SOFT 2004 Meeting Abstracts - Society of Forensic Toxicologists

C33
NOVEL ASPECTS OF IN VITRO METABOLISM OF BUPRENORPHINE
Van Chang* and David E. Moody
Center for Human Toxicology, University of Utah, Salt Lake City, UT
Buprenorphine is mainly metabolized by cytochrome P450 (P450) 3A4 mediated N-dealkylation to
norbuprenorphine; both buprenorphine and norbuprenorphine conjugate with glucuronic acid. Previously,
tentative 6-0-desmethyl norbuprenorphine in free and conjugated form and some unknown polar
metabolites were observed in rats; while no other metabolites were observed in human. We find higher
buprenorphine elimination compared to norbuprenorphine formation in human liver microsomes (HLM),
which supports the hypothesis that other metabolic pathways might exist. In the current study, the phase I
and phase II metabolism of buprenorphine was investigated in HLM and eDNA-expressed P450s.
Metabolites were screened by LC-MS/MS, coupled with precursor-ion scan, product-ion scan and neutralloss
scan.
The incubation for phase I metabolism was performed in a mixture of 0.1 M phosphate buffer (pH 7.4, 1.0
mM EDTA and 5.0 mM MgCh); the NADPH generating system (10 mM glucose-6-phosphate, 1.2 mM
NADP, and 1.2 units gluscose-6-phosphate); and 0.5 mglml microsomal protein. The reaction was initiated
by the addition ofNADPH generating system in a 37°C shaking water bath and continued for 30 min. The
incubations ofbuprenorphine in cDNA- expressed P450s were performed as described above except that 25
pmol eDNA-expressed P450s was used and the incubation time was 20 min. The incubation for phase II
metabolism was conducted in the presence of 25 Ilg/ml alamethicin and 2 mM UDPglucuronosyltransferases.
LC-MSIMS utilized a Finnigan TSQ 7000 equipped with a triple-quadrupole
mass spectrometer and an electrospray ionization source.
During phase I metabolism of buprenorphine, 5 metabolites (M 1-M5) in addition to norbuprenorphine were
identified. MI and M2 are hydroxylations of buprenorphine at the tert-butyl group and ring moiety,
respectively. M3-M5 are secondary hydroxylation metabolites ofnorbuprenorphine. The hydroxyl groups
are on the tert-butyl group (M3) and ring moiety (M4 and M5), respectively. The metabolism of
buprenorphine in eDNA-expressed P450s showed that P450 3A5 had highest catalytic activity in Ml
formation, with the involvement of 2C8 and 3A7, but their activity is only 5.9% and 3.3% of 3A5,
respectively; P450 3A4 has the highest catalytic activity in M3 formation, with contributions of 3A7, 3A5
and IA2 to a lesser extent, approximately 12.5%, 2.7% and 2.4% of 3A4, respectively. No detectable
signal was observed for M2, M4 and M5 by selected reaction monitoring on LC-MSIMS. M3 is the major
metabolite of norbuprenorphine in HLM, which is mediated mainly by P450 3A4, with the contribution of
3A5 and 3A 7 no more than 6% of 3A4. Norbuprenorphine metabolism suggested that secondary
metabolite M3 of buprenorphine was formed through norbuprenorphine. The primary study on phase II
metabolism of buprenorphine showed the presence of conjugated hydroxyl buprenorphine and hydroxyl
norbuprenorphine, the structural identification will be processed in details in the future work.
Keywords: Buprenorphine, Metabolism, P450
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