CYP2C8 and CYP3A4 are the principal enzymes involved in the human in vitro biotransformation of the insulin secretagogue repaglinide.
OBJECTIVE
To identify the principal human cytochrome P450 (CYP) enzyme(s) responsible for the human in vitro biotransformation of repaglinide. Previous experiments have identified CYP3A4 as being mainly responsible for the in vitro metabolism of repaglinide, but the results of clinical investigations have suggested that more than one enzyme may be involved in repaglinide biotransformation.


METHODS
[14C]-Repaglinide was incubated with recombinant CYP and with human liver microsomes (HLM) from individual donors in the presence of inhibitory antibodies specific for individual CYP enzymes. Metabolites, measured by high-performance liquid chromatography (HPLC) with on-line radiochemical detection, were identified by liquid chromatography-mass spectrophotometry (LC-MS) and LC-MS coupled on-line to a nuclear magnetic resonance spectrometer (LC-MS-NMR).


RESULTS
CYP3A4 and CYP2C8 were found to be responsible for the conversion of repaglinide into its two primary metabolites, M4 (resulting from hydroxylation on the piperidine ring system) and M1 (an aromatic amine). Specific inhibitory monoclonal antibodies against CYP3A4 and CYP2C8 significantly inhibited (> 71%) formation of M4 and M1 in HLM. In a panel of HLM from 12 individual donors formation of M4 and M1 varied from approximately 160-880 pmol min-1 mg-1 protein and from 100-1110 pmol min-1 mg-1 protein, respectively. The major metabolite generated by CYP2C8 was found to be M4. The rate of formation of this metabolite in HLM correlated significantly with paclitaxel 6alpha-hydroxylation (rs = 0.80; P = 0.0029). Two other minor metabolites were also detected. One of them was M1 and the other was repaglinide hydroxylated on the isopropyl moiety (M0-OH). The rate of formation of M4 in CYP2C8 Supersomes was 2.5 pmol min-1 pmol-1 CYP enzyme and only about 0.1 pmol min-1 pmol-1 CYP enzyme in CYP3A4 Supersomes. The major metabolite generated by CYP3A4 was M1. The rate of formation of this metabolite in HLM correlated significantly with testosterone 6beta-hydroxylation (rs = 0.90; P = 0.0002). Three other metabolites were identified, namely, M0-OH, M2 (a dicarboxylic acid formed by oxidative opening of the piperidine ring) and M5. The rate of M1 formation in CYP3A4 Supersomes was 1.6 pmol min-1 pmol-1 CYP enzyme but in CYP2C8 Supersomes it was only approximately 0.4 pmol min-1 pmol-1 CYP enzyme.


CONCLUSIONS
The results confirm an important role for both CYP3A4 and CYP2C8 in the human in vitro biotransformation of repaglinide. This dual CYP biotransformation may have consequences for the clinical pharmacokinetics and drug-drug interactions involving repaglinide if one CYP pathway has sufficient capacity to compensate if the other is inhibited.
Bidstrup
Tanja Busk
TB
Clinical Pharmacology, University of Southern Denmark, Winsløwparken 19, 5000 Odense C, Denmark. tbbi@novonordisk.com
Bjørnsdottir
Inga
I
Sidelmann
Ulla Grove
UG
Thomsen
Mikael Søndergård
MS
Hansen
Kristian Tage
KT
D000911:Antibodies, Monoclonal; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000502:physiology*; D001711:Biotransformation; D002219:Carbamates / Q000378:metabolism* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D065727:Cytochrome P-450 CYP2C8; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D004347:Drug Interactions; D006801:Humans; D007004:Hypoglycemic Agents / Q000378:metabolism* / Q000493:pharmacokinetics; D008862:Microsomes, Liver / Q000201:enzymology; D010880:Piperidines / Q000378:metabolism* / Q000493:pharmacokinetics
D000911:Antibodies, Monoclonal; D002219:Carbamates; D065607:Cytochrome P-450 Enzyme Inhibitors; D007004:Hypoglycemic Agents; D010880:Piperidines; C072379:repaglinide; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; C104464:CYP3A protein, human; D065727:Cytochrome P-450 CYP2C8; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human