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Approaches to Mitigating the Bioactivation Potential of Compounds in Lead Optimization Deepak Dalvie Pharmacokinetics, Dynamics and Metabolism Department Pfizer San Diego. Question. Is covalent binding or GSH Adduct Formation a kill shot for a candidate??.

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  1. Approaches to Mitigating the Bioactivation Potential of Compounds in Lead OptimizationDeepak Dalvie Pharmacokinetics, Dynamics and Metabolism DepartmentPfizer San Diego

  2. Question Is covalent binding or GSH Adduct Formation a kill shot for a candidate??

  3. Adverse Drug Reactions (ADR) A Problem?? • A common cause for drug recalls or black box warnings • Unpredicted or Idiosyncratic ADR (IADR) – more problematic • Normally not observed until phase III or post launch • Frequency of occurrence – 1 in 10000 to 1 in 100000 • Responsible for drug withdrawal • Very expensive - law suits etc. • Patients have been deprived of several good drugs

  4. Drug Safety - One of the leading causes for candidate attrition ADR Liver toxicity Agranulocytosis Other Interaction with Macromolecules Haptenization Reaction with proteins Reactive Metabolite Formation Drug Metabolism • Not easy to predict IADR • No animal models • Lack of specific biomarkers • Circumstantial evidence linksmetabolic activation to IADR • Several examples demonstrate that bioactivation liability and IADR are linked • Kalgutkar AS and Soglia JR (2005). Exp. Opin. Drug Metab. & Toxicol. 1:91-141)

  5. Drugs Associated With IADRs Drugs Withdrawn Temp. Withdrawn or Withdrawn in other Countries Marketed Drugs Isoniazid (antibacterial) Hepatitis (can be fatal) Phenytoin (anticonvulsant) Agranulocytosis, cutaneous ADRs Procainamide (antiarrhythmic) Hepatitis, agranulocytosis Sulfamethoxazole (antibacterial) Agranulocytosis, aplastic anaemia Terbinafine (antifungal) Hepatitis, cutaneous ADRs Ticlopidine (antithrombotic) Agranulocytosis, aplastic anaemia Tolcapone (antiparkinsons) Hepatitis (fatal) Trazodone (antidepressant) Hepatitis Trimethoprim (antibacterial) Agranulocytosis, aplastic anaemia, cutaneous ADRs Thalidomide (immunomodulator) Teratogenicity Valproic acid (anticonvulsant) Hepatitis (fatal), teratogenicity Abacavir (antiretroviral) Cutaneous ADRs Acetaminophen (analgesic) Hepatitis (fatal) Captopril (antihypertensive) Cutaneous ADRs, agranulocytosis Carbamazepine (anticonvulsant) Hepatitis, agranulocytosis Clozapine (antipsychotic) Agranulocytosis Cyclophosphamide (anticancer) Agranulocytosis, cutaneous ADRs Dapsone (antibacterial) Agranulocytosis, cutaneous ADRs, aplastic anaemia Diclofenac (antiinflammatory) Hepatitis Felbamate (anticonvulsant) Hepatitis (fatal), aplastic anaemia (fatal), severe restriction in use Furosemide (diurectic) Agranulocytosis, cutaneous ADRs, aplastic anaemia Halothane (anesthetic) Hepatitis Imipramine (antidepressant) Hepatitis Indomethacin (antiinflammatory) Hepatitis Aclcofenac (antiinflammatory) Hepatitis, rash Alpidem (anxiolytic) Hepatitis (fatal) Amodiaquine (antimalarial) Hepatitis, agranulocytosis Amineptine (antidepressant) Hepatitis, cutaneous ADRs Benoxaprofen (antiinflammatory) Hepatitis, cutaneous ADRs Bromfenac (antiinflammatory) Hepatitis (fatal) Carbutamide (antidiabetic) Bone marrow toxicity Ibufenac (antiinflammatory) Hepatitis (fatal) Iproniazid (antidepressant) Hepatitis (fatal) Metiamide (antiulcer) Bone marrow toxicity Nomifensine (antidepressant) Hepatitis (fatal), anaemia Practolol (antiarrhythmic) Severe cutaneous ADRs Remoxipride (antipsychotic) Aplastic anaemia Sudoxicam (antiinflammatory) Hepatitis (fatal) Tienilic Acid (diuretic) Hepatitis (fatal) Tolrestat (antidiabetic) Hepatitis (fatal) Troglitazone (antidiabetic) Hepatitis (fatal) Zomepirac (antiinflammatory) Hepatitis, cutaneous ADRs Aminopyrine (analgesic) Agranulocytosis Nefazodone (antidepressant) Hepatitis (> 200 deaths) Trovan (antibacterial) Hepatitis Zileuton (antiasthma) Hepatitis For most of these drugs, bioactivation to reactive metabolites has been demonstrated in vitro or in vivo Kalgutkar AS and Soglia JR (2005). Exp. Opin. Drug Metab. & Toxicol. 1:91-141)

  6. Role of Drug Metabolism in Addressing IADR • Identify toxicophores – “Structural Alerts” early on • Especially those undergoing ‘metabolic activation’ • One approach adopted by many pharmaceutical companies • Eliminate metabolic activation of a candidate • Screen for ability to form reactive metabolites very early • Avoid chemical functionalities that undergo bioactivation • No guarantee that this will make compounds safer • But, preventing reactive metabolite formation  chances of IADR • TALL ORDER BUT AVOIDS RISK! • Saves $$ • Saves time and effort

  7. Most definitive Useful in quantitation of the reactive metabolite Helps to detect all reactive metabolites Limited by availability of radiolabel Methods to Assess Bioactivation Potential • Most popular in a discovery setting • Nucleophiles used to trap are: • Glutathione • N-Acetylcysteine • Other nucleophiles - cyanide • Acts as a surrogate marker of covalent binding Evans D. C. et. al. Chem. Res. Toxicol. 2004

  8. General Method for Assessing Reactive Intermediates Drug (10 – 50 mM) + Human Liver Microsomes + GSH (3 mM) NADPH regenerating system (+/-) Incubation at 37º C for 1.0 hr Samples analyzed for GSH adducts using various LC-MS methods

  9. What Does a Positive Signal in These Assays Mean? Some Known Facts: • No assay predicts potential of a new drug to cause IADR • Important to Weigh the Benefits versus Risks • Is it a prototype or a back up? • Indication • Is the drug being developed for an unmet medical need? • First in class? • Dosing regimen of the drug • Acute or chronic? • Drugs that are used chronically are more prone to IADRs

  10. Relationship Between Dose and Frequency of Idiosyncratic Reactions • Dose • A lower dose reduces the risk •  Dose •  • Lesser reactive metabolite formed •  • Lower Risk Issue: • Dose is unknown in the early stages

  11. Other Factors from a Drug Metabolism Perspective • Contribution of pathways that detoxify reactive metabolites • Contribution of competing metabolic routes “After all it is all about body burden of RM”

  12. Case Studies • Paroxetine – an antidepressant SSRI • Raloxifene – A selective estrogen receptor modulator (SERM) • A Case Study from Pfizer

  13. Paroxetine Case Carbene Methylenedioxy Moiety • Paroxetine contains themethylenedioxy group - atoxicophore • Results in inactivation of CYP2D6 • clinical pharmacokinetic interactions with substrates well established An Carbene – Iron Complex §Bertelsen KM, Venkatakrishnan K, Von Moltke LL, Obach RS and Greenblatt DJ (2003) Drug Metab. Dispos. 31:289-293

  14. Paroxetine Also Undergoes Metabolic Activation Incubation with HLM in the presence of GSH and NADPH Zhao SX, Dalvie DK, Kelly JM, Soglia JR, Frederick KS, Smith EB, Obach RS and Kalgutkar AS (2007) Chem. Res. Toxicol. 20:1649-1657.

  15. Incubation of Radiolabeled Paroxetine with HLM Incubation of radiolabel with HLM/NADPH [14C]Paroxetine + Human Liver Microsomes + NADPH Covalent Binding to Microsomal Proteins Zhao SX, Dalvie DK, Kelly JM, Soglia JR, Frederick KS, Smith EB, Obach RS and Kalgutkar AS (2007) Chem. Res. Toxicol. 20:1649-1657.

  16. Inference? • Paroxetine is prone to bioactivation • Covalently binds to microsomal protein • Would one nominate Paroxetine if it was to be developed as an antidepressant to date? • Paroxetine is a commonly prescribed antidepressant • IADRs (especially hepatotoxicity) are extremely rare

  17. Effect of GSH on Covalent Binding of Paroxetine Incubation with Human Liver Microsomes Covalent Binding Glutathione Adducts GSH and UDPGA Mops Reactive Intermediate Reduces covalent binding! Zhao SX, Dalvie DK, Kelly JM, Soglia JR, Frederick KS, Smith EB, Obach RS and Kalgutkar AS (2007) Chem. Res. Toxicol. 20:1649-1657.

  18. Covalent Binding of Paroxetine in Human S9 S-Adenosyl methionine /COMT GSH Conjugates Sulfate and Glucuronide Conjugates Catechol detoxified via methylation O-Methoxy derivatives formed Reduces covalent binding! This is in addition to GSH conjugation! Zhao SX, Dalvie DK, Kelly JM, Soglia JR, Frederick KS, Smith EB, Obach RS and Kalgutkar AS (2007) Chem. Res. Toxicol. 20:1649-1657.

  19. Analysis of the Data • Paroxetine was bioactivated but: • o-Quinone is detoxified by Glutathione • Catechol detoxified by methylation and glucuronidation • Additional factors that may contribute to less IADRs with paroxetine • Low daily dose (20 mg QD) • Reactive metabolite burden readily handled by endogenous glutathione pool in the mammal • CYP2D6 inactivation by paroxetine following chronic dosing may inhibit its own metabolism • Decreases the body burden of the catechol ando-benzoquinone GSH Conjugates Quinone Drug Catechol Sulfate & Glucuronide Conjugates Methylation

  20. Lesson • Thorough knowledge of ALL metabolism pathways is important • GSH conjugate detection needs to be put into right context • GSH is a detoxication pathway! • Acts as a electrophile mopping agent in the body • Early assessment of dose can help

  21. Raloxifene Case G1 G1 250000 250000 uAU uAU 200000 200000 M1 M1 Raloxifene Raloxifene Absorbance Absorbance 150000 150000 G2 G2 M2 M2 100000 100000 Time (min) Time (min) Incubation with HLM/NADPH and GSH • A second generation SERM approved for osteoporosis • Bioactivated by CYP3A4 to quinonoid intermediates • A mechanism-based inactivator of CYP3A4 • No IADRs or DDIs reported GSH Conjugates So would one nominate this compound for further development?

  22. Why is Raloxifene is Devoid of any IADRs or DDI • Primary route of raloxifene metabolism – Glucuronidation Covalent Binding Incubation of [14C]Raloxifene with HLM % bound relative to only NADPH 72 68 50 Glucuronidation of raloxifene 50% reduction in covalent binding observed in the presence of GSH and UDPGA! Dalvie D, Kang P, Zientek M, Xiang C, Zhou S, and Obach RS (2008) Chem. Res. Toxicol. 21, 2260.

  23. Impact of Intestinal Metabolism on Bioactivation of Raloxifene Control (no UDPGA) • Glucuronidation primarily catalyzed by 1A8 and 1A10 • UGT1A1 also catalyzes the glucuronidation but to a minor extent • UGT 1A8 and 1A10 are exclusively present in the small intestine Preincubation Experiment Preincubation with Human Intestinal Microsomes results in significant decrease in covalent binding! Daniel C. Kemp, Peter W. Fan, and Jeffrey C. Stevens Drug Metab. Dispos. 30, 694.2002 Dalvie D, Kang P, Zientek M, Xiang C, Zhou S, and Obach RS (2008) Chem. Res. Toxicol. 21, 2260.

  24. Efficiency of Raloxifene Glucuronidation versus Oxidation Efficiency of detoxification pathway explains the impact of glucuronidation on hepatic bioactivation Dalvie D, Kang P, Zientek M, Xiang C, Zhou S, and Obach RS (2008) Chem. Res. Toxicol. 21, 2260.

  25. What does this data tell us! GI Tract Systemic Circulation Reduces the body burden Liver Site of metabolism Low exposure of Raloxifene Highly Efficient process • Intestinal glucuronidation limits the amount of raloxifene undergoing bioactivation • Dose of raloxifene – 60 mg QD • Other pathways of clearance are responsible for reducing the body burden

  26. Lesson! • The compound would have been “withdrawn from development” in the absence of all the relevant metabolism data to date • Understand the impact/contribution of other metabolic pathways • Identify enzymes responsible – maybe non-hepatic • Especially the intestine Bottomline Understand the Metabolism of the candidate thoroughly prior to any Major Decisions

  27. A Pfizer Case Mitigating the Bioactivation Risk of 11--HSD-1 Inhibitor PF-915275

  28. 11-b-HSD-1 Inhibitors 11βHSD1 inhibitor inhibits conversion of cortisone to cortisol • Hepatic Glucose Cortisol (F) Active glucocorticoid Cortisone (E) Inactive glucocorticoid Project Goal • Develop an 11βHSD1(11β-HydroxySteroid Dehydrogenase type-1) inhibitor for the treatment of Type II diabetes

  29. Lead Candidate in the 11-HSD1 Program • Has great physicochemical properties • Good potency • Pharmacokinetic Attributes • Key Issues from a metabolism perspective • Risk of bioactivation • No Structural Alerts – yet +ve signal in RM assay PF-915275 Natilie Hosea Sajiv Nair

  30. Metabolism of PF-915275 Site of bioactivation and adduct formation No metabolism at this site HLM/NADPH GSH Bis-conjugate Adduct Unique GSH Adduct Reactive metabolites trapped as Novel GSH Adducts

  31. Risk Assessment for PF-915275 • Preliminary dose prediction was 30 – 50 mg • Uncertainties in clearance (low) and Ceff • Oxidation leading to reactive metabolites • a primary route • Question? • Do other competing metabolic pathways or detoxication of the reactive metabolite play a role?

  32. Risk Analysis: Assessment of Reactive Metabolite Formation in LM/NADPH/UDPGA No oxidative metabolites observed in the absence of GSH or UDPGA! Minor Pathway UDPGA UDPGA [O]/GSH Deepak Dalvie Natilie Hosea GSH Adducts • 14C-PF-915275 synthesis accelarated to see the impact on covalent binding • Assess the covalent binding in vitro and in vivo

  33. Covalent Binding Studies with [14C]-PF-915275Using Human Liver S9 M3 RM RM RM RM RM RM RM RM M4 Drug Drug Drug Drug Drug Drug Drug Drug M1 M1 M1 M1 M1 M1 M1 M1 S9 Assay M2 M2 M2 M2 M2 M2 M2 M2 • 915275 – was bioactivated but: • Metabolites were detoxified by GSH, sulfation and glucuronidation • In vitro covalent binding near background when other co-factors are used Ping Kang Sue Zhou

  34. Further De-risking Factors to Move PF-915275 into Development • Rat studies performed • Very low binding to liver protein in vivo (0.21 pmol/mg; <0.05% of dose) • No GSH conjugates or related metabolites observed • Proper PK/PD studies helped to refine the dose • Refined Dose =0.3 to 3 mg using monkey PK/PD data • Investigative toxicology studies performed • Compound dosed to GSH depleted rats

  35. Overall Conclusions From the 3 Cases • Covalent binding to microsomal proteins may not be predictive of toxicity • Some refs by Obach et. al. further confirm this • Thorough assessment of metabolism is pivotal in early discovery • Important to consider the contribution of other metabolic pathways (hepatic or extrahepatic) in light of a positive signal • Some idea of total daily dose/exposure helps • Get a range to see if the dose is lower than 20 – 50 mg • Use in vitro potency and clearance from HLM as first cut • Refine the dose prediction from the efficacy model • Estimate the body burden of the reactive metabolite: • DRM = D x fa x fm x fRM • Gan and co-workers Chem. Res. Toxicol. 2009, 22, 690.

  36. Is Covalent Binding or GSH Adduct Formation A Kill Shot?? • Clearly, a positive signal by itself ‘is not’ a kill shot • Not good predictors of IADR • Needs to be considered as a ‘flag’ to trigger additional studies • GSH conjugation is a detoxication pathway –need to put in right context • However, helps to elucidate mechanisms of bioactivation • Useful in circumventing the liability through iterative design • Liver microsomal assays do not always address all metabolic pathways for a compound • Use of hepatocytes or S-9 – supplemented with co-factors gives a better picture of all metabolic pathways

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