Abstract
Previous investigations of solid organ transplant patients treated with tacrolimus showed that individuals carrying a CYP3A5*1 allele have lower dose-adjusted trough blood concentrations compared with homozygous CYP3A5*3 individuals. The objective of this investigation was to quantify the contribution of CYP3A5 to the hepatic and renal metabolic clearance of tacrolimus. Four primary tacrolimus metabolites, 13-O-desmethyl tacrolimus (13-DMT) (major), 15-O-desmethyl tacrolimus, 31-O-desmethyl tacrolimus (31-DMT), and 12-hydroxy tacrolimus (12-HT), were generated by human liver microsomes and heterologously expressed CYP3A4 and CYP3A5. The unbound tacrolimus concentration was low (4–15%) under all incubation conditions. For CYP3A4 and CYP3A5, Vmax was 8.0 and 17.0 nmol/min/nmol enzyme and Km,u was 0.21 and 0.21 μM, respectively. The intrinsic clearance of CYP3A5 was twice that of CYP3A4. The formation rates of 13-DMT, 31-DMT, and 12-HT were ≥1.7-fold higher, on average, in human liver microsomes with a CYP3A5*1/*3 genotype compared with those with a homozygous CYP3A5*3/*3 genotype. Tacrolimus disappearance clearances were 15.9 ± 9.8 ml/min/mg protein and 6.1 ± 3.6 ml/min/mg protein, respectively, for the two genotypes. In vitro to in vivo scaling using both liver microsomes and recombinant enzymes yielded higher predicted in vivo tacrolimus clearances for patients with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. In addition, formation of 13-DMT was 13.5-fold higher in human kidney microsomes with a CYP3A5*1/*3 genotype compared with those with a CYP3A5*3/*3 genotype. These data suggest that CYP3A5 contributes significantly to the metabolic clearance of tacrolimus in the liver and kidney.
Footnotes
-
Financial support for this work was provided in part by grants from the National institutes of Health: GM63666, GM32165, and ES07033.
-
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
-
doi:10.1124/dmd.105.008680.
-
ABBREVIATIONS: 13-DMT or M-I, 13-O-desmethyl tacrolimus; 15-DMT or M-III, 15-O-desmethyl tacrolimus; 31-DMT or M-II, 31-O-desmethyl tacrolimus; 12-HT or M-VI, 12-hydroxy tacrolimus; rCYP3A4, CYP3A4 Supersomes; rCYP3A5, CYP3A5 Supersomes; rCYP3A4+b5, CYP3A4 + cytochrome b5 Supersomes; rCYP3A5+b5, CYP3A5 + cytochrome b5 Supersomes; rCYP3A7+b5, CYP3A7 + cytochrome b5 Supersomes; , unbound intrinsic clearance of heterologously expressed CYP3A enzymes; , unbound intrinsic clearance of human liver microsomes; , unbound substrate disappearance clearance of human liver microsomes; , plasma hepatic clearance; , unbound intrinsic clearance of human liver microsomes predicted from heterologously expressed CYP3A enzymes; P450, cytochrome P450; CLint, intrinsic clearance; LC/MS, liquid chromatography-mass spectrometry; LC/MS-MS, liquid chromatography/tandem mass spectrometry; TFA, trifluoroacetic acid; ESP+, positive electrospray; BSA, bovine serum albumin; AM1, 1η(8′)-hydroxy cyclosporine A; AM9, 9γ-hydroxy cyclosporine A; AM4N, 4-N-desmethyl cyclosporine A; SIR, single ion reaction.
-
↵1 Current affiliation: Center for Organ and Cell Transplantation, Scripps Green Hospital, La Jolla, California.
- Received November 30, 2005.
- Accepted February 14, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|