Structural elucidation of human oxidative metabolites of muraglitazar: use of microbial bioreactors in the biosynthesis of metabolite standards.
Muraglitazar (Pargluva), a dual alpha/gamma peroxisome proliferator-activated receptor activator, is currently in clinical development for treatment of type 2 diabetes. This study describes the structural elucidation of the human oxidative metabolites of muraglitazar through the use of a combination of microbial bioreactors, NMR and accurate mass analyses, and organic synthesis. Plasma, urine, and feces were collected from six healthy subjects following oral administration of 14C-labeled muraglitazar (10 mg, 100 microCi) and pooled samples were analyzed. Approximately 96% of the recovered radioactive dose was found in the feces and 3.5% in the urine. The parent compound represented >85% of the radioactivity in plasma. The fecal radioactivity was distributed among 16 metabolites (M1-M12, M14-M16, and M8a) and the parent drug, of which hydroxylation and O-demethylation metabolites (M5, M10, M11, M14, and M15) represented the prominent human metabolites. The urinary radioactivity was distributed into several peaks including muraglitazar glucuronide (M13) and the parent drug. Low concentrations of metabolites in human samples prevented direct identification of metabolites beyond liquid chromatographic (LC)-mass spectrometric analysis. Microbial strains Cunninghamella elegans and Saccharopolyspora hirsuta produced muraglitazar metabolites that had the same high performance liquid chromatography retention times and the same tandem mass spectrometric (MS/MS) properties as the corresponding human metabolites. The microbial metabolites M9, M10, M11, M14, M15, and M16 were isolated and analyzed by NMR. Based on these LC-MS/MS and NMR analyses, and organic synthesis, the structures of 16 human oxidative metabolites were identified. The oxidative metabolism of muraglitazar was characterized by hydroxylation, O-demethylation, oxazolering opening, and O-demethylation/hydroxylation, as well as O-dealkylation and carboxylic acid formation. This study demonstrated the utility of microbial bioreactors for the identification of metabolites.
Zhang
Donglu
D
Pharmaceutical Candidate Optimization, Pharmaceutical Research Institute, Bristol-Myers Squibb, P.O. Box 4000, Princeton, NJ 08543, USA. Donglu.Zhang@BMS.com
Zhang
Haiying
H
Aranibar
Nelly
N
Hanson
Ronald
R
Huang
Yande
Y
Cheng
Peter T
PT
Wu
Shung
S
Bonacorsi
Samuel
S
Zhu
Mingshe
M
Swaminathan
Arun
A
Humphreys
W Griffith
WG
D019149:Bioreactors; D001711:Biotransformation; D020093:Cunninghamella / Q000378:metabolism*; D005243:Feces / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics / Q000652:urine; D006801:Humans; D010080:Oxazoles / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics* / Q000652:urine; D010084:Oxidation-Reduction; D047493:PPAR alpha / Q000819:agonists; D047495:PPAR gamma / Q000819:agonists; D016930:Saccharopolyspora / Q000378:metabolism*
D010080:Oxazoles; D047493:PPAR alpha; D047495:PPAR gamma; D005998:Glycine; C500085:muraglitazar