Role of Metabolite Elucidation in Support of Drug Discovery and Development: Strategy and Techniques Ala F. Nassar

1. Role of Metabolite Elucidationin Support of Drug Discovery and Development: Strategy and Techniques Ala F. Nassar

2. Today’s Topics • Introduction • Utilization of in-silico methods and automation techniques for rapid metabolite elucidation • Online H-D exchange method • Presentation of technique capable of detection down to 20 cpm 14C peaks • Using stable isotopes for metabolite elucidation • Case studies

3. Questions for Drug Metabolism Scientists • What are the chemical reactions involved in the metabolism of foreign compounds? • Where do these reactions take place in the body? • Which enzyme systems catalyze the metabolism of foreign compounds? • What are the biochemical mechanisms of these processes? • What are the biological consequences of xenobiotic metabolism? Outcome Guiding drug candidate selection by taking advantage of metabolic reactions to design more effective and safer drugs

4. Typical Screening Cascade In Vitro Enzyme Assay Cell Based Assay Selectivity Exposure screening Microsomal stability Metabolites ID IV/PO PK studies CYP inhibition screening Caco-2 permeability solubility In parallel or In series Expanded PK (definitive, TK, formulations, etc.) Chronic efficacy model Acute in vivo efficacy model Select Drug Candidate

5. Techniques for ADME Studies • Microsomal Stability • Hepatocyte Stability • Caco-2 Permeability • Metabolic Profiling • Protein Binding • Whole Body Autoradiography • P450 Inhibition and Induction • Pharmacokinetic Analysis • Metabolite Elucidation

6. Significance of Metabolite Elucidation • Assistance with chemical synthesis efforts to block • or enhance metabolism • Prediction of metabolites likely to be formed in • vivo  • Determination of metabolic differences between • species • Identification of potential pharmacologically-active • or -toxic metabolites • Synthesis of metabolites for toxicology testing • Prediction of drug-drug interactions

7. Method for Characterization of Metabolites in Drug Discovery Utilizing in-silico Prediction, GENESIS Workstation and QTOF-MS

8. The automated assay system consists of: Pallas MetabolExpert software to predict possible metabolites Robotic liquid handler (Genesis workstation) to generate and process samples QTOF-MS coupled with liquid chromatography to analyze samples MetaboLynx software to find potential metabolites Exact mass measurement to help for Met ID Advanced Chemistry Development/MS (ACD/MS) software to predict hypothetical metabolite chemical structures

10. Prediction of possible metabolites for propranolol using Pallas MetabolExpert 10.0 software

11. Prediction of possible metabolites for propranolol using Pallas MetabolExpert 10.0 software

12. TOF-MS/MS spectrum of (A) dextromethorphan and (B) dextromethorphan-M1

13. TOF-MS/MS spectra of (A) propranolol and (B) propranolol-M1

15. Mass accuracy measurements of selected drugs and their metabolites, the exact mass difference between each metabolite and parent drug

17. Proposed metabolic pathways of dextromethorphan in hepatic microsomal incubations based on MS/MS and ACD data

18. Proposed metabolic pathways of propranolol in hepatic microsomal incubations based on MS/MS and ACD data

19. Proposed metabolic pathways of alprenolol in hepatic microsomal incubations based on MS/MS and ACD data

21. Hydrogen-deuterium Exchange and QTOF-MS for Metabolite Elucidation in Drug Metabolism

22. H-D exchange methods are useful for determination of: • N- or S-oxide formation and mono- • hydroxylation • Conjugation such as glucuronidation • Dehydrogenation or dealkylation

25. Proposed metabolic pathways of nimodipine in hepatic microsomal incubations in H2O

26. Proposed metabolic pathways of nimodipine in hepatic microsomal incubations in D2O

27. CONCLUSIONS • One advantage of the H-D exchange method is that, with • LC-MS/MS, it offers an easy estimation of the number of • labile hydrogen atoms in such groups as -OH, -NH, -NH2 • and -COOH • This number is useful in comparing the metabolite structure • with that of the parent drug to determine the presence or • absence of the above groups • H-D exchange experiments have facilitated structural • elucidation and interpretation of fragmentation processes • as well • Our results indicated that this method should be • particularly desirable for identification of metabolites • produced by dehydrogenation, oxidation, and dealkylation

28. NOVEL APPROACH TO PERFORMING METABOLITE IDENTIFICATION IN DRUG METABOLISM

29. AIMS • Validate capability of on-line radioactivity • detector coupled with MS to measure • radiolabeled compounds • Enhance the sensitivity of radioisotope • measurement for metabolite identification

30. PC Sampler Column Agilent 1100 Pump StopFlow Controller Packard 500 Detector Waste Cocktail Fraction Collector Laptop PC ARC Control Lines ARC Data System LCQ Control Lines LCQ MS LC-ARC-MS-FC System

32. HPLC-MS chromatograms of [14C]dextromethorphan following incubation with HLM in the presence of NADPH showing M-1, M-2, M-3 and dextromethorphan

33. LC-MS spectra of [M+H]+ m/z 274, nonmetabolized [14C]dextromethorphan, (A) MS2, (B) MS3 and (C) MS4 following incubation of [14C]dextromethorphan with HLM

35. Proposed metabolic pathways of dextromethorphan in hepatic microsomal incubations

37. Stable Isotope Approach for Metabolite Elucidation in Drug Metabolism

38. Case Studies

39. Considerations to Enhance Metabolic Stability. • One of the most important keys to successful drug design and development is a process of finding the right combination of multiple properties such as activity, toxicity and exposure. Optimize these three properties for drug candidates, and thus their suitability for advancement to development. • The responsibility of the drug metabolism scientist is to optimize plasma T1/2 (clearance compound), drug/metabolic clearance, metabolic stability, and the ratio of metabolic to renal clearance. • Another concern is to minimize or eliminate the following: • gut/hepatic-first-pass metabolism, • inhibition/induction of drug-metabolizing enzymes by metabolites, • biologically active metabolites, • metabolism by polymorphically expressed drug-metabolizing enzymes • formation of reactive metabolites.

40. Advantages of Enhancing Metabolic Stability • Increased bioavailability and longer half-life, which in turn should allow lower and less frequent dosing thus promoting better patient compliance. • Better congruence between dose and plasma concentration, thus reducing or even eliminating the need for expensive therapeutic monitoring. • Reduction in metabolic turnover rates from different species which, in turn, may permit better extrapolation of animal data to humans. • Lower patient-to-patient and intra-patient variability in drug levels, since this is largely based on differences in drug metabolic capacity. • Diminishing the number and significance of active metabolites and thus lessening the need for further studies on drug metabolites in both animals and man.

41. Strategies to Enhance Metabolic Stability • In general, metabolism can be reduced by incorporation of stable functions (blocking groups) at metabolically vulnerable sites. Substrate structure activity relationships of metabolizing enzymes have to be accommodated within the structure activity relationships of the actual pharmacological target. • The following strategies have been used: • Deactivating aromatic rings towards oxidation by substituting them with strongly electron withdrawing groups (e.g., CF3, SO2NH2, SO3-). • Introducing an N-t-butyl group to prevent N-dealkylation. • Replacing a labile ester linkage with an amide group. • Constraining the molecule in a conformation which is unfavorable to the metabolic pathway, more generally, protecting the labile moiety by steric shielding • The phenolic function has consistently been shown to be rapidly glucuronidated. Thus, avoidance of this moiety in a sterically unhindered position is advised in any compound intended for oral use.

42. Strategies to Enhance Metabolic Stability (cont’d) • Avoidance of other conjugation reactions as primary clearance pathways, would also be advised in the design stage in any drug destined for oral usage. • Sometimes the best strategy is to anticipate a likely route of metabolism and prepare the expected metabolite if it has adequate intrinsic activity. For example, often N-oxides are just as active as the parent amine, but won't undergo further N-oxidation.

43. H O N O O N O N N H H O N O O F F H O N O O O N N N H H O N O O Examples from literature to enhance metabolic stability in the molecular design Reduce the overall lipophilicity (logP, logD) of the structure EC50 = 0.078mM, clogP = 2.07, C7hr = 0.012mM EC50 = 0.058mM, clogP = 0.18, C7hr = 0.057mM Dragovich, P. et al (2003). Journal of Medicinal Chemistry, 46(21), 4572-4585.

44. Remove or block the vulnerable site of metabolism (Benzylic oxidation) Ki = 66 nM, AUC 0-6h = 40 ng/ml hr Ki = 2.1 nM, AUC 0-6h = 6500 ng/ml hr Ki = 2 nM, AUC 0-6h = 1400 ng/ml hr Palani, A. et al (2001) Journal of Medicinal Chemistry, 44(21), 3339-3342.

45. Remove or block the vulnerable site of metabolism (Allylic oxidation) IC50 = 0.06 mg/ml, Cmax = 14-140 ng/ml IC50 = 0.02 mg/ml, Cmax = 70-300 ng/ml Victor F et al (1997). Journal of medicinal chemistry, 40(10), 1511-8.

46. Remove or block the vulnerable site of metabolism (Glucuronidation) Effect of linker UDPGA rate (nmol/min/mg protein) = 0.19, t1/2 = 4.7 hr UDPGA rate (nmol/min/mg protein) = 0.05, t1/2 = 5.5 hr Effect of template UDPGA rate (nmol/min/mg protein) = 0.05, t1/2 = 5.5 hr UDPGA rate (nmol/min/mg protein) = 0.012, t1/2 = 14.5 hr Effect of stereochemistry UDPGA rate (nmol/min/mg protein) = 0.02, t1/2 = 7.7 hr UDPGA rate (nmol/min/mg protein) = 0.01, t1/2 = 8.7 hr Bouska J J. et al (1997) Drug metabolism and disposition: biological fate of chemicals, 25(9), 1032-8.

47. Examples to improve PK properties through structural modification of drug candidates Minimize First-pass effect/prodrug approach • Oral dosage of propranolol (Hasegawa et al 1978)produces a low bioavailability and a wide variation from patient to patient when compared to intravenous administration; this difference is attributed to first-pass elimination of the drug. • Hemisuccinate ester of propranolol was selected as a potential prodrug with the hypothesis that propranolol hemisuccinate ester administration would avoid glucuronide formation during absorption and subsequently be released in the blood by hydrolysis. Propranolol Hydrolysis Glucuronidation Propranolol AUC 0-6 = 132 ng/ml.h Hemisuccinate ester of propranolol AUC 0-6 = 1075 ng/ml.h

48. Examples to improve PK properties through structural modification of drug candidates (cont’d) Half-life • ABT-418, an analogue of (S)-nicotine in which the pyridine ring is replaced by the 3-methyl-5-isoxazole moiety, has been shown to possess cognitive-enhancing and anxiolytic-like activities in animal models with an improved safety profile compared to that of nicotine (Lin et al. 1997). • One shortcoming of ABT-418 was its very poor bioavailability (%F = 1.2), with a short plasma half-life (t1/2 = 0.21 h). Research on structural modification led to the identification of ABT-089, 2-Methyl-3-(2(S)-pyrrolidinylmethoxy)pyridine, with a vastly improved oral bioavailability (%F = 61.5) with t1/2 = 1.6 h. 5 pyrollidine ABT-418 t1/2 = 0.2 h ABT-089 t1/2 = 1.6 h

49. Conclusions • In-silico and in vitro techniques are available to screen compounds for key ADME characteristics. • Structural information on metabolites is a great help in enhancing as well as streamlining the process of developing new drug candidates. • By improving our ability to identify both helpful and harmful metabolites, suggestions for structural modifications will optimize the likelihood that other compounds in the series are more successful. • Structural modifications to solve a metabolic stability problem may not necessarily lead to a compound with an overall improvement in PK properties. • Solving metabolic stability problems at one site could result in the increase in the rate of metabolism at another site, a phenomenon known as metabolic switching. Further, reduction in hepatic clearance may lead to increased renal or biliary clearance of a parent drug or inhibition of one or more drug-metabolizing enzymes. Therefore, it is advisable that in vitro metabolic stability data be integrated with other ADME screening.

50. Conclusions (cont’d) • An accurate measurement of the pharmacokinetic parameters and a good understanding of the factors that affect the pharmacokinetics will guide drug design. • High metabolic liability usually leads to poor bioavailability and high clearance, and formation of active or toxic metabolites will have an impact on the pharmacological and toxicological outcomes. • Drug candidates should have little or none of the following: • gut/hepatic-first-pass metabolism • inhibition/induction of drug-metabolizing enzymes • biologically active metabolites • metabolism by polymorphically expressed drug-metabolizing enzymes • formation of reactive metabolites • Also, it is important to have the most desirable plasma half-life and ratio of metabolic to renal clearance.