Pregnancy Increases Norbuprenorphine Clearance in Mice by Induction of Hepatic Glucuronidation

Abstract

Norbuprenorphine (NBUP) is the major active metabolite of buprenorphine (BUP) that is commonly used to treat opiate addiction during pregnancy; it possesses 25% of BUP's analgesic activity and 10 times BUP's respiratory depression effect. To optimize BUP's dosing regimen during pregnancy with better efficacy and safety, it is important to understand how pregnancy affects NBUP disposition. In this study, we examined the pharmacokinetics of NBUP in pregnant and nonpregnant mice by administering the same amount of NBUP through retro-orbital injection. We demonstrated that the systemic clearance (CL) of NBUP in pregnant mice increased ∼2.5-fold compared with nonpregnant mice. Intrinsic CL of NBUP by glucuronidation in mouse liver microsomes from pregnant mice was ∼2 times greater than that from nonpregnant mice. Targeted liquid chromatography tandem-mass spectrometry proteomics quantification revealed that hepatic Ugt1a1 and Ugt2b1 protein levels in the same amount of total liver membrane proteins were significantly increased by ∼50% in pregnant mice versus nonpregnant mice. After scaling to the whole liver with consideration of the increase in liver protein content and liver weight, we found that the amounts of Ugt1a1, Ugt1a10, Ugt2b1, and Ugt2b35 protein in the whole liver of pregnant mice were significantly increased ∼2-fold compared with nonpregnant mice. These data suggest that the increased systemic CL of NBUP in pregnant mice is likely caused by an induction of hepatic Ugt expression and activity. The data provide a basis for further mechanistic analysis of pregnancy-induced changes in the disposition of NBUP and drugs that are predominately and extensively metabolized by Ugts.

Quantitative Proteomics Reveals Changes in Transporter Protein Abundance in Liver, Kidney and Brain of Mice by Pregnancy

“…Mice were maintained under 12-h light/dark cycles, and food and water were provided ad libitum . Pregnant mice on gestation day (gd) 15 were obtained and maternal tissues (liver, kidney and brain) were collected as previously described [7, 14]. Gd 15 was selected as we previously showed that mRNA levels of most drug-metabolizing enzymes and transporters in the liver and kidney reached the maximal changes on gd 15 and remained relatively unchanged after gd 15 [10].…”

“…Protein quantification of transporters in the liver, kidney and brain of pregnant and non-pregnant mice by quantitative LC-MS/MS proteomics analysis. Quantification of changes in protein levels of Abcb1a (a P-gp isoform), Abcb1b (a P-gp isoform), Abcg2 (Bcrp), Abcb11 (Bsep), Abcc2 (Mrp2), Abcc3 (Mrp3), Abcc4 (Mrp4), Slco1a1 (Oatp1a1), Slco1a4 (Oatp1a4), Slco2b1 (Oatp2b1) and Slc22a3 (Oct3) in the liver, kidney and brain (Table 1 ) of pregnant and non-pregnant mice was carried out using a surrogate peptide-based LC-MS/MS method as previously described [7, 14, 15]. Transporter protein was quantified by the surrogate peptide LC-MS/MS method (https://ascpt.onlinelibrary.wiley.com/doi/abs/10.1002/cpt.819).…”

“…The corresponding heavy peptides containing labeled [ 13 C 6 15 N 2 ]-lysine or [ 13 C 6 15 N 4 ]-arginine were used as internal standards (Table ). Total membrane proteins were isolated from mouse tissues, reduced, denatured, alkylated, desalted and digested as previously described [7, 13, 14]. Bovine serum albumin (BSA) and heavy peptide internal standard cocktail were added to each sample before trypsin digestion [16].…”

“…The chromatographic conditions and instruments used were the same as previously described [7, 14]. Relative protein levels of individual transporters were presented as the peak area ratios of analyte peptides to the corresponding IS peptides, and then the ratios were normalized to BSA and the ratios of respective transporters in pooled tissue homogenates.…”

“…To the best of our knowledge, few previous studies have offered a comprehensive evaluation of the effects of pregnancy on protein abundance of a variety of drug transporters important for drug disposition simultaneously in multiple organs. In the present study, we chose pregnant mice as the animal model to conduct such analyses as we have previously shown that the Pharmacokinetic (PK) changes of several drugs in pregnant women can be replicated in the pregnant mouse model [5, 7, 11]. Knowledge about the changes in drug transporter abundance by pregnancy is important for understanding of pregnancy-induced alternations in drug/xenobiotic PK, efficacy and safety.…”