Abstract
Structures of in vivo secondary metabolites of a norbornane-containing drug candidate with multiple prochiral centers were triangulated, in a regio- and stereospecific fashion, using in vitro metabolism data from synthetic primary metabolites and in vivo metabolism data from the separate administration of a radiolabeled primary metabolite, [14C]-(S)-2-((1R,2S,4R,5S)-5-hydroxybicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (M1). A mass balance study on the 11β hydroxysteroid dehydrogenase type 1 enzyme inhibitor [14C]-(S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221) in rats was dosed at 2 mg/kg. Radioactivity was excreted mainly in urine. Metabolites of AMG 221 were quantified by high-performance liquid chromatography with radiometric detection and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS revealed at least 38 metabolites. Seven monohydroxylated metabolites mediated formation of the other 31 metabolites. Twenty-eight metabolites were identified regio- and stereo-specifically. Little parent drug was observed in urine or feces. Monohydroxy metabolite M1 was the major metabolite comprising 17 to 24% of excreted dose, and seven monohydroxy metabolites comprised 29 (male) and 37% (female) of dose. Of 11 quantifiable isobaric dihydroxy metabolites that comprised 8.3 (male) and 24% (female) of dose, 10 were identified regio- and stereospecifically by triangulation. A single trihydroxy metabolite comprised approximately 10% of dose. Complex secondary metabolism of drugs with multiple prochiral centers can be elucidated in a regio- and stereospecific fashion without NMR through synthesis and in vitro and in vivo studies on the metabolism of chiral primary oxidation products.
Footnotes
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ABBREVIATIONS:
- AMG 221
- (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one
- 11βHSD1
- 11β hydroxysteroid dehydrogenase type 1
- P450
- cytochrome P450
- ADME
- absorption, distribution, metabolism, and elimination
- HPLC
- high-performance liquid chromatography
- LC-MS
- liquid chromatography-mass spectrometry
- M1
- (S)-2-((1R,2S,4R,5S)-5-hydroxybicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one
- M2
- (S)-2-((1R,2S,4R,5R)-5-hydroxybicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one
- M3
- (S)-2-((1S,2S,4R,6R)-6-hydroxybicyclo [2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one
- M4
- (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-((R)-1-hydroxypropan-2-yl)-5-methylthiazol-4(5H)-one
- M6
- (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-((S)-1-hydroxypropan-2-yl)-5-methylthiazol-4(5H)-one
- M7
- (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-(2-hydroxypropan-2-yl)-5-methylthiazol-4(5H)-one
- M8
- (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-methyl-5-(prop-1-en-2-yl)thiazol-4(5H)-one
- M10
- (S)-5-isopropyl-5-methyl-2-((1R,2S,4R)-5-oxobicyclo[2.2.1]heptan-2-ylamino)thiazol-4(5H)-one
- MS/MS
- tandem mass spectrometry
- BDC
- bile duct-cannulated
- β-RAM
- radiometric detector
- RLM
- rat liver microsomes
- CID
- collision-induced dissociation
- MH+
- protonated molecular ion
- SULT
- sulfotransferase
- D2624
- N-(2,6-dimethylphenyl)-5-methyl-3-isoxazolecarboxamide.
- Received October 3, 2011.
- Accepted January 31, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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