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DMD 35:139–149, 2007  Overall Metabolic Scheme for MuraglitazarMuraglitazarDual / PPAR activatorGlucose and lipid lowering...
Oxidation of Muraglitazar in Humans    minor
Muraglitizar Metabolism (in vivo)
Enterohepatic Recirculation
Materials•   Radiolabled muraglitazar (MGZ, 99.6% pure, 8.4    µCi/mg)•   CYP Inhibitors:     – Furafylline (1A2), 8-MOP (...
HLMs•   A:     – 360 pmol/mg CYP     – 320 pmol/mg CYP reductase     – 570 pmol/mg cytochrome b5     – Activities of probe...
Incubations•   Run in duplicate•   14C-MGZ(26.6 µM, 25 µM, or 0-150 µM)•   CYP Baculosomes (40 or 200 pmol/mL) or     – HL...
Kinetic Parameters for MGZ glucuronidation                 by UGTs
ID of major MGZ metabolites by MS/MS
Inhibition of MGZ depletion•   14C-MGZ (2.5 µM)•   HLM-B (0.5 mg/mL)•   Chemical and Antibody Inhibitors     –   Monteluka...
Inhibition of MGZ metabolite formation•   14C-MGZ (5.17 mM)•   HLM-A (1 mg/mL)•   Chemical Inhibitors•   1 mM NADPH•   100...
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...
HPLC•   Radioactivity Profiling     –   Shimadzu Class VP system     –   Diode array detector (SPD-M10A)     –   YMC ODS A...
Metabolic Profile of MGZLC/radiomatic analysis                            26.6 µM MGZ                            1 mg/mL H...
Metabolic profile of MGZ by CYPs                        26.6 µM MGZ                        200 pmol/mL CYP                ...
Metabolic Profiles of MGZ by CYPsCYPs involved        2C8, 2C9, 2C19, 2D6, 3A4CYPs not involved:       1A2, 2A6, 2B6, 2C18...
Inhibition of MGZ depletion by MAbs and                 chemicals       2.5 µM MGZ (plasma concentration)       0.5 mg/mL ...
Metabolic Profile of MGZ in HLM-BIdentify which metab CYP 2C8 (approx 38% of total) forms                                 ...
Metabolic Profile of MGZ                     Compare to                     HLM-B
Inhibition of MGZ metabolism in HLMs25 or 26.6 µM MGZ   Inhibitors of CYPs 1A2 and 2B6 showed minor inhibition   Inhibitor...
Km/Vmax determinations for MGZ oxidation               by CYPs                             0-150 µM MGZ                   ...
Kinetic Parameters for MGZ oxidation by                CYPs     Michaelis-Menten analysis               Formation         ...
Possible Problems with the Calculations  Vs = A3A4v3A4 + A2C9v2C9 + A2D6v2D6 + …..  A = relative abundance of each CYP in ...
Relative contributions of CYPs to MGZ                    oxidationValues for overall metabolism don’t change much from 1-2...
Overall Metabolic Scheme for MGZCoadministration of ketoconazole (CYP3A4 inhibitor) or       gemfibrozil (CYP2C and UGT1A1...
Glucuronidation of MGZHLM-A or UGT26.6 µM30 minThe MGZ acyl glucuronide was stable at RT if the sample        was acidifie...
Summary/Conclusions•   MGZ is not extensively metabolized in vitro (16%) by    CYPs but is metabolized at several site by ...
Involvement of multiple P450s and UDP-GTs in the in vitro metabolism of Muraglitazar
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Involvement of multiple P450s and UDP-GTs in the in vitro metabolism of Muraglitazar

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Involvement of multiple P450s and UDP-GTs in the in vitro metabolism of Muraglitazar

  1. 1. DMD 35:139–149, 2007 Overall Metabolic Scheme for MuraglitazarMuraglitazarDual / PPAR activatorGlucose and lipid lowering effectsExtensively oxidized and glucuronidatedFecal 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. 2. Oxidation of Muraglitazar in Humans minor
  3. 3. Muraglitizar Metabolism (in vivo)
  4. 4. Enterohepatic Recirculation
  5. 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. 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. 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. 8. Kinetic Parameters for MGZ glucuronidation by UGTs
  9. 9. ID of major MGZ metabolites by MS/MS
  10. 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. 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. 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. 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. 14. Metabolic Profile of MGZLC/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
  15. 15. 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%)
  16. 16. Metabolic Profiles of MGZ by CYPsCYPs involved 2C8, 2C9, 2C19, 2D6, 3A4CYPs not involved: 1A2, 2A6, 2B6, 2C18, 2E1, 3A5
  17. 17. 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%
  18. 18. Metabolic Profile of MGZ in HLM-BIdentify 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)
  19. 19. Metabolic Profile of MGZ Compare to HLM-B
  20. 20. Inhibition of MGZ metabolism in HLMs25 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
  21. 21. Km/Vmax determinations for MGZ oxidation by CYPs 0-150 µM MGZ 40 pmol CYP 30 min
  22. 22. 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
  23. 23. Possible Problems with the Calculations Vs = A3A4v3A4 + A2C9v2C9 + A2D6v2D6 + ….. A = relative abundance of each CYP in HLMs Vmax for isoform specific rxn in HLMsRAF = -------------------------------------------------------------------- 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
  24. 24. Relative contributions of CYPs to MGZ oxidationValues for overall metabolism don’t change much from 1-25 µM CYP2Cs account for 60% CYP2D6 accounts for < 1% CYP3A4 account for ~ 40%
  25. 25. Overall Metabolic Scheme for MGZCoadministration of ketoconazole (CYP3A4 inhibitor) or gemfibrozil (CYP2C and UGT1A1 inhibitor) did not affect the clearance of muraglitazar
  26. 26. Glucuronidation of MGZHLM-A or UGT26.6 µM30 minThe MGZ acyl glucuronide was stable at RT if the sample was acidified immediately and stored at -20°CNext: kinetic analysis (0-30 µM MGZ)Km 3 µM in HLMs and for the 3 UGTsCannot estimate relative contributions since the [UGTs] in the liver are not known
  27. 27. 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.

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