1. DMD 35:139–149, 2007
Overall Metabolic Scheme for Muraglitazar
Muraglitazar
Dual / PPAR activator
Glucose and lipid lowering effects
Extensively oxidized and glucuronidated
Fecal elimination was major pathway (>95% of recovered dose)
Purpose of the study:
To determine the CYPs and UGTs involved in human
metabolism of MGZ

2. Oxidation of Muraglitazar in Humans
minor

3. Muraglitizar Metabolism (in vivo)

4. Enterohepatic Recirculation

5. Materials
• Radiolabled muraglitazar (MGZ, 99.6% pure, 8.4
µCi/mg)
• CYP Inhibitors:
– Furafylline (1A2), 8-MOP (2A6), orphenadrine (2B6),
sulfaphenazole (2C9), tranylcypromine (2C19), quinidine
(2D6), ketoconazole (3A4), montekulast (2C8),
benzylnirvanol (2C19), 1-ABT (general CYP)
• Baculosomes of:
– CYPs 1A2, 2A6, 2B6, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4
and 3A5
– UGTs 1A1, 1A3, 1A4, 1A6, 1A8, 1A9, 1A10, 2B4, 2B7,
and 2B15
• Monoclonal Antibodies for:
– CYPs 1A2, 2A6, 2B6, 2C9, 2Cs, 2D6, 2E1, and 3A4

6. HLMs
• A:
– 360 pmol/mg CYP
– 320 pmol/mg CYP reductase
– 570 pmol/mg cytochrome b5
– Activities of probe substrates
• CYP2C8 – 240 pmol/min/mg (paclitaxel)
• CYP2C9 – 3100 pmol/min/mg (diclofenac)
• CYP2C19 – 48 pmol/min/mg (S-mephenytoin)
• CYP2D6 – 71 pmol/min/mg (bufuralol)
• CYP3A4 – 6800 pmol/min/mg (testosterone)
• B:
– 240 pmol/mg CYP ( 70%)
– 340 pmol/mg CYP reductase ( 100%)
– 500 pmol/mg cytochrome b5 ( 90%)
– Activities of probe substrates
• CYP2C8 – 500 pmol/min/mg ( 200%)
• CYP2C9 – 1700 pmol/min/mg ( 40%)
• CYP2C19 – 26 pmol/min/mg ( 40%)
• CYP2D6 – 70 pmol/min/mg ( 100%)
• CYP3A4 – 4200 pmol/min/mg ( 60%)

7. Incubations
• Run in duplicate
• 14C-MGZ(26.6 µM, 25 µM, or 0-150 µM)
• CYP Baculosomes (40 or 200 pmol/mL) or
– HLM-A (1 mg/mL) or
– baculosomes + heat inactivated HLM-A (1 mg/mL)
– Human hepatocytes (2 X 106 cells/mL)
• 1 mM NADPH
• 100 mM NaPO4 (pH 7.4)
• 0.25 mL or
– 2 mL (hepatocyte studies)
• 20 or 30 min at 37 C with shaking at 100 rpm or
– 3 hr at 37 C (hepatocyte studies)
• Kill w/ 0.25 mL ACN or
– 2 mL (hepatocyte studies)
• Centrifuge 10 min at 2000g
• Inject 75 µL or 100 µL onto HPLC

8. Kinetic Parameters for MGZ glucuronidation
by UGTs

9. ID of major MGZ metabolites by MS/MS

10. Inhibition of MGZ depletion
• 14C-MGZ (2.5 µM)
• HLM-B (0.5 mg/mL)
• Chemical and Antibody Inhibitors
– Montelukast (3 µM)
– Sulfaphenazole (10 or 20 µM)
– Benzylnirvanol (1 µM)
– Tranylcypromine (30 µM)
– Quinidine (1 or 15 µM)
– Ketoconazole (1 or 10 µM)
– 1-ABT (1000 µM)
– Anti-CYP monocolonal antibodies (2-5 µL)
• 1.2 mM NADPH
• 50 mM NaPO4 (pH 7.4)
• 5 mM MgCl2
• 0.2 mL
• 30 min at 37 C with shaking at 100 rpm
• Kill w/ 0.4 mL ACN containing 3% AA and 1.5 µg/mL IS
• Centrifuge 15 min at 2000g
• Dilute supernatant with ACN/water (2:1, v/v)
• Analyze by LC/MS

11. Inhibition of MGZ metabolite formation
• 14C-MGZ (5.17 mM)
• HLM-A (1 mg/mL)
• Chemical Inhibitors
• 1 mM NADPH
• 100 mM NaPO4 (pH 7.4)
• 5 mM MgCl2
• 1 mL
• 15 min at 37 C with shaking at 100 rpm or
– Preincubate with MBIs + NADPH for 10 min
• Kill w/ 1.5 mL ACN
• Centrifuge 15 min at 2000g
• Dry supernatant under N2
• Reconstitute in 300 µL water ACN (2:1, v/v)
• Inject 75 µL on HPLC

12. UGT Incubations with HLM-A
• Run in triplicate for kinetic studies
• 14C-MGZ (4 µM, 26.6 µM or 0-30 µM)
• HLM-B (0.8 mg/mL) or
– UGT enzyme (0.1 mg/mL)
• 25 µg/mL alamethicin
• 25 mM Tris-HCl (pH 7.5)
• 10 mM MgCl2
• 0.25 mL
• Preinubate at 37 C for 5 min with shaking at 100 rpm
• Initiate reaction with UDPGA (2.5 mM)
• 30 min at 37 C
• Kill w/ 0.5 mL ice-cold ACN
• Centrifuge 15 min at 2000g
• Inject 100-200 µL onto HPLC

13. HPLC
• Radioactivity Profiling
– Shimadzu Class VP system
– Diode array detector (SPD-M10A)
– YMC ODS AQ C-18 (5 µm, 4.6 X 150 mm, Waters)
– 1 mL/min
– 96-well LumaPlates used to collect 0.26 min fractions
– Gilson model 202 fraction collector
– 70 min run
– Dry plates on a SpeedVac
– Analyze on a TopCount
– Solvent A = 0.06% TFA, Solvent B = ACN + 0.06% TFA
– Subtract out the average cpm value from first 8 samples
– Total amount of each metabolite was calculated based on
the percentage distribution and the total amount of parent
compound used in the incubation

14. Metabolic Profile of MGZ
LC/radiomatic analysis
26.6 µM MGZ
1 mg/mL HLM-A
30 min
Phase 1
4 µM MGZ
Alamethicin
30 min
Phase 2
25 µM MGZ
3 hr
Phase 1 + 2
16 oxidative metabs
In vivo

16. Metabolic profile of MGZ by CYPs
26.6 µM MGZ
200 pmol/mL CYP
30 min
Parent drug is the
Major peak
M10, M11, M14,
M15 were very minor
Metabolites (<0.5%)

17. Metabolic Profiles of MGZ by CYPs
CYPs involved
2C8, 2C9, 2C19, 2D6, 3A4
CYPs not involved:
1A2, 2A6, 2B6, 2C18, 2E1, 3A5

18. Inhibition of MGZ depletion by MAbs and
chemicals
2.5 µM MGZ (plasma concentration)
0.5 mg/mL HLM-B
Inhibitor
30 min
(5 µM)
(montelukast, 3 µM)
(sulfaphenazole, 10 µM)
(benzylnirvanol, 1 µM ) (21.1%)
(quinidine, 1 µM) (21.4%)
(ketoconazole, 1 µM) (39.9%)
(1-ABT, 1000 µM)
mABs of CYPs 1A2, 2A6, 2B6, and 2E1 inhibited < 10%

19. Metabolic Profile of MGZ in HLM-B
Identify which metab CYP 2C8 (approx 38% of total) forms
HLM-B
25 µM MGZ
30 min
Formation of M15:
HLM-A << HLM-B
2X more 2C8
Activity in HLM-B
(2C8 inhibitor)

20. Metabolic Profile of MGZ
Compare to
HLM-B

21. Inhibition of MGZ metabolism in HLMs
25 or 26.6 µM MGZ
Inhibitors of CYPs 1A2 and 2B6 showed minor inhibition
Inhibitor of CYP 2A6 showed minor activation
pure CYP HLM-B
CYP2C8 M10, M15 M10, M15
CYP2C9 M10 M10, M11, M14
CYP2C19 M10, M11 M10, M11, M15, M15
CYP2D6 M10 M11, M14
CYP3A4 M10, M14 M10, M11, M14, M15

22. Km/Vmax determinations for MGZ oxidation
by CYPs
0-150 µM MGZ
40 pmol CYP
30 min

23. Kinetic Parameters for MGZ oxidation by
CYPs
Michaelis-Menten analysis
Formation mg/mic Relative
of each protein contribution
metab by of each CYP
each CYP
[CYPs] in mics (pmol/mg):
2C8: 64
2C9: 96
2C19: 19
2D6: 10
3A4: 108

24. Possible Problems with the Calculations
Vs = A3A4v3A4 + A2C9v2C9 + A2D6v2D6 + …..
A = relative abundance of each CYP in HLMs
Vmax for isoform specific rxn in HLMs
RAF = --------------------------------------------------------------------
Vmax for isoform specific rxn by pure CYP system
(1) RAF was from a group at Merck
(2) Assumes the same amount of uncoupling with every
substrate/CYP combination
(3) Used different isoform-selective substrates
(4) Used different microsomes
different levels of CYP reductase, CYP, b5
polymorphisms
(4) Used different pure CYP systems
lymphoblasts vs. baculosomes

25. Relative contributions of CYPs to MGZ
oxidation
Values for overall metabolism don’t change much from 1-25 µM
CYP2Cs account for 60%
CYP2D6 accounts for < 1%
CYP3A4 account for ~ 40%

26. Overall Metabolic Scheme for MGZ
Coadministration of ketoconazole (CYP3A4 inhibitor) or
gemfibrozil (CYP2C and UGT1A1 inhibitor) did not affect
the clearance of muraglitazar

27. Glucuronidation of MGZ
HLM-A or UGT
26.6 µM
30 min
The MGZ acyl glucuronide was stable at RT if the sample
was acidified immediately and stored at -20°C
Next: kinetic analysis (0-30 µM MGZ)
Km 3 µM in HLMs and for the 3 UGTs
Cannot estimate relative contributions since the [UGTs] in
the liver are not known

28. Summary/Conclusions
• MGZ is not extensively metabolized in vitro (16%) by
CYPs but is metabolized at several site by several
different CYPs.
• Selective inhibitors and mAbs were used to support the
results from metabolism experiments using individual
CYPs.
• MGZ appears to be glucuronidated by UGTs
(glucuronides account for 80% of radioactivity in vivo).
• The extensive metabolism observed for MGZ should
lead to similar clearance among patients since >> one
enzyme is involved in its metabolism.
• The results from the individual CYP and selective CYP
inhibition do not seem to match up. Might be due to
submaximal inhibition of the compounds, non-specific
inhibition.
• Possible calculation issues.