Risk Assessment of Reactive Metabolites in Drug Discovery: A Focus on Acyl Glucuronides and Acyl-CoA Thioesters

Transcription

1 Risk Assessment of Reactive Metabolites in Drug Discovery: A Focus on Acyl Glucuronides and Acyl-CoA Thioesters The Delaware Valley Drug Metabolism Discussion Group 2017 Rozman Symposium HUMAN BITRANSFRMATIN: THRUGH THE MIST Langhorne, PA, June 13, 2017 Mark Grillo, PhD Drug Metabolism & Pharmacokinetics MyoKardia South San Francisco, CA mgrillo@myokardia.com

2 Commercialization Drug Process Discovery verview & Development Therapeutic Area/Disease pportunity Assessment Lead Selection & Pre-clinical Testing Product Strategy Development Market Readiness & Regulatory Approval Launch / Post-Launch Lifecycle Management Discovery Screen Hit-to- Lead Lead ptimization Pre-clinical Phase 1 Phase 2 Phase 3 Filing Launch Post- Launch Preclinical Pharmacokinetics & Drug Metabolism Assays Discovery Target Selection & Validation Screen ptimize Pharmacokinetics Liver microsomal stability Membrane permeability Efflux/transport assays Plasma stability Rat PK Protein binding Blood to plasma ratio Early DDI Assessment Enzyme induction PXR activation CYP inhibition Hit-to-Lead Lead compound Selection Lead ptimization Characterize Preclinical Candidate Intrinsic clearance Excretion balance Definitive metabolite ID Higher species PK Human PK prediction Assess DDI Potential Competitive CYP IC 50 Time-dependent CYP inhibition Hepatocyte induction CYP reaction phenotyping Detection and Assessment of Chemically-Reactive Metabolites 2

3 Circumstantial Evidence Links Reactive Metabolites to Adverse Drug Reactions 3 Toxicity is a frequent cause of drug withdrawal from the market. Reactive drug metabolites may mediate liver injury via covalent modification of biological macromolecules. Reactive metabolites of carboxylic acidcontaining drugs, acyl glucuronides and acyl-coa thioesters, might contribute to idiosyncratic drug toxicity. Underlying mechanisms not clear. Believed to be immune-mediated. Liver Injury

4 Drugs Withdrawn and Associated with Idiosyncratic Drug Toxicity Alcofenac (anti-inflammatory) Hepatitis, rash Alpidem (anxiolytic) Hepatitis (fatal) Amodiaquine (antimalarial) Hepatitis, agranulocytosis Amineptine (antidepressant) Hepatitis, cutaneous ADRs Benoxaprofen (anti-inflammatory) Hepatitis, cutaneous ADRs Bromfenac (anti-inflammatory) Hepatitis (fatal) Carbutamide (antidiabetic) Bone marrow toxicity Ibufenac (anti-inflammatory) Hepatitis (fatal) Iproniazid (antidepressant) Hepatitis (fatal) Metiamide (antiulcer) Bone marrow toxicity Nomifensine (antidepressant) Hepatitis (fatal), anemia Practolol (antiarrhythmic) Severe cutaneous ADRs Remoxipride (antipsychotic) Aplastic anaemia Sudoxicam (anti-inflammatory) Hepatitis (fatal) Tienilic Acid (diuretic) Hepatitis (fatal) Tolrestat (antidiabetic) Hepatitis (fatal) Troglitazone (antidiabetic) Hepatitis (fatal) Zomepirac (anti-inflammatory) Hepatitis, cutaneous ADRs In many cases, metabolic activation reactive metabolites leading to covalent binding to protein demonstrated in vitro and/or in vivo. 4 Carboxylic acid drugs Kalgutkar and Soglia (2005) Exp. pin. Drug Metab. Toxicol. 1,

5 Danger Hypothesis Illustration for Immune-mediated Idiosyncratic Hepatotoxicity 5 Kaplowitz (2005) Idiosyncratic drug hepatotoxicity. Nature Reviews 4,

6 Assessing Formation of / Exposure to Reactive Drug Metabolites Formation of adducts with nucleophiles In vitro trapping studies with glutathione (GSH) or CN -, and others In vivo metabolic profiling studies (e.g., GSH-adducts in bile, NACadducts in urine) Covalent binding studies with radiolabeled drug Definitive Measures total burden of protein bound-drug residue Helpful compliment to trapping studies Trapping studies with glutathione and covalent binding studies with radiolabel carboxylic acid-containing drugs to examine the importance of acyl glucuronides vs. acyl-coa thioesters as reactive intermediates in vitro and in vivo. 6 Park et al. (2011) Nature Rev. Drug Discov., 10: Evans et al. (2004) Chem. Res. Toxicol., 17:3-16.

7 Relationship between daily-dose of oral medications and idiosyncratic drug-induced liver injury (DILI) Drugs dosed at <10 mg/day extremely rare to lead to idiosyncratic drug toxicity (whether or not these drugs are prone to metabolic activation). 7 Kalgutkar (2009) Chem. Biodiver. 6, ; Lammert et al., Hepatology 47, (2008)

8 Assessing the Potential for Risk of Idiosyncratic Drug Toxicity Caused by Candidate Drugs (AstraZeneca, 2012) 8 Drugs that demonstrated high covalent binding (CVB) burden (>1 mg/day, Zones 3 and 4) correlated with many of the toxic compounds. CVB Burden (#9) Ibufenac 24 (#32) Ibuprofen 5.2 (#20) Diclofenac 1.8 (#29) Caffeine <0.23 f cvb = (CVB 0.26 mg)/(turnover 4000 pmol) CVB burden = fcvb dose Recommendation: <1 mg/day exposure (CVB burden) to reactive metabolites Thompson et al. (2012) Chem. Res. Toxicol. 25:

9 uau uau uau uau Cross-species Liver Microsomes Metabolite Profile: LC/UV detection Identification of GSH-adduct as Major Metabolite in HLM fortified with NADPH and GSH RT: SM: 11G UV 280 nm Rat UV 280 nm Dog GSH-adduct hydroxylated ndr Drug metabolite metabolite NL: 5.38E4 nm= PDA 3509rlm+n+g NL: 5.94E4 nm= PDA 3509dlm+n+ g UV 280 nm Cyno monkey NL: 5.16E4 nm= PDA 3509clm+n+ g UV 280 nm Human NL: 5.92E4 nm= PDA 3509hlm+n+ g ndr, not drug related Time (min) GSH-adduct major metabolite formed in HLM (1.5 % relative peak area to parent, ~50% of total metabolite peak area) Decision to perform reactive metabolite/toxicity risk assessment prior to advancement. 9 Unpublished observations

10 C o v a le n t B in d in g to P ro te in (p m o l e q./m g /p ro te in ) C o v a le n t B in d in g to P ro te in (p m o l e q./m g /p ro te in ) Covalent Binding (CVB) of Radiolabeled Test Drug and [ 14 C]Diclofenac to Protein in Human Hepatocytes in Suspension: Prediction of CVB Burden in Human Compound Test Compound Diclofenac Diclofenac (Thompson, et al.) Covalent Binding In c u b a tio n T im e (h ) [ 1 4 C ]D ic lo fe n a c [ 1 4 C ]D ic lo fe n a c (lite ra tu re ) R a d io la b e le d T e s t C o m p o u n d R a d io la b e le d Metabolic Stability In c u b a tio n T im e (h ) T e s t C o m p o u n d (0 % tu rn o v e r) [ 1 4 C ]D ic lo fe n a c (8 7.5 % tu rn o v e r) [ 1 4 C ]D ic lo fe n a c (lite ra tu re ) CVB (pmol/mg Incubation Turnover Daily CVB Burden f protein) time (h) (%) cvb Dose (mg) (mg) 24.0 ± ± * ± ± * ± ± * ± ± ± ± ± ± Substrate concentration (10 µm), incubation volume (2.5 ml); *no loss of drug detected (assume 1% turnover); Preliminary prediction of drug dose in human based on rat PK/PD and simple allometric scaling (4-species) for human PK. 10

11 Metabolites in Safety Testing (MIST) (FDA, 2008) Acyl Glucuronides Ref. 5 Faed, EM, 1984, Properties of Acyl Glucuronides. Implications for Studies of the Pharmacokinetics and Metabolism of Acidic Drugs, Drug Metab Rev, 15:

12 Partial list of carboxylic-acid containing drugs withdrawn from clinical use and/or also associated with allergic reactions B r C l C l Alclofenac Bromfenac Ibufenac H 3 C Zomepirac N Withdrawn NH 2 C H 3 H H H F H S C l C l C l N Tienilic Acid Benoxaprofen N C H 3 H Flunoxaprofen N Indoprofen H C H 3 H C H 3 H H H H H 3 C C l Tolmetin Allergic reactions H 3 C Ibuprofen C l NH Diclofenac H H 3 C Fenoprofen H H H C H 3 N H H C l F F N S H H H H C H 3 C H 3 Clofibric Acid Probenecid Diflunisal Valproic Acid H 12 Fung et al. (2001) Drug Information Journal, 35, Stepan et al. (2011) Chem Res Toxicol, 24,

13 Relative Intensity (%) % R e m a in in g Drug Acyl Glucuronide: Instability (Primarily by Acyl Migration) Time = 20 min ph 7.4, 37 o C Time = 0 min ph 7.4, 37 o C 1--bisomer 1--bisomer *Acyl migration isomers * * * t 1 /2 ~ 1 0 m in In c u b a tio n T im e (m in ) Retention Time (min) * * Incubation of 1--βdrug-acyl glucuronide in buffer (ph 7.4, 37 o C) to determine degradation rate/ degradation half-life. LC-MS/MS analysis: Xue et al. ptimization to eliminate interference of migration isomers for measuring 1--βacyl glucuronide with out extensive chromatographic separation. Rapid Commun. Mass Spectrom. 2008; 22:

14 14 Mechanisms for A) the transacylation of protein nucleophiles by 1-β--acyl-linked glucuronides and B) for stable adduct formation with lysine-residues on proteins by reaction with open-chain acyl glucuronide migration isomers.

15 Covalent Binding (mole/mole protein x e-3) Predictability of Covalent Binding of Acidic Drugs in Man Zomepirac Cl H 3C C H 3 N H Tolmetin H 3C C H 3 N H Carprofen Cl H N H 3 C H H Fenoprofen H 3C H H Increasing the degree of substitution at the alphacarboxy-carbon correlates with increasing stability (decreasing reactivity with protein) of the acyl glucuronide. Benet et al. (1993) Life Sci, 53:141-6 Smith et al. (1986) J Clin. Invest. 77: H 3 C H H H 3 C H N Furosemide Beclobric Acid S H 2 N Cl Cl Tolmetin Zomepirac (R)-Fenoprofen 2 (S)-Fenoprofen (R)-Carprofen 1 (S)-Carprofen Furosemide ( R,S )-Beclobric Acid Degradation Rate Constant (k, 1/hrs)

16 16 The data provide conclusive evidence for the covalent binding of tolmetin glucuronide to HSA by Schiff-base formation with lysine ε-amino groups. In all of the identified tolmetin-containing peptides, the glucuronic acid moiety was present. Ding et al., Proc. Natl. Acad. Sci. 90 (1993). ur results establish that the binding of these reactive metabolites to nucleophilic sites of proteins occur via two different mechanisms: one involving imine (Schiff base) formation and the other involving nucleophilic displacement of glucuronic acid. Ding et al., Drug Metab Dispos., 23 (1994). Zomepirac Zia-Amirhosseini et al. (1995) Biochem. J. 311: Benoxaprofen Qiu et al. (1998) Drug Metab. Dispos. 26: Diclofenac Hammond et al. (2014) J Pharmacol. Exper. Therap. 350: Fevipiprant Pearson et al. (April, 2017) DMD Fast Forward

17 Predictability of Idiosyncratic Drug Toxicity Risk for Carboxylic Acid-Containing Drugs Based on the Chemical Stability of Acyl Glucuronide The classification value of the degradation halflife which separated the safe drugs from the withdrawn drugs was calculated to be 3.6 h. The KPB system was considered to be the best for evaluating the stability of AGs, and the classification value of the half-life in KPB serves as a useful key predictor for the IDT risk. 17 Sawamura et al. (2010) Drug Metab. Dispos., 38:

18 Covalent Binding: Bioactivation of Valproic acid (VPA) Microvesicular steatosis proposed to be due to irreversible inhibition of fatty acid metabolism leading to a build-up of lipids. Covalent binding studies in rat hepatocytes showed that inhibition of P450 or Glucuronidation had no effect on covalent binding to protein, whereas inhibition of acyl-coa formation led to a 90% decrease in covalent binding to protein. VPA-acyl-CoA implicated in CB to protein 18 (UW) Porubek et al. (1989) Drug Metab Dispos. 2:

19 Proposed Metabolism Routes of Carboxylic Acid-Containing Drugs to Reactive Derivatives and Non-Toxic Products Remains intracellular, not excreted, doesn t circulate in plasma 1 2 Excretion in urine, bile, detection in plasma Conjugation Reactions: , 3 -, A c y l Glucuronides Glycine Amides Taurine Amides Carnitine Esters Hybrid Triglycerides Choline Esters Fatty Acylation? Acyl-CoA Thioester Covalent Binding to Protein A c y l G l u cu r o n i d e 6 5 pen-chain Aldehydes 1. Acyl-CoA Formation 2. Acyl Glucuronidation 3. Acyl Migration 4. Conjugation Reactions 5. Ring-Chain Tautomerism 6. Schiff-Base Formation With Proteins Potential Toxicity 19

20 Acyl-CoA Synthetase-mediated Formation of S-acyl-CoA Thioesters Leading to the Transacylation of GSH and Protein nucleophiles Detoxification? Adverse Drug Reactions? 20 Detoxification

21 Acyl-CoA Thioesters Shown to Acylate Proteins In Vitro (early reports) Salicyl-SCoA Arachidonyl-SCoA Palmitoyl-SCoA Tishler and Goldman (1970) Properties and reactions of salicyl-coenzyme A. Biochem. Pharmacol. 19: Yamashita A. et al. (1995) Acyl-CoA binding and acylation of UDP-glucuronosyltransferase isoforms of rat liver: their effect on enzyme activity. Biochem. J. 312: Duncan, J. A. and Gilman, A. G. (1996) Autoacylation of G protein alpha subunits. J. Biol. C hem. 271: Nafenopin-SCoA S allustio, B. C. (2000) In vitro covalent binding of nafenopin-c oa to human liver proteins. Toxicol. App. Pharmacol. 163: Clofibryl-SCoA 21 Grillo, M.P. and Benet L.Z. (2002) Studies on the reactivity of clofibryl-sacyl-coa thioester with glutathione in vitro. Drug Metab. Dispos. 30:55-62.

22 Enantioselective Covalent Binding Studies with a Model Profen, 2-Phenylpropionic Acid (2-PPA), in Rat Hepatocytes 22 (UCSF) Li et al. (2002) Chem. Res. Toxicol. 15:

23 Enantioselective Covalent Binding Studies with 2-Phenylpropionic Acid in vitro in Rat Hepatocytes Covalent binding to protein correlates closely with acyl-coa formation but not acyl glucuronidation. Corresponding studies in vivo in rat showed CB to liver protein in favor of the acyl-coa thioester. Li et al., JPET, 305, (2003) 23 (UCSF) Li et al. (2002) Chem. Res. Toxicol. 15:

24 [ 14 C]Phenylacetic Acid: Reversible Covalent Binding to Protein in Rat Hepatocytes Covalent Binding (pmol equivalents/mg protein) [PA-CoA], M S N H H Phenylacetic Acid (PAA) Acyl-CoA Synthetase N H H 3 C CH 3 H H P P H H Phenylacetyl-S-Acyl-CoA (PA-CoA) Transacylation of protein Glycine nucleophiles conjugation Protein adducts ATP, CoASH NH Phenylacetylglycine (PA-gly) H N H H H P H N H H H NH 2 N N 350 PA-CoA 300 Covalent Binding Incubation Time (min) KDa PAA-acyl-CoA formation in rat hepatocytes leads to the highly selective, but reversible, covalent binding to hepatocyte proteins, but not to the transacylation of glutathione KDa (Amgen) Grillo and Lohr (2009) Covalent binding of phenylacetic acid to protein in rat hepatocytes. Drug Metab. Dispos

25 In vitro covalent binding/hlm Ibuprofen, Ibufenac, Zomepirac, Tolmetin, Suprofen, Tienilic acid, Fenclozic acid all formed acyl glucuronides, but did not lead to covalent binding to liver microsomal protein. Ibuprofen, Ibufenac, Fenclozic acid, and Tolmetin Acyl-CoA-mediated covalent binding Suprofen and Tienilic acid P450-mediated covalent binding 25 Darnel et al. (2015) Chem. Res. Toxicol. 28:

26 Metabolism of Nicotinic Acid Liver Dysfunction Cases of severe hepatic toxicity, including fulminant hepatic necrosis. Dosing: Recommended dietary allowance = mg/day. Tolerable upper intake level = 35 mg/day (to avoid flushing). Hepatotoxicity observed at 500 mg/day and higher. Flushing and hepatotoxicity are important adverse effects of nicotinic acid. 26 Stern (2007) The role of nicotinic acid metabolites in flushing and hepatotoxicity. J. Clin. Lipidology 1:

27 C o v a le n t B in d in g (p m o l/m g p ro te in ) In Vitro Covalent Binding Studies with [ 14 C]Nicotinic Acid and [ 14 C]Diclofenac in Human Hepatocytes [ 1 4 C ]-N ic o tin ic a c id (1 0 M ) a n d [ 1 4 C ]D ic lo fe n a c (1 0 M ) T im e -d e p e n d e n t C o v a le n t B in d in g to P ro te in in In c u b a tio n s w ith H u m a n H e p a to c y te s (1 m illio n v ia b le c e lls /m L ) in S u s p e n s io n (W illia m s M e d ia E ) D ic lo fe n a c C o v a le n t B in d in g % R e m a in in g D ic lo fe n a c M e ta b o lism N ic o tin ic A c id C o v a le n t B in d in g N ic o tin ic A c id M e ta b o lism 27 Compound Nicotinic Acid Diclofenac In c u b a tio n T im e (h ) CVB (pmol/mg protein) Incubation Time (h) 0 f cvb = (CVB 0.26 mg)/(turnover 4000 pmol) CVB burden = f cvb *dose Daily Dose Turnover f cvb (mg) CBV Burden (mg) *140 *2 *1.0 * *1.8 Unpublished observations *Thompson et al. (2012) Chem. Res. Toxicol. 25:

28 Acyl-CoAs reported to be 20- to 100-fold more reactive toward GSH than the corresponding acyl glucuronides in buffer (ph 7.4, 37 C) Ibuprofen 20-fold Clofibric acid 40-fold 2-Phenylproprionic 70-fold Naproxen 100-fold 28 Grillo (2011) Current Drug Metab. 12:

29 Acyl-S-CoA Reactivity with Glutathione: Influenced by both Steric and Electronic Effects Phenoxyacetic Salicylic (ortho-hydroxy-benzoic) Increasing the degree of substitution at the alphacarboxy-carbon correlates with increasing stability (decreasing reactivity with GSH) of the acyl-coa. 29 Skonberg et al.(2008) Metabolic Activation of Carboxylic Acid Drugs. Expert pin. Drug Metab. Toxicol. 4:

30 Reactivity of S-Acyl-Glutathione Thioesters (0.1 mm) with N-Acetylcysteine (NAC, 1 mm) at 25 o C and ph 7.4 Increasing the degree of substitution at the alpha-carboxy-carbon correlates with increasing stability (decreasing reactivity with NAC) of the acyl-sg. 30 (UCSF) Grillo and Benet (2002) Drug Metab Dispos. 30:

31 Enantioselective Studies with (R)- and (S)-Ibuprofen/Hepatocytes Proposal that Ibuprofenacyl-CoA thioester, and not ibuprofen acyl glucuronide, mediates the transacylation of GSH in rat hepatocytes. 31 (Pharmacia/Pfizer) Grillo and Hua (2008) Chem. Res. Toxicol. 21:

32 Enantioselective Bioactivation of Ibuprofen by Acyl-CoA Formation Favoring the R-Ibuprofen Isomer in Rat Hepatocytes Acyl-CoA Formation Acyl-SG Formation 32 (Pharmacia/Pfizer) Grillo and Hua (2008) Chem. Res. Toxicol. 21:

33 Carboxylic Acids Bioactivated by Acyl-CoA Formation Leading to S-Acyl-Glutathione Adducts Zomepirac Ibuprofen Mefenamic Acid Tolmetin Flunoxaprofen Cholic Acid analogues Diclofenac Benoxaprofen 33 Grillo (2011) Current Drug Metab. 12:

34 In vivo (Advil, 800 mg) 34 (Amgen) Grillo et al. (2013) Interaction of Ƴ-Glutamyltranspeptidase with Ibuprofen-S-Acylglutathione in vitro and in vivo in human. Drug Metab. Dispos. 41:

35 Relative Abundance In Vivo Formation of Ibuprofen-N-Acyl-Cysteine in Rats dosed with Pseudoracemic Deuterated D 3 -(R)- and D 0 -(S)-Ibuprofen LC-MS/MS Analysis of Rat Urine Extracts 100 A) Ibuprofen Psuedoracemate Dosed Rat Urine Extract > B) Ibuprofen Psuedoracemate Dosed Rat Urine Extract 313 > C) D3-(R)-Ibuprofen-N-Acyl-Cystiene Synthetic Standard 313 > D) D0-(R,S)-Ibuprofen-N-Acyl-Cystiene Synthetic Standard 310 > Retention Time (min) D 3 C H H D 3 -(R)-Ibuprofen H 3 C H H H 3 -(S)-Ibuprofen D 3 C H N H SH H Ibuprofen-N-Acyl-Cysteine 35 (Pharmacia/Pfizer) Grillo and Hua (2008) Unpublished observations

36 Benoxaprofen: Mechanisms of Benoxaprofen-induced Cholestasis A number of cases of hepatotoxicity in elderly patients led to the withdrawal of the drug from the market. the history of this drug and its adverse effects is an important chapter in the annals of hepatotoxicity. Hepatotoxicity observed mostly in elderly females. Morphologic features consisted of severe hepatic cholestasis accompanied by slight to moderate necrosis. No evidence for immunebased hypersensitivity. Bioactivation by P450 not observed. 36 Benoxaprofen glucuronide Insoluble metabolite? Metabolic idiosyncratic reaction proposed. Proposed to be due to insoluble metabolite ( acyl glucuronide ) excreted in bile leading to blockage of bile flow, cholestasis. Boelsterli et al. (1995) Critical. Rev. Toxicol. 25: Boelsterli (2007) Mechanistic Toxicology 2 nd ed. CRC Press.

37 Benoxaprofen: Mechanisms of Benoxaprofen-induced Cholestasis Incubation of benoxaprofen-s-acyl-glutathione with bovine gamma-glutamyltransferase (Ƴ- GT) leads to precipitate formation in vitro. Identity of precipitate not yet known. Ƴ-GT is located in the bile canaliculus membrane. Benoxaprofen -S-acyl Glutathione Benoxaprofen -S-acyl Glutathione Species differences in hepatic Ƴ-GT levels. -Rat low, human high. Ƴ-GT levels increase with age. Proposal that insoluble precipitate formed from the Ƴ-GT-mediated breakdown of benoxaprofen-acyl-sg may play a role in hepatic cholestasis. 37 Ƴ-GT Insoluble precipitate? Unpublished observations, Hinchman and Ballatori (1990) Glutathione-degrading capacities of liver and kidney in different species. Biochem. Pharmacol. 40:

38 7th International Conference on Early Toxicity Screening ETS-2008, Seattle, WA Metabolism-Based Toxicity in Drug Development Role of Drug Metabolism in Drug-induced Liver Toxicity (Sid Nelson, University of Washington; Seattle, WA) Drug-induced liver injury is one of the most common reasons for failure of drugs in development, and for either removal of drugs from the market or "black-box" warnings on marketed drugs. This presentation will focus on the role of reactive metabolites as a major cause of drug-induced liver injury, on structure-toxicity relationships, and on methods to assess risk from reactive metabolites. The role of acyl glucuronides in carboxylic acid drug toxicity may have been oversold. 38

39 Summary The chemical reactivity, covalent binding, and toxicity of acyl glucuronides has been widely-studied, although a clear link has not been established. Acyl-CoA thioesters are substantially more reactive than their corresponding acyl glucuronides, and increasing evidence demonstrates that acyl-coa thioesters contribute more importantly than acyl glucuronides to the covalent modification of hepatic protein in vitro and in vivo. The importance of chemically-reactive acyl-coa metabolites in drug-induced hepatotoxicity may be underestimated. 39

40 40 Acknowledgements UW Seattle Tom Baillie, PhD, DSc Dave Porubek, PhD UC San Francisco Chunze Li, PhD Leslie Z. Benet, PhD MyoKardia Jim Driscoll Pharmacia/Pfizer PDM Amgen PKDM