Pregnancy Increases Norbuprenorphine Clearance in Mice by Induction of Hepatic Glucuronidation

1521-009X/46/2/100–108$25.00 https://doi.org/10.1124/dmd.117.076745 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 46:100–108, February 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics Pregnancy Increases Norbuprenorphine Clearance in Mice by Induction of Hepatic Glucuronidation Michael Z. Liao, Chunying Gao, Brian R. Phillips, Naveen K. Neradugomma, Lyrialle W. Han, Deepak Kumar Bhatt, Bhagwat Prasad, Danny D. Shen, and Qingcheng Mao Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington Received May 5, 2017; accepted November 17, 2017 ABSTRACT Norbuprenorphine (NBUP) is the major active metabolite of bupre- proteomics quantification revealed that hepatic Ugt1a1 and norphine (BUP) that is commonly used to treat opiate addiction Ugt2b1 protein levels in the same amount of total liver membrane ’ ∼ during pregnancy; it possesses 25% of BUP s analgesic activity proteins were significantly increased by 50% in pregnant mice Downloaded from and 10 times BUP’s respiratory depression effect. To optimize versus nonpregnant mice. After scaling to the whole liver with BUP’s dosing regimen during pregnancy with better efficacy and consideration of the increase in liver protein content and liver safety, it is important to understand how pregnancy affects NBUP weight, we found that the amounts of Ugt1a1, Ugt1a10, Ugt2b1, disposition. In this study, we examined the pharmacokinetics of and Ugt2b35 protein in the whole liver of pregnant mice were NBUP in pregnant and nonpregnant mice by administering the significantly increased ∼2-fold compared with nonpregnant mice. same amount of NBUP through retro-orbital injection. We demon- These data suggest that the increased systemic CL of NBUP in dmd.aspetjournals.org strated that the systemic clearance (CL) of NBUP in pregnant mice pregnant mice is likely caused by an induction of hepatic Ugt increased ∼2.5-fold compared with nonpregnant mice. Intrinsic CL expression and activity. The data provide a basis for further of NBUP by glucuronidation in mouse liver microsomes from mechanistic analysis of pregnancy-induced changes in the dispo- pregnant mice was ∼2 times greater than that from nonpregnant sition of NBUP and drugs that are predominately and extensively mice. Targeted liquid chromatography tandem-mass spectrometry metabolized by Ugts. at ASPET Journals on October 3, 2021 Introduction BUP (Fig. 1) is a semisynthetic m-opioid receptor agonist and k d Opioid addiction cases in the United States continue to escalate -opioid and -opioid receptor antagonist. BUP is metabolized by as a top national health crisis. Opioid addiction as a result of cytochrome P450 3A through N-dealkylation to norbuprenorphine prescription opioid use during pregnancy accounted for 28% of (NBUP) (Fig. 1). NBUP is the major active metabolite of BUP in ∼ ’ admissions of pregnant women to treatments of drug abuse from humans and rodents with 25%ofBUPs intrinsic analgesic activity 1992 to 2012 (Martin et al., 2015). Untreated substance abuse during (Brown et al., 2012). Both BUP and NBUP can be further metabolized b pregnancy increases the risk for adverse pregnancy outcomes and primarily to buprenorphine-3- -D-glucuronide and norbuprenorphine- b 9 fetal anomalies. Due to the dual risk to the mother and fetus, opioid 3- -D-glucuronide (NBUP-G, Fig. 1), respectively, by uridine 5 - addiction treatment is an increasing priority for this understudied diphospho-glucuronosyltransferases (UGTs) (Brown et al., 2011). A population. Although methadone as a medication-assisted treatment mass balance study on BUP revealed that NBUP and NBUP-G exposure . of opioid addiction is the gold standard, recent research supports the accounted for 10% of the total BUP plasma exposure (Jones, 1997). use of buprenorphine (BUP) for opioid treatment during pregnancy. As per US Food and Drug Administration Safety Testing of Drug . Randomized clinical trials comparing methadone and BUP indicate Metabolite Guidance for Industry, human metabolites formed at 10% that both medications are effective in preventing relapse in opioid- of total drug-related exposure at steady state could raise safety concerns dependent patients (Stotts et al., 2009). Moreover, increasing clinical (US Food and Drug Administration, 2016). Given the fact that NBUP research suggests that the use of BUP results in more favorable can produce respiratory depression 10 times more potent than BUP in maternal and fetal outcome (shorter hospital stay, shorter duration of rodents (Yassen et al., 2007) and possibly in infants (Kim et al., 2012), treatment of neonatal abstinence syndrome) compared with metha- drug-drug interactions with BUP leading to elevated systemic concen- done (Jones et al., 2010). trations of NBUP may contribute to an increased risk of neurorespiratory depression. Therefore, to optimize BUP’s dosing regimen with better efficacy and safety, we need to understand the disposition and This work was supported by the National Institutes of Health [Grant DA032507] pharmacokinetics (PK) of not only BUP but also NBUP. Although the and the National Center for Advancing Translational Sciences of the NIH [Grant disposition of BUP has been extensively studied (Hand et al., 1990; TL1TR000422]. Chiang and Hawks, 2003; Kacinko et al., 2008) and the dose-normalized https://doi.org/10.1124/dmd.117.076745. plasma AUC of BUP has been shown to be significantly decreased about ABBREVIATIONS: AUC, area under plasma concentration-time curve; BUP, buprenorphine; CL, clearance; CLint, intrinsic clearance; EDTA, ethylenediaminetetraacetic acid; gd, gestation day; LC-MS/MS, liquid chromatography tandem-mass spectrometry; NBUP, norbuprenorphine; NBUP-G, buprenorphine-3-b-D-glucuronide; PK, pharmacokinetics; UDPGA, UDP-glucuronic acid; UGT/Ugt, uridine 59-diphospho-glucuronosyltransferases. 100 Effect of Pregnancy on Norbuprenorphine Disposition in Mice 101 Fig. 1. Chemical structures of buprenorphine, norbuprenorphine, and norbuprenorphine-3-b-D-glucuronide. twofold by pregnancy in pregnant women (Bastian et al., 2017), much with pregnant mice throughout this study with respect to the PK of NBUP and its less is known about NBUP in this regard, particularly during pregnancy. derived glucuronidated metabolite NBUP-G. Pregnancy causes drastic changes in hormone production, such as The animal study protocol was essentially the same as previously described elevated levels of estrogens, progesterone, cortisol, and growth hor- (Liao et al., 2017). Briefly, NBUP was dissolved in a solution that contained 0.5% mones, which may be responsible for altered expression and activity of (v/v) dimethyl sulfoxide, 10% (v/v) ethanol, 39.5% (v/v) saline, and 50% (v/v) polyethylene glycol 400 at a concentration of 0.5 mg/ml. Under 2%–5% drug metabolizing enzymes (Anderson, 2005). As a result, some UGTs Downloaded from isoflurane anesthesia, each pregnant or nonpregnant mouse was administered may be upregulated by pregnancy (Jeong et al., 2008; Chen et al., 2009; thesamedoseof33mg of NBUP by retro-orbital injection. This dose was selected Wen et al., 2013). Given that NBUP is extensively metabolized by based on literature data about elicitation of a significant decrease in respiratory UGTs (Chang and Moody, 2009; Rouguieg et al., 2010), whether the rate in mice and the achievement of plasma concentration of NBUP in mice disposition of NBUP is altered by pregnancy due to induction of (Brown et al., 2012) that was comparable to that observed in pregnant women in glucuronidation warrants investigation. No such studies have been the late second and early third trimesters (Concheiro et al., 2011). Previous reported so far. pharmacokinetic studies showed that the half-life of NBUP in rodents was dmd.aspetjournals.org Therefore, in this study, we first determined the PK of NBUP and approximately 0.9 hours (Ohtani et al., 1994). We therefore administered NBUP NBUP-G in pregnant and nonpregnant mice. We then demonstrated to pregnant and nonpregnant female mice and collected blood at time points up to whether the intrinsic clearance of NBUP by glucuronidation and Ugt 720 minutes, which is longer than 10 half-lives. Thus, at various times (2, 30, 120, 240, 480, and 720 minutes) after NBUP administration, mice (n = 3/time point) protein expression in the mouse liver were increased by pregnancy. The were euthanized under anesthesia by cardiac puncture. Blood samples were data obtained will facilitate mechanistic understanding of changes in the collected in heparinized microcentrifuge tubes (BD Bioscience, San Jose, CA) disposition of NBUP during pregnancy, which could be important to and centrifuged at 1000 g for 10 minutes at 4°C. Plasma was collected and stored inform therapeutic strategies in pregnant women for BUP and drugs that at 280°C until use. At the first two time points, blood samples were collected are primarily metabolized by UGTs. under the same anesthetic used for the NBUP administration. Liver tissues were at ASPET Journals on October 3, 2021 collected at the same time points as blood samples. NBUP and NBUP-G in plasma samples were quantified by a validated LC-MS/MS assay as previously described Materials and Methods (Liao et al., 2017). Materials. NBUP and NBUP-G used for animal studies were provided by the Plasma Protein Binding of NBUP. Protein binding of NBUP in mouse National Institute on Drug Abuse (Bethesda, MD). NBUP, norbuprenorphine-d3 plasma was determined by ultrafiltration using Microcon-10 kDa Centrifugal (NBUP-d3),

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