NON-P450-MEDIATED METABOLISM: IDENTIFICATION AND IMPLICATION DURING COMPOUND SELECTION AND OPTIMIZATION

Xiaoliang ZhuoMetabolism and PharmacokineticsPreclinical Candidate Optimization Bristol-Myers SquibbWallingford, CT

2017 Nanjing International Conference of Drug Metabolism

Cerny, et al., DMD, 2016, 44:1246

(Aldehyde oxidase)

P450s Still the Main Contributors to Metabolism of Approved Oral and Intravenous Small Molecule Drugs (2006 – 2015)

• Multiple classes of non-P450 enzymes play a significant role in drug metabolism • Hydrolysis of amide, etc.

• Approved drugs possess optimized ADME profiles• Unfavorable properties, e.g.

exclusive AO-mediated metabolism, likely eliminated during discovery phase‒ Prodrugs excluded

‒ Secondary pathways excluded‒ Based on human ADME data‒ ≥ 10% of dose = major pathways

(sulfotransferases, cytidine deaminase, dehydrogenase, etc.)

PART I Aldehyde Oxidase (AO): An Emerging Focus As A Result of SAR Intended To Mitigate P450-mediated Clearance

Only one isoform in human

Expressed in cytosol• In vitro metabolism in liver microsomes

does not reveal AO-mediated pathways • Appropriate subcellular fractions or

hepatocytes required

Predominantly present in liver• Also expressed in kidney

No cofactor required• Used to differentiate oxidation mediated

by CYPs and FMOs

Optimize physical-chemical properties: decreasing lipophilicityand electron density of aryls, etc.

Mitigated metabolic clearance by P450s leads to ….

… increased metabolism by AO

AO Differs from P450 in Many Respects

AO Targets Electron-deficient Carbons

HN

S

O

2-quinolinone-SGX523

Diamond, et al., DMD, 2010, 38:1277

Nucleophilic attack by Mo-OH and utilization of water as a source of oxygen

– Oxidation of electron-deficient carbons in aza-heterocycles

– Reduction of N-O or N-S bond– Amide hydrolysis

S

O

N N

NH

N

N

O

O

HN

GDC-0843(Bruton’s tyrosine kinase inhibitor)* Carboxylase also involved Sodhi, et al., DMD, 2015, 43:908

Hydrolysis *

N

S NN

NN

NN

SGX523

Oxidation

S

Mn

S

OH

SVI

Molebdenum center

(c-MET tyrosine kinase inhibitor)

Complications Associated with Prediction of Metabolic Clearance of AO Substrates in Human

Profound variation in expression levels across species • Monkey ~ human ~ SD rat > mouse >> dog • Strain difference in rats, gender difference in mice• Selection of species suitable for PK projection and toxicity studies

Substantial inter-individual variations in human • Single nucleotide polymorphisms identified • Environmental factors • AO contents vary among vendors and batches

Unique expression pattern• Selection of appropriate in vitro metabolic

systems for IVIVC critical

Discontinued development of AO substrates due to:

• Under-prediction of human clearance, low bioavailability: Carbazeran, BIBX1382,

• Safety issue: SGX523 (obstructive nephropathy due to the AO metabolite)

Hutzler et al., Expert Opinion in Drug Metabolism and Toxicology 2013, 9(2):153-168

A Paradigm to Ensure a Comprehensive Evaluation of Metabolic Pathways

• Structural features/alerts

• Poor IVIVC

Estimation of Fm(AO)

Identification of AO-mediated MetabolismMetabolic systems +/- cofactors Findings and conclusionsLiver microsomes with or without NADPH

Metabolism by CYPs

Liver cytosol without cofactor or hepatocytes

New +16 Da products: non-CYP mediated pathway

Liver cytosol, liver S9 or hepatocytes + AO selective inhibitor (hydralazine) *

To confirm metabolism by AO* (1) A positive control should be used to confirm the result(2) Hydralazine also inhibits CYP2D6(3) Use of multiple batches is recommended

Identification of the role of AO- Estimation of Fm(AO)- If exclusively or predominantly by AO

Structural modification - Block AO sites

- Rearrange N atoms - Other heteroaryls- Maintain potency + other ADME properties

Extensive Metabolism by AO Represents An Unfavorable ADME Property, Here IS How to Avoid it ….

Advance compoundwith diverse

metabolic clearance mechanisms

Human CL projection

- Allometric scaling not suitable

- Simple scaling from human in vitro systems likely results in under-prediction

- Compared with benchmark compounds *

Toxicological considerations

- AO products, e.g. lactams, less soluble than parents

- Toxicological species with a comparable metabolite profile as human

- Mitigated DDI

* low CL: Zaleplon; high CL: zoniporide, 6-deoxypenciclovir DMD, 2010, 38:1322

PART II Glutathione S-Transferase-Mediated Metabolism

Glutathione S-Transferases Catalyze Nucleophilic Attack to Electrophilic Atoms

Expression• Primarily present in cytosol• Also in endoplasmic reticulum (membrane-

bound)• High concentrations in liver, kidney, and

lung

Isoforms• Multiple members

Mechanisms• Substrates include a wide array of

electrophilic xenobiotics and their intermediates

Conjugation via arene oxide

Cl

Cl

NO2

GS-

Cl- Cl

SG

NO21,2-Dichloro-4-nitrobenzene

NH

O

F3CCl

O

OSO3H

P450NH

O

F3CCl

O

OSO3H

OH

GS-, H+

NH

O

F3CCl

O

OSO3H

HO

SG

Efavirenz metabolite

Direct replacement of an electron-withdrawing group

Addition

OOH

SG

SG

OHP450

GS-, H+

Naphthalene

Rat or human No cofactor added + GSH

Liver cytosol Stable > 80% of the parent formed GSH conjugates

+ NADPH + GSH or NADPH + GSH

Liver microsomes ~1% oxidative metabolites

6% GSH conjugates

Potassium phosphate buffer (pH 7.4) + GSH

~ 2% GSH conjugates, indicating a chemicalreaction.

Suggesting involvement of membrane-bound GST,but to a lesser extent

Suggesting a role of cytosolic GST

Case I: GSH Conjugation Mediated by Cytosolic GST Led toRapid Depletion of an mGluR5 Allosteric Modulator

N

OHN

O

F

N

N

A D

C

B

N

O

HN

O

F

NNA

D

C

B

GS H

In vitro scale-up productionIsolation + NMR analysis

GSH

GST

(multiple region- and configurationalisomers identified)

• Moderate-high CL in rats• But stability ~95% in RLM• Any pathways mediated by

non-CYPs?

N

OHN

O

N

SG

N

OHN

O

C

N

SG

N

OHN

O

N

N

OHN

O

CN SG

GS

H+

H

Compound 2 N

OHN

O

C

N

SGH

F

F

F

F

F

F

F

F

FF

GSH

GST

Proposed Mechanism of GSH Conjugation: Nucleophilic Attack Onto Acetylene Moiety by Thiolate Anion

The negatively charged intermediate stabilized via delocalization to the adjacent heteroaryl nitrogens via resonance

GST: GSH S-transferase

GS Thiolate anion

Zhuo, et al., DMD, 2015, 43:578

mGluR5 compounds featuring two heterocycles proximal to an acetylene

Extensive GSH addition to the acetylene moiety through GST and chemical reactivity

• Rapid clearance of compounds• Depletion of GSH

• May leave cells less protected from damage by reactive oxygen species

• May lead to formation of protein adducts

N

OHN

O

F

N

N

A D

C

B

Extensive Reaction with GSH Is Not A Favorable Property

A. Flanked by two heteroarylsB. Flanked by one heteroarylC. Flanked by no heteroarylD. Nitrogens to support delocalizationE. Nitrogens NOT to support delocalization

N

N

N

N

N

FNN

F

1

3

2

N

5

4

N

6

Extent of GSH Conjugation in LS9

A, D A, E

C, E

B, E

Presence of two heteroaryls and proximity of nitrogens to the acetylene determined reactivity toward GSH

Structure and Metabolism Relationships Were Used toGuide Chemotype Optimization and Compound Selection

Identified compounds with mitigated risk, retained potency and improved PK profile • Select appropriate in vitro systems• Conduct mechanistic biotransformation studies• Collaborate with medicinal chemists

PART III Drug Metabolism Mediated By An Enzyme, Whose Primary Function Is TransformationOf Endogenous Substrates

• Superior potency• Acceptable PK and ADME profiles • No alerts from in vitro metabolism

In vivo BDC rat (IV) results• Biliary excretion of metabolites as main clearance pathway• Extensive hepatic metabolism• Bicyclic hydroxylation-associated products ~ 40%• An unknown metabolite (P + 181 Da) (~ 60%)

LC/MS, NMR of isolated metabolite

Case II: An HCV Inhibitor Metabolized Byan Unexpected Pathway in Rat Liver

Zhuo, et al., DMD, 2016, 44:1332

F

N O

NHO

OHN

X

F3C

Compound 2

Hydroxylation (in vitro)

Candidate Characterization

?

• Superior potency• Acceptable PK and ADME profiles • No alerts from in vitro metabolism

In vivo BDC rat (IV) results• Biliary excretion of metabolites as main clearance pathway• Extensive hepatic metabolism• Bicyclic hydroxylation-associated products ~ 40%• An unknown metabolite (P + 181 Da) (~ 60%)

LC/MS, NMR of isolated metabolite

F

N O

NHO

OHN

X

F3C

Phosphocholine conjugate

O

PO

O O

N

Case II: An HCV Inhibitor Metabolized Byan Unexpected Pathway in Rat Liver

Zhuo, et al., DMD, 2016, 44:1332

F

N O

NHO

OHN

X

F3C

Compound 2

Hydroxylation (in vitro)

Candidate Characterization

Choline Phosphotransferase Catalyzes A Final Reaction for Phospholipid Synthesis And Xenobiotic Metabolism

OHN

P

N

OO

O

CYPs OHN

OH

OHN

O

CDP-choline CMP

Choline

phosphotransferase(s)

Phosphocoline conjugate(a prerequisite step)

O OH

OOR'

O

O

R O O

OOR'

O

O

RP

O

O

O

N

1,2-Diacylglycerol Phosphatidylcholine

Glycerol-3-phosphate + Fatty acyl Co-A

Compound 2

Unexpected results • HCV inhibitors do not resemble the

endogenous substrates• Could not be recapitulated in

hepatocytes or in LS9 with a cofactor

Endogenous pathway

Metabolism ofxenobiotics

Uncertainties led to termination of the bicyclic seriesInterference with the de novo synthesis and function of phospholipids?Relevance to human?More extensive via PO administration?

A Precedent for Phosphocholine Conjugation in Drug Metabolism

O

O

O

O

O

N O

OO

HO

O

OO

O

OH

P

OO

O

NOH

O

O

O

O

N O

OO

HO

O

OO

O

OHCDP-choline CMP

Choline

phosphotransferase(s)

Everolimus Everolimus phosphocholine ester

Zollinger, et al., DMD, 2008, 36:1457

A potent immunosuppressant Detected in human and preclinical species

A primary alcohol

CONCLUSIONS

Focusing solely on CYPs will likely lead to significant disconnects

Selection of appropriate in vitro and in vivo systems greatly assists in identifying the causes of unfavorable DMPK properties

Benchmarking representative and lead compounds will reveal potential ADME-associated liabilities, and will provide valuable evidence in lead optimization and selection

• Structural features may suggest potential pathways• Updated list of known pathways keeps potential metabolites on the radar

Comprehensive ADME characterization of clinical candidates will guide selection of • toxicological species with adequate exposures of human metabolites for safety testing• species undergoing comparable clearance pathways with human

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