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

Transcription

1 2017 anjing International Conference of Drug Metabolism -P450-MEDIATED METABLISM: IDETIICATI AD IMPLICATI DURIG CMPUD SELECTI AD PTIMIZATI Xiaoliang Zhuo Metabolism and Pharmacokinetics Preclinical Candidate ptimization Bristol-Myers Squibb Wallingford, CT

2 P450s Still the Main Contributors to Metabolism of Approved ral and Intravenous Small Molecule Drugs ( ) Multiple classes of non-p450 enzymes play a significant role in drug metabolism ydrolysis of amide, etc. Cerny, et al., DMD, 2016, 44:1246 (Aldehyde oxidase) (sulfotransferases, cytidine deaminase, dehydrogenase, etc.) Prodrugs excluded Secondary pathways excluded Based on human ADME data 10% of dose = major pathways Approved drugs possess optimized ADME profiles Unfavorable properties, e.g. exclusive A-mediated metabolism, likely eliminated during discovery phase

3 PART I Aldehyde xidase (A): An Emerging ocus As A Result of SAR Intended To Mitigate P450-mediated Clearance

4 A Differs from P450 in Many Respects ptimize physical-chemical properties: decreasing lipophilicity and electron density of aryls, etc. Mitigated metabolic clearance by P450s leads to. increased metabolism by A nly one isoform in human Expressed in cytosol In vitro metabolism in liver microsomes does not reveal A-mediated pathways Appropriate subcellular fractions or hepatocytes required Predominantly present in liver Also expressed in kidney o cofactor required Used to differentiate oxidation mediated by CYPs and Ms

5 A Targets Electron-deficient Carbons S ydrolysis * GDC-0843 (Bruton s tyrosine kinase inhibitor) * Carboxylase also involved Sodhi, et al., DMD, 2015, 43:908 xidation S SGX523 (c-met tyrosine kinase inhibitor) S 2-quinolinone-SGX523 Diamond, et al., DMD, 2010, 38:1277 ucleophilic attack by Mo- and utilization of water as a source of oxygen xidation of electron-deficient carbons in aza-heterocycles Reduction of - or -S bond Amide hydrolysis S Mn VI S S Molebdenum center

6 Complications Associated with Prediction of Metabolic Clearance of A Substrates in uman 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 A contents vary among vendors and batches Unique expression pattern Selection of appropriate in vitro metabolic systems for IVIVC critical Discontinued development of A substrates due to: Under-prediction of human clearance, low bioavailability: Carbazeran, BIBX1382, Safety issue: SGX523 (obstructive nephropathy due to the A metabolite) utzler et al., Expert pinion in Drug Metabolism and Toxicology 2013, 9(2):

7 A Paradigm to Ensure a Comprehensive Evaluation of Metabolic Pathways Structural features/alerts Poor IVIVC Identification of A-mediated Metabolism Metabolic systems +/- cofactors indings and conclusions Liver microsomes with or without ADP Liver cytosol without cofactor or hepatocytes Metabolism by CYPs ew +16 Da products: non-cyp mediated pathway * (1) A positive control should be used to confirm the result (2) ydralazine also inhibits CYP2D6 (3) Use of multiple batches is recommended Liver cytosol, liver S9 or hepatocytes + A selective inhibitor (hydralazine) * To confirm metabolism by A Estimation of m(a)

8 Extensive Metabolism by A Represents An Unfavorable ADME Property, ere IS ow to Avoid it. uman CL projection Identification of the role of A - Estimation of m(a) - If exclusively or predominantly by A Structural modification - Block A sites - Rearrange atoms - ther heteroaryls - Maintain potency + other ADME properties - Allometric scaling not suitable - Simple scaling from human in vitro systems likely results in under-prediction - Compared with benchmark compounds * Advance compound with diverse metabolic clearance mechanisms Toxicological considerations - A 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

9 PART II Glutathione S-Transferase-Mediated Metabolism

10 Glutathione S-Transferases Catalyze ucleophilic Attack to Electrophilic Atoms Expression Primarily present in cytosol Also in endoplasmic reticulum (membranebound) igh concentrations in liver, kidney, and lung Isoforms Multiple members Conjugation via arene oxide aphthalene Cl P450 Direct replacement of an electron-withdrawing group Cl - GS - Cl Cl GS -, + SG SG SG Mechanisms Substrates include a wide array of electrophilic xenobiotics and their intermediates 2 1,2-Dichloro-4-nitrobenzene Cl Addition 3 C Cl 3 C 2 GS -, + Cl 3 C SG S 3 Efavirenz metabolite P450 S 3 S 3

11 Case I: GS Conjugation Mediated by Cytosolic GST Led to Rapid Depletion of an mglur5 Allosteric Modulator Moderate-high CL in rats But stability ~95% in RLM Any pathways mediated by non-cyps? A A GS GS GST B B C In vitro scale-up production Isolation + MR analysis C D D Rat or human o cofactor added + GS Liver cytosol Stable > 80% of the parent formed GS conjugates Liver microsomes Potassium phosphate buffer (p 7.4) + GS + ADP + GS or ADP + GS ~1% oxidative metabolites Suggesting a role of cytosolic GST 6% GS conjugates Suggesting involvement of membrane-bound GST, but to a lesser extent ~ 2% GS conjugates, indicating a chemical reaction. (multiple region- and configurational isomers identified)

12 Proposed Mechanism of GS Conjugation: ucleophilic Attack nto Acetylene Moiety by Thiolate Anion GS GST SG C SG GS + Compound 2 SG C SG GST: GS S-transferase GS Thiolate anion C The negatively charged intermediate stabilized via delocalization to the adjacent heteroaryl nitrogens via resonance Zhuo, et al., DMD, 2015, 43:578

13 Extensive Reaction with GS Is ot A avorable Property mglur5 compounds featuring two heterocycles proximal to an acetylene Extensive GS addition to the acetylene moiety through GST and chemical reactivity A B C D Rapid clearance of compounds Depletion of GS May leave cells less protected from damage by reactive oxygen species May lead to formation of protein adducts

14 Structure and Metabolism Relationships Were Used to Guide Chemotype ptimization and Compound Selection Presence of two heteroaryls and proximity of nitrogens to the acetylene determined reactivity toward GS 1 2 A, D A, E Extent of GS Conjugation in LS9 B, E 6 C, E A. lanked by two heteroaryls B. lanked by one heteroaryl C. lanked by no heteroaryl D. itrogens to support delocalization E. itrogens T to support delocalization Identified compounds with mitigated risk, retained potency and improved PK profile Select appropriate in vitro systems Conduct mechanistic biotransformation studies Collaborate with medicinal chemists

15 PART III Drug Metabolism Mediated By An Enzyme, Whose Primary unction Is Transformation f Endogenous Substrates

16 Case II: An CV Inhibitor Metabolized By an Unexpected Pathway in Rat Liver ydroxylation (in vitro) X Superior potency Acceptable PK and ADME profiles o alerts from in vitro metabolism 3 C Compound 2? Candidate Characterization 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 Da) (~ 60%) LC/MS, MR of isolated metabolite Zhuo, et al., DMD, 2016, 44:1332

17 Case II: An CV Inhibitor Metabolized By an Unexpected Pathway in Rat Liver ydroxylation (in vitro) X Superior potency Acceptable PK and ADME profiles o alerts from in vitro metabolism X P 3 C Compound 2 Candidate Characterization 3 C Phosphocholine conjugate 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 Da) (~ 60%) LC/MS, MR of isolated metabolite Zhuo, et al., DMD, 2016, 44:1332

18 Choline Phosphotransferase Catalyzes A inal Reaction for Phospholipid Synthesis And Xenobiotic Metabolism Endogenous pathway Glycerol-3-phosphate + atty acyl Co-A R R' 1,2-Diacylglycerol CDP-choline CMP R P R' Phosphatidylcholine Metabolism of xenobiotics Compound 2 CYPs (a prerequisite step) Choline phosphotransferase(s) P Phosphocoline conjugate Unexpected results CV inhibitors do not resemble the endogenous substrates Could not be recapitulated in hepatocytes or in LS9 with a cofactor Uncertainties led to termination of the bicyclic series Interference with the de novo synthesis and function of phospholipids? Relevance to human? More extensive via P administration?

19 A Precedent for Phosphocholine Conjugation in Drug Metabolism A primary alcohol P CDP-choline CMP Choline phosphotransferase(s) Everolimus A potent immunosuppressant Everolimus phosphocholine ester Detected in human and preclinical species Zollinger, et al., DMD, 2008, 36:1457

20 CCLUSIS ocusing 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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