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

DMD 35:139–149, 2007

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 (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

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%)

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

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

– 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

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

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

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

Metabolic Profile of MGZ

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

LC/radiomatic analysis

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%)

Metabolic Profiles of MGZ by CYPs

CYPs involved

2C8, 2C9, 2C19, 2D6, 3A4

CYPs not involved:

1A2, 2A6, 2B6, 2C18, 2E1, 3A5

(montelukast, 3 µM)

(sulfaphenazole, 10 µM)

(benzylnirvanol, 1 µM )

(quinidine, 1 µM)

(ketoconazole, 1 µM)

(1-ABT, 1000 µM)

(21.1%)

(21.4%)

(39.9%)

(5 µM)

Inhibition of MGZ depletion by MAbs and

chemicals

2.5 µM MGZ (plasma concentration)

0.5 mg/mL HLM-B

Inhibitor

30 min

mABs of CYPs 1A2, 2A6, 2B6, and 2E1 inhibited < 10%

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)

Metabolic Profile of MGZ

Compare to

HLM-B

Inhibition of MGZ metabolism in HLMs

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

25 or 26.6 µM MGZ

Km/Vmax determinations for MGZ oxidation

by CYPs

0-150 µM MGZ

40 pmol CYP

30 min

Kinetic Parameters for MGZ oxidation by

CYPs

[CYPs] in mics (pmol/mg):

2C8: 64

2C9: 96

2C19: 19

2D6: 10

3A4: 108

mg/mic

protein

Formation

of each

metab by

each CYP

Michaelis-Menten analysis

Relative

contribution

of each CYP

Possible Problems with the Calculations

(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

Vmax for isoform specific rxn in HLMs

RAF = --------------------------------------------------------------------

Vmax for isoform specific rxn by pure CYP system

Vs = A3A4v3A4 + A2C9v2C9 + A2D6v2D6 + …..

A = relative abundance of each CYP in HLMs

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%

Overall Metabolic Scheme for MGZ

Coadministration of ketoconazole (CYP3A4 inhibitor) or

gemfibrozil (CYP2C and UGT1A1 inhibitor) did not affect

the clearance of muraglitazar

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

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.