2014/10/02 SAPA-GP Webinar: Introduction to drug metabolism case studies for its impacts on drug discovery and development Zhoupeng Zhang Dept of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism Merck Research Laboratories Sino-American Pharmaceutical Professionals Association (SAPA) – A lecture for Medicinal Chemists (October 2, 2014)

1. Introduction to Drug metabolism: case studies
for its impacts on drug discovery and development
Zhoupeng Zhang
Dept of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism
Merck Research Laboratories
Sino-American Pharmaceutical Professionals Association (SAPA)
– A lecture for Medicinal Chemists
(October 2, 2014)

2. R&D Spending trend of 8 US pharmaceutical companies*
35
30
25
20
15
10
5
0
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006
R&D Spending ($ billions)
C&E News July 2, 2007 issue
* Abbott, BMS, Eli Lilly, J&J, Merck, Pfizer, Schering-Plough, and Wyeth.

3. Numbers of drugs approved by FDA (1996 – 2007)
Hughes B, Nature Reviews Drug Discovery 7, 107, 2008

4. Drug development processes
Dickson M and Gagnon J P, Nature Reviews Drug Discovery 3, 417, 2004

5. Success rate by phase of development
Kola I and Landis J, Nature Reviews Drug Discovery 3, 711, 2004

6. Trends in times for development of a NCE
Dickson M and Gagnon J P, Nature Reviews Drug Discovery 3, 417, 2004

7. Estimated cost and time for a NCE
• > $ 800 millions !
• ~ 8 – 12 years !
Dickson M and Gagnon J P, Nature Reviews Drug Discovery 3, 417, 2004

8. Reasons for termination of drug candidates in development
Human Human Animal Lack of
PK AEs Toxicity efficacy
Years Financial Others
1964-1985 39% 10% 11% 29% 6% 5%
Prentis, R.A., 1988

9. Reasons for termination of drug candidates in development
Kola I and Landis J, Nature Reviews Drug Discovery 3, 711, 2004

10. Involvement of drug metabolism in drug
discovery and development processes

11. ADME
Drug
Absorption
Distribution
Metabolism
Excretion Elimination
Disposition

12. ADME
Absorption – Process by which drug proceeds from site of
administration to the general circulation (plasma)
Disposition – All processes after absorption of drug
- Distribution – Reversible transfer of drug to and from the
circulation
- Elimination – Irreversible loss of drug from the circulation
- Metabolism – Conversion of drug to another chemical
species
- Excretion – Irreversible loss of unchanged drug

13. ADME
Drug release &
dissolution
Absorption Drug in systemic
circulation
Drug in
tissues
Metabolism &
Excretion
Types of metabolism:
- Phase I metabolism – oxidation, reduction and hydrolysis.
- Phase II metabolism – glucuronidation, sulfation, conjugations
with GSH, amino acids, acetylation,
methylation, etc.
- Phase III (?) metabolism – transport of drugs

14. Metabolism and drug design
• Hard drugs – non-metabolizable. They are
excreted primarily through either the bile or
kidney.
• Soft drugs are pharmacologically active, and
undergo a predictable and controllable
metabolism to nontoxic and inactive
metabolites.
• Active metabolites are pharmacologically
active, and are generated from metabolism.
• Not all metabolites are nontoxic.
Academic interest

15. Pharmacokinetics and drug design
• Absorption - influenced by solubility, lipophilicity
(cell peameability), involvement of transporters, etc.
• Prodrug – inactive prodrug is converted to
an active metabolite via in vivo metabolism.
• Distribution - influenced by lipophilicity,
protein binding, P-glycoprotein, etc.
• Plasma half-life – determined by the volume of
distribution and elimination clearance.

16. DMPK in drug development
• In vitro studies of drug metabolism
- Determination of metabolic pathways
- Identification of drug-metabolizing enzymes
- Drug-drug interaction potential
- prediction of in vivo metabolic clearance
• In vitro studies of drug absorption
• In vitro studies of protein binding
• Polymorphism in drug metabolism

17. Requirements of a successful new drug
• Satisfies an unmet medical need
• Exhibits superiority over existing treatments
– new mechanism of action
– improved potency or selectivity
– improved safety profile
– superior pharmacokinetics
– improved metabolic characteristics

18. Criteria for an “ideal” drug from a
drug metabolism point of view
• Good aqueous solubility (oral absorption / i.v. formulation)
• Acceptable (linear) PK for intended route / frequency of dosing
• “Balanced” clearance (renal, biliary, metabolism)
• Oxidative metabolism catalyzed by several enzymes (CYPs)
• Minimal dependence on polymorphically-expressed enzymes
• Low propensity to inhibit drug-metabolizing
enzymes/transporters
• Low first-pass effect, high oral bioavailability
• Moderate plasma protein binding
• Minimal conversion to chemically reactive metabolites
– Concerns over potential toxicity

19. Criteria for an “ideal” drug from a
drug metabolism point of view?
• Good aqueous solubility (oral absorption / i.v. formulation)
• Acceptable (linear) PK for intended route / frequency of dosing
• “Balanced” clearance (renal, biliary, metabolism)
• Oxidative metabolism catalyzed by several enzymes (CYPs)
• Minimal dependence on polymorphically-expressed enzymes
• Low propensity to inhibit drug-metabolizing
enzymes/transporters
• Low first-pass effect, high oral bioavailability
• Moderate plasma protein binding
• Minimal conversion to chemically reactive metabolites
– Concern over potential toxicity
Metabolite identification

20. Metabolite Identification and Profiling in Drug Design

21. Outline
1. Optimization of Pharmacokinetics (PK) and
Pharmacokinetics (PD) Properties
- Identification of Metabolic Soft Spots
- Identification of Active Metabolites
2. Improvement of Safety Profile
- Minimization of Bioactivation Potential
- Minimization of Drug-drug Interaction and Polymorphic
Drug Metabolizing Enzyme-related Risk
- Evaluation of Species Differences in Metabolism

22. 1. Optimization of PK and PD Properties
- Identification of Metabolic Soft Spots
• After oral administration, drugs absorbed by intestine pass through the gut wall and liver
prior to reaching the systemic circulation.
• GI tract and liver contain high levels of drug metabolizing enzymes, and extensive metabolism
in GI tract and liver following oral dosing (first-pass metabolism) would lead to low oral
bioavailability, and short t1/2 (drug coverage shorter than a desired duration).
• Optimize the rate of metabolism of an NCE would eventually realize its PD effect both in
animal models and humans.
• Liver often is the major organ responsible for drug metabolism. Thus, metabolic stability
studies generally are performed in vitro with liver microsomes/hepatocytes for screening.
• In vitro metabolite profiling studies of selected metabolically unstable compounds are
conducted in order to identify metabolic soft spots which represent metabolically liable
functional groups or the positions on a functional group (likely responsible for fast clearance )
based on the characterization of major metabolites.
• Blockage of metabolic soft spots via chemical modification of compounds is one of the
approaches to improve metabolic stability of compounds, particularly when metabolism is
catalyzed by P450s, leading to the improved PK properties (t1/2 and oral bioavailability).

23. Case #1: Metabolic stability of compound A (1 μM) in rat liver
microsomes (0.5 mg protein/mL) in the presence of NADPH
100
10
0 10 20 30 45
Time (min)
% of parent remaining

24. Metabolynx report of metabolite profile of compound A
M1
Cpd A M1
Cpd A
in incubation with rat liver microsomes

25. Radiochromatogram of a sample from incubation of
[3H]compound A with rat liver microsomes.
M1
Compound A
Rat liver microsomes (60 min)
Time (min)
Radioactivity (DPM)

26. Case #2: Identification of Metabolic Soft Spots of FK788
O
HO O
O
O
N
OH
FK788
• A prostanoid PGI2 receptor agonist.
• t1/2: 1.5 h in human, 1.8 h in rat.

27. O
HO O
O
N
OH
O
FK788
O
HO O
O
N
OH
O
M1
OH
O
HO O
O
N
OH
O
Compound 1
F
O
HO O
OH F
O
N
O
F
Compound 2
FK788 Compound 1 Compound 2
Clint: 31.9 mL/min/kg Clint: 10.3 mL/min/kg Clint: 4.6 mL/min/kg
(In RLM)
AUC: 601 ng*h/mL AUC: 961 ng*h/mL AUC: 1607 ng*h/mL
F: 9.6% F: 19.2% F: 29.5%
T1/2: 1.8 h T1/2: 1.6 h T1/2: 3.0 h
(In rats)

28. 1. Optimization of PK and PD Properties
- Identification of Active Metabolites
• Drug metabolites could have no pharmacological activity or the activity less than, or
equivalent to, or more potent than that of the parent molecules.
• Metabolites with equivalent or better pharmacological activity are active metabolites.
• Metabolite identification and profiling identifies a new structural template with
increased potency/selectivity or reduced adverse.
• A hint of the presence of active metabolite may come from a lack of PK-PD
correlation wherein the in vivo efficacy exceeds the in vitro potency of an NCE.
• Consequently, the NCE would be characterized for its metabolism, including
identification of major circulating metabolites in preclinical PD models.
• The metabolites would be synthesized and tested for their potency against the
pharmacological target of interest.

29. Identification of Active Metabolites – a Case Study
N
O
O
NH
O
Cl
LM-4108
N
O
O
OH O
NH
O
Cl
2'-Hydroxy-LM-4108
N
O
O
NH
O
Cl
2'-Oxo-LM-4108
+
• A COX-2 inhibitor.
• Two major metabolites detected in LM of mouse, rat and human,
• Metabolites are equipotent as LM-4108 in the in vitro assays
• Metabolites are also present in plasma of rats, may contribute to
the PD effects.
• Further investigation of these two metabolites is warranted.

30. Acetaminophen
HO
HN O
Phenacetin
O
HN
O
HN
O
Acetanilide

31. N
Cl
O O
N
Loratadine (Claritin)
N
Cl
HN
Descarboethoxyloratadine
(Desloratadine)
IC50 for IL-6 (nM) : 300 0.0026
IC50 for IL-8 (nM) : 200 0.001
Clinical dose (mg): 10 5

32. 2. Improvement of Safety Profile
- Minimization of Bioactivation Potential

33. O
O
O
S Protein S Protein
OH
S Protein
OH
OH
S Protein
O
S Protein
OH
H2O
S Protein
Formation of drug-protein adducts
Idyosyncratic drug reactions Tissue damages

34. Biomarkers commonly used for studying the
mechanism of metabolic activation of drugs
O N H O
S H NH
O
OH
O
OH
NH2
HO O
S H NH
O
CN-O
Glutathione (GSH) N-acetylcysteine (NAc) Cyanide
O
O
- R
S R S
OH
OH
R S
OH
Detection by LC/MS/MS
and NMR
-CN
CN
+
N N

35. Minimization of Bioactivation Potential – Case Study
R
O
R OH
OH
R O
O
[O] H2O
[O]
R OH
OH
GSH
GS
R
F
O
R
OH
SG
-HF
R O
SG
[O]
R OH
SG
GSH
F F
F
F F
R
Compound 3
1490 pmol/mg protein
in rat liver microsomes
R F
F
Compound 4
841 pmol/mg protein
in rat liver microsomes

36. Covalent Protein Binding of [3H]Compounds in Liver Microsomes
of Rats (RLM) and human (HLM) (pmol/mg protein)
R
Compound 3
R F
F
Compound 4
R N
Compound 5
R N
Cl
Compound 6
R N
CF3
Compound 7
RLM 1490 841 535 190 111
HLM 3870 1690 911 303 88

37. 2. Improvement of Safety Profile
- Minimization of Drug-Drug Interaction and Polymorphic
Drug Metabolizing Enzyme-related Risk
• Phenotyping metabolizing enzyme(s):
- In vitro metabolite profiling with recombinant human enzymes
- In vitro metabolite profiling with human liver microsomes in the presence of
selective chemical inhibitors and/or antibodies.
• If a single and/or polymorphic enzyme is responsible for the clearance of an NCE:
- A clinical DDI study is warranted;
- If there is a significant safety concern, the development of the NCE could be
terminated prior to the clinical study.
• Thus, it is important to minimize DDI potentials and the dependence of
metabolism on a single polymorphic enzyme early in drug discovery.

38. Minimization of Drug-drug Interaction and Polymorphic
Drug Metabolizing Enzyme-related Risk – a Case Study
HN
O OH
O
Metoprolol
65%
10%
10%
• A beta-1-adrenoceptor antagonist (blocker) for hypertension.
• Low oral bioavailability and short duration of action in vivo,
due to extensive hepatic metabolism.
• Metabolized mainly by CYP2D6, a polymorphic enzyme.
• A 100-fold variation for plasma exposure of metoprolol and its
α-OH metabolite and up to 36-fold difference in Cl in 91 patients
with cardiovascular diseases.

39. HN
O OH
O
Betaxolol
• Slow metabolism was observed.
• Metabolized by CYP2D6 and CYP1A2, and CYP2D6 only accounts
for 40% of metabolism in human.
• Betaxolol posses a reduced dependence of polymorphic
CYP2D6-mediated metabolism, and therefore may exhibit a smaller
individual variation in its clinical PK as compared to metoprolol.

40. 2. Improvement of Safety Profile
- Evaluation of Species Differences in Metabolism
• For a drug candidate, it is important to know which preclinical species
is capable of producing metabolite profiles similar to that of humans.
• In vitro and in vivo metabolite profiling provides the data that enable
a comparison of the metabolic fate of compounds in different species,
allowing for selection of an appropriate preclinical model for:
- human pharmacokinetic prediction, and
- preclinical safety evaluation
• If significant difference of metabolism is observed between human and
an animal species, or a human specific major metabolite is detected,
there may be a need to perform additional studies to evaluate the
safety of the metabolite.

41. Evaluation of Species Differences in Metabolism – a Case Study
O
O
N
R1
R2
R4
R3
CDP-840
O
O
N
R1
R2
R4
R3
M3 OH
O
O
N+
R1
R2
R4
R3
M2
Gluc
+
• An inhibitor of phosphodiesterase type IV Major in HH, not in RH Major in RLM,
O
O
N
R1
R2
R4
R3
Cl
CT2412
O
O
N+
R1
R2
R4
R3
O-CT2481
Cl
Not in HLM and HH.
• Similar metabolite profiles in LM
and HP from various species

42. Workflow for a Centralized Metabolite Identification Assay
On-line submission for
MetID of compounds
Automated in vitro
incubations using a
robotic liquid handler
Submission of a consolidated
list of compounds to a central
compound repository
DMPK scientists
Delivery of compounds
in a 96-well plate
Dilution of compounds
in 96-well plate
MetID software for post
acquisition processing
and reporting
LC-accurate
MS (HRMS)
runs

43. Major Utilities of Metabolite Identification and Profiling in Drug Discovery
• Minimization of bioactivation potential
• Improvement of PK and PD
Lead identification NCE characterization
Hit Lead
Lead optimization
NCE
candidate
NCE
Lead characterization
• Finding of active metabolite
or new structural template
• Selection of preclinical safety species
• Finding of active metabolite
or new structural template
• Minimization of DDI potential
• Selection of preclinical safety species

44. Role of DMPK in drug development
Baillie T A, Chem Res Toxicol 21, 129, 2008

45. Roles of DMPK
in drug discovery and development
Baillie T A, Chem Res Toxicol 21, 129, 2008

46. Acknowledgements
Mingshe Zhu, Wei Tang
Colleagues at Merck