliva

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Example Case Studies of Metabolism Based DDIs

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• Mibefradil is an antihypertensive Ca channel blocker approved in 1997

• After approval a serious DDI with β-blockers was observed

• Mibefradil was voluntarily withdrawn in 1998

• Turns out mibefradil was a potent TDI of CYP3A4

Example 1: Inhibition by a perpetrator drug

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• Terfenadine is a prodrug of the active drug fexofenadine

• Terfenadine is converted to its active form predominantly by CYP3A4

• Taking terfenadine with CYP3A4 inhibitors can lead to QT prolongation

• Terfenadine was withdrawn in 1997

Example 2: Terfenadine and CYP3A4 inhibitors

Terfenadine(Seldane® - withdrawn)

Fexofenadine(Allegra®)

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Regulatory Requirements and in vitro Studies to Predict Metabolism Based DDIs

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Regulatory GuidanceFDA: Final January 2020 EMA: Final 2013 PMDA: Final 2019

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Additional GuidanceFDA “MIST”: Rev 2

March 2020FDA / ICH: Final 2010

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In vitro ADME & DDI study typesADME component Type of in vitro study

Drug Metabolism (M, E) 1. Inter-species comparative metabolism

2. Metabolite ID – Qualitative analysis of metabolite profile

3. Reaction phenotyping – Determine which CYPs are metabolizing

Drug Metabolizing Enzymes (M, E) 1. CYP Inhibition – Profile specific CYP inhibitions

2. CYP induction – Induction potential for specific CYPs

Drug Transporters (A, D, E) 1. Transporter substrate – Determine Transporter substrate profile

2. Transporter inhibition – Profile specific inhibition of major Transporters

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Test Systems for in vitro DDI Experiments• Subcellular fractions – non-living, cell/tissue lysates that have been

fractionated to enrich for certain enzyme activities. • S9 • Microsomes • Cytosol• These subcellular fractions are derived from many relevant drug

metabolizing organs/tissues. May require co-factors.• Cryopreserved primary hepatocytes – living, isolated directly from

living liver tissue and frozen for use at a later time. From multiple small animal models and human livers.

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Drug Metabolism Studies

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Drug Metabolism: Inter-Species Comparative Metabolism• Design: Drug incubations with hepatocytes or subcellular fractions from various species• Typical species: Human, Rat, Mouse, Dog, Rabbit, Monkey, Pig Rat Dog

Time (min)0 60 120 180 240

Perc

ent r

emai

ning

0

20

40

60

80

100

t1/2 = > 240 min

Time (min)0 60 120 180 240

Perc

ent r

emai

ning

0

20

40

60

80

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t1/2 = 97.6 min

Monkey Human

Time (min)0 60 120 180 240

Perc

ent r

emai

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60

80

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t1/2 = 139 min

Time (min)0 60 120 180 240

Perc

ent r

emai

ning

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20

40

60

80

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t1/2 = 187 min

Metabolic Stability (Hepatocytes)

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0 60 120 180 240

Perc

ent r

emai

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(%)

Incubation time (min)

RatHumanMonkeyDog

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CoumarinHuman Rat

7-Hydroxycoumarin

No toxicity

Coumarin-3,4-epoxide

Hepatotoxicity

Goals: • Complete profile of metabolites• Are there human specific

metabolites? • Which other species have a similar

metabolic profile?

Drug Metabolism: Inter-Species Comparative Metabolite ID

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Metabolite IDLC-MS/MS analysis – Qualitative identification of the metabolites

6.00 8.00 10.00 12.00 14.00 16.00

AU

0.0

1.0e-3

2.0e-3

3.0e-3

4.0e-3

5.0e-3

6.0e-3

7.0e-3

8.0e-3

9.0e-3

1.0e-2

1.1e-2

Range: 1.9.11

8.88 10.28

Repaglinide

Hydroxyrepaglinide Repaglinide dicarboxylicacid metabolite (M2)

UnknownRepaglinide glucuronide (RG)

Hydroxyrepaglinide LC-MS/MS

50 µM Repaglinide; Human hepatocytes; 60 minutes; 37°C

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Cross-Species Metabolite IDComponent Retention time

(min) Mass shift Proposed biotransformation Mouse Rat Dog Pig Human

C1 3.43 255.9889 Sulfation + glucuronidation + + + + +

C2 3.63 354.0783 Di-glucuronidation + hydrogenation + + + + +

C3 3.78 159.9135 Di-sulfation + + + + +

C4 4.00 258.0045 Sulfation + glucuronidation + hydrogenation + + + + +

C5 4.41 161.9298 Di-sulfation + hydrogenation + + + + +

C6 4.44 194.0428 Glucuronidation + oxygenation + hydrogenation ND ND ND + +

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• Design: Incubate drug + recombinant human CYPs or human liver microsomes or hepatocytes ± selective inhibitors

• Goal: Determine which CYPs drive the metabolism of the drug• Unique CYP metabolism is of concern High DDI potential: few enzymes involved

Follow-up studies:• Confirm with selective inhibitors• Evaluate non-CYP pathways in HLM or

hepatocytes

0102030405060708090

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Recombinant human CYP enzyme

Rate

of m

etab

olis

m

Terfenadine(Seldane® - withdrawn)

Fexofenadine(Allegra®)

Drug Metabolism: CYP Reaction Phenotyping (Victim potential)

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Drug Metabolizing Enzymes(Perpetrator Potential)

Enzyme Inhibition

Enzyme Induction

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CYP Activity AssayCYP1A2 Phenacetin O-dealkylationCYP2B6 Bupropion hydroxylationCYP2C8 Amodiaquine N-dealkylationCYP2C9 Diclofenac 4´-hydroxylationCYP2C19 S-Mephenytoin 4´-hydroxylationCYP2D6 Dextromethorphan O-dealkylationCYP3A4 Testosterone 6β-hydroxylationCYP3A4 Midazolam 1´-hydroxylation

Drug Metabolizing Enzymes: CYP Inhibition

Design:Drug incubations with HLM + marker substrate ±pre-incubation

Goal:Predict clinically relevant inhibition of CYP enzymes

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• Design: Drug incubations with HLM + marker substrate ± pre-incubation• Goal: Assess inhibition of CYP enzymes

Ketoconazole:Potent inhibitor of 3A4 precludes co-administration of other drugsMibefradilRemoved from market in 1998 due to potential for fatal DDIs

In Vitro Enzyme Inhibition

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In Vitro CYP Induction

[Rifampin] (µM)

0.001 0.01 0.1 1 10 100

Effe

ct(F

old

Indu

ctio

n)

1

1.5

2

2.5

3

3.5

Parameter Value Std. Error

Emax 3.3378 0.1200EC50 0.1362 0.0528

[Rifampin] (µM)

0.001 0.01 0.1 1 10 100

Effe

ct(F

old

Indu

ctio

n)

2

4

6

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Parameter Value Std. Error

Emax 6.0649 0.1981EC50 0.5795 0.1244

[Rifampin] (µM)

0.001 0.01 0.1 1 10 100

Effe

ct(F

old

Indu

ctio

n)

1

2

3

4

Parameter Value Std. Error

Emax 3.2417 0.3163EC50 0.1371 0.1427

Donor 1 Donor 2 Donor 3

• Design: Drug incubations in cultured human hepatocytes, measure mRNA (or activity) of various CYPs

• Goal: Assess induction of CYP enzymes

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Drug Transporters

Transporter Inhibition(Perpetrator)

Substrate Potential(Victim)

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Drug Transporter Studies

• Drug absorption, distribution, tissue-specific drug targeting, and elimination

• Drug-drug interactions• Clearance of transporter substrates (Victims) can be impacted by transporter

inhibitors or inducers (Perpetrators)• Toxicity or loss of efficacy

• Real world example - Statins• Hepatic uptake transporter (OATPs) substrates: taken up in the liver, reduce

cholesterol• Cyclosporine inhibits OATPs: up to 10-fold increase in statin exposure• Toxic side effect: rhabdomyolysis (skeletal muscles break down, cells released

into bloodstream, can lead to kidney failure and possibly death)

ADME

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Areas of concern

for in vitro DDI studies: study

design and data

interpretation

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Areas of concern: CYP Inhibition study designFalse negative results arise from poorly designed studies

• Example: Clinically relevant time-dependent inhibition of CYP2C19 by omeprazole missed with high [protein] and long marker substrate incubation

[Omeprazole] (µM)0.01 0.1 1 10 100 1000

S-M

ephe

nyto

in 4

´-hyd

roxy

latio

n(P

erce

nt o

f con

trol)

0

20

40

60

80

100

120

140

160zero-min preincubation IC50 = 6.9 µM

30-min preincubation +NADPH IC50 = 1.7 µM

30-min preincubation -NADPH IC50 = 8.7 µM

[Omeprazole] (µM)0.01 0.1 1 10 100 1000

S-M

ephe

nyto

in 4

´-hyd

roxy

latio

n(P

erce

nt o

f con

trol)

0

20

40

60

80

100

120

140

160zero-min preincubation IC50 = 8.3 µM

30-min preincubation +NADPH IC50 = 6.3 µM

1 mg/mL protein, 30 min substrate incubation0.1 mg/mL protein, 5 min substrate incubation Time-dependent inhibition of CYP2C19 by omeprazole is readily detectable with HLM at 0.1 mg/mL with a 5-min substrate incubation period (left) but not at 1.0 mg/mL with a 30-min incubation period (right)

1.3-fold shift4-fold shift

Detected Missed

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CYP induction studies: positive controls with very large induction

0

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12000

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0 5 10 15 20 25 30 35 40

Rank order based on control rates

Test

oste

rone

6β-

hydr

oxyl

atio

n(p

mol

/mg/

min

)

Rifampin-treated (20 µM) hepatocytes CYP3A4 activity

Rank order of control rates

Test

oste

rone

6β-

hydr

oxyl

atio

n(p

mol

/mg/

min

)

Control hepatocytes CYP3A4 activity

0

1000

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0 5 10 15 20 25 30 35 40

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Fold

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Rank order based on control rates

Fold induction

• When it comes to induction, more is not always better

• A high fold-induction (>20 fold) of CYP3A4 activity by rifampin is a sign of incomplete hepatocellular differentiation of the cultured human hepatocytes

Areas of concern: CYP Induction study design

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Metabolism studies: Choose the right test system based on the structure• Ezetimibe is oxidized by CYP3A4 however results with HLM & NADPH alone can be

misleading.

Oxidation does not occur clinically due to rapid phenolic glucuronidation. Recombinant human UGTs or human hepatocytes would be a better test system. CYPs are not the only enzyme system.

Areas of concern: Reaction Phenotyping

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Timing of ADME studies

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Type of drug Lead optimization Pre-IND Phase I to NDA

Typical small molecule

1. Comparative metabolism

2. Metabolite ID3. Screening for

others

1. CYP inhibition2. CYP Induction3. Transporter

inhibition4. Limited transporter

substrate

1. Reaction phenotyping2. Additional transporter

substrate (dependent on routes of elimination)

Small molecule with orphan, breakthrough

status, etc.

1. Comparative metabolism May be able to defer

1. Metabolite ID2. CYP inhibition3. Transporter inhibition4. Reaction phenotyping5. CYP induction

Peptides, oligos, ADCs, other biologics May be able to defer May be able to defer

1. Metabolite ID2. CYP inhibition3. Transporter inhibition4. Reaction phenotyping5. CYP induction

Priority depends on strategy for each drug & need for de-risking at each stage

Drug Development Pipeline: timing in vitro DDI studies

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“Why conduct these studies? Is this just box checking?”

No. The information in aggregate has real utility: 1. Provide deeper understanding of the molecule

• Metabolism, enzymes involved in metabolism, etc.• The information generated from DDI studies goes on the drug label• From the pharma company’s perspective these studies can inform go/no-go decisions for a drug candidate• Predictive toxicology and dose selection for certain non-clinical in vivo studies

2. Prepare for clinical studies• Prediction of FIH dose and DDI risk

3. Satisfy regulatory expectations and comply with regulatory guidance

These studies may appear deceptively simple – maximizing insight can be complex

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Conclusions: In vitro ADME & DDI studies • Provide understanding of drug characteristics and insight concerning future

performance in in vivo systems; notably concerning predictive toxicology, dose/species selection for IND enabling studies, and FIH trial considerations.

• Satisfaction of regulatory interests is critical for prevention of delays

• Prioritization varies based on drug class and program de-risking needs

• Conduct and interpretation can be deceptively simple; they both benefit expert design and understanding

• Provide as much information of the drug as possible for appropriate guidance

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Seeking further information? Check out our new site, detailing how we help drug developers

understand their compound’s ADME/PK & DDI potential