Glucuronidation of the Antiretroviral Drug Efavirenz by UGT2B7 and an in Vitro Investigation of Drug-Drug Interaction with Zidovudine

Abstract

ABSTRACT:The non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) is directly conjugated by the UDP-glucuronosyltransferase (UGT) pathway to form EFV-N-glucuronide (EFV-G), but the enzyme(s) involved has not yet been identified. The glucuronidation of EFV was screened with UGT1A and UGT2B enzymes expressed in a heterologous system, and UGT2B7 was shown to be the only reactive enzyme. The apparent K m value of UGT2B7 (21 M) is similar to the value observed for human liver microsomes (24 M), whereas the variant allozyme UGT2B7*2 (Tyr 268 ) displayed similar kinetic parameters. Because 3-azido-3-deoxythymidine (AZT), one of the most current nucleotide reverse transcriptase inhibitors prescribed in combination with EFV, is also conjugated by UGT2B7, the potential metabolic interaction between EFV and AZT has been studied using human liver microsomes. Glucuronidation of both drugs was inhibited by one another, in a concentrationdependent manner. At K m values (25 and 1000 M for EFV and AZT, respectively), EFV inhibited AZT glucuronidation by 47%, whereas AZT inhibited EFV glucuronidation by 23%. With a K i value of 17 M for AZT-glucuronide formation, EFV appears to be one of the most selective and potent competitive inhibitor of AZT glucuronidation in vitro. Moreover, assuming that concentrations of EFV achieved in plasma (C max ‫؍‬ 12.9 M) are in a range similar to its K i value, it was estimated that EFV could produce a theoretical 43% inhibition of AZT glucuronidation in vivo. We conclude that UGT2B7 has a major role in EFV glucuronidation and that EFV could potentially interfere with the hepatic glucuronidation of AZT.Efavirenz [(EFV) (Sustiva); Bristol-Myers Squibb Co., Princeton, NJ] is the preferred non-nucleoside reverse transcriptase inhibitor (NNRTI) agent for initial therapy for HIV infection. A typical antiretroviral treatment regimen consists of two nucleoside reverse transcriptase inhibitors (NRTIs) plus a protease inhibitor or an NNRTI. The 2008 IAS-USA guidelines recommend either of two basic threedrug regimens for treatment of antiretroviral-naive patients: 1) EFV plus two NRTIs; or 2) a ritonavir-boosted protease inhibitor (lopinavir, atazanavir, fosamprenavir, darunavir, or saquinavir) plus two NRTIs [tenofovir and emtricitabine; abacavir and lamivudine; or 3Ј-azido-3Ј-deoxythymidine (zidovudine; AZT) and lamivudine] (Hammer et al., 2008). A recent study on HIV-infected adults of Southern Africa further revealed that among patients who began nevirapine-based or EFV-based antiretroviral therapy between January 1998 and September 2004, 1321 of 1822 EFV-treated patients (72.5%) received it in combination with the NRTI pair AZT plus lamivudine (Nachega et al., 2008). Thus, a significant proportion of HIV-infected, highly active antiretroviral therapy-naive adults receive EFV and AZT in combination.A large interpatient variability was found to affect EFV bioavailability (coefficient of variation up to 118%) (Marzolini et al., 2001), which might be explained in part by its extensive metabolism....

Modulation of the UGT2B7 Enzyme Activity by C-Terminally Truncated Proteins Derived from Alternative Splicing

“…UGT2B7 is an enzyme responsible for detoxification of xenobiotics, including drugs such as morphine, efavirenz, zidovudine, and fenofibric acid, and is involved in the homeostasis of endogenous molecules like eicosanoids, bile acids, and sex steroids (Jin et al, 1997;Barbier et al, 2000;Stone et al, 2003;Turgeon et al, 2003;Thibaudeau et al, 2006;Mano et al, 2007;Bélanger et al, 2009;Tojcic et al, 2009;Trottier et al, 2013). Its expression is high in liver and kidney, but is also detectable in the gastrointestinal tract and peripheral tissues (Nakamura et al, 2008;Court et al, 2012).…”

Pharmacogenetics of CYP2B6, CYP2A6 and UGT2B7 in HIV treatment in African populations: focus on efavirenz and nevirapine

“…In another study involving Ghanaian patients, no association was found between the *1c and *2 alleles and efavirenz concentration. Elens et al (2010) as well as Bélanger et al (2009) also concluded that the *1c and *2 alleles were not associated with variations in efavirenz plasma concentrations. Likewise, no association was found between UGT2B7 allele frequencies and efavirenz plasma concentrations in a Zimbabwean population (Maimbo et al, 2012).…”

“…Efavirenz is mainly metabolized by CYP2B6 into 8-hydroxyefavirenz and to a lesser extent by CYP2A6 into 7-hydroxyefavirenz, with CYP3A4/5 and CYP1A2 playing a minor role in this step (Ward et al, 2003;Whirl-Carrillo et al, 2012). N-Glucuronide-efavirenz is formed when efavirenz undergoes conjugation by UGT2B7 (Bélanger et al, 2009). It has been proposed however that CYP2A6 and UGT2B7 only play a significant role in the efavirenz pathway when CYP2B6 is impaired (di Iulio et al, 2009).…”

Quantification of Glucuronide Metabolites in Biological Matrices by LC-MS/MS

“…Sometimes, a separate incubation of a commercially obtainable substrate (for example 4-methylumbelliferone glucuronide) in the same matrix is used to monitor the -glucuronidase activity (Blount, Milgram et al 2000). Each incubation should contain 200 -25 000 units of -glucuronidase per 1 mL of incubation medium (Gu, Laly et al 2005;Belanger, Caron et al 2009). Note that vendors define the glucuronidase activity in Roy or in Fishman units, where 1 Fishman unit releases 1 µg phenolphthalein from phenolphthalein-β-glucuronide in 1 h at 38°C.…”