Buprenorphine, Norbuprenorphine and Methadone Enantiomers Induce ABCB1/P‐gp and ABCG2/BCRP Expression and are Potent Aryl‐hydrocarbon Receptor Agonists in Human Placental Cells
Authors: Neraudgomma Naveen Kumar , Mao Qingcheng 
Published: November 2023

Opioid dependence in women during pregnancy is on a rise in the US. US public health survey data indicates a 15% increase in opiate use during pregnancy between 2009–2013. Several strategies are used to manage pregnancy drug abuse, and current consensus is to maintain opioid‐addicted pregnant women on a long acting opioid receptor agonist to avoid adverse health implications to the mother or fetus/baby. Methadone (MET), a potent μ‐opioid receptor agonist, and buprenorphine (BUP), a partial μ‐opioid receptor agonist and a potent κ‐opioid receptor antagonist, are widely used in opiate‐maintenance therapy. Methadone, given as an oral tonic, exists as racemic mixture of R (RMET) and S (SMET) enantiomers and RMET is 10 times more potent than SMET. BUP, administered sublingually, is metabolized to the major active metabolite, norbuprenorphine (NBUP) which has high affinity to μ‐ and κ‐opioid receptors. Placenta, a transient organ of fetal origin, develops during pregnancy and is critical for maternal‐to‐fetal exchange of nutrients and hormones. The ABC efflux transporters ABCB1/P‐gp and ABCG2/BCRP highly expressed on the apical membrane of placental syncytiotrophoblasts play a critical role in limiting fetal exposure to drugs and xenobiotics, thus protecting the fetus. In this study, we examined the effects of BUP, NBUP, RMET and SMET on expression of ABCB1 and ABCG2 in human placental cell lines. Data obtained will have important clinical implications as to how the use of these opiate‐maintenance medications may influence the protective role of human placenta.

The human placental choriocarcinoma cell line JEG3 was used in this study. The cells were treated with BUP and NBUP (0.001, 0.01 and 0.1 μM) and RMET and SMET (0.1, 1.5, 15 μM) for 12 h and 24 h, after 24 h of serum starvation. These concentrations represent the unbound Cmax, actual Cmax and 10 times Cmax, found in human plasma. MTT assay was used to evaluate the effect of these drugs on cell survival. Real‐time PCR and western blots were used to study changes in gene and protein expression, respectively, of ABCB1 and ABCG2. Mechanism of gene induction was examined using promoter‐reporter gene assays.

Drug treatment led to a 3–4 fold induction of ABCG2 mRNA and a 5–30 fold induction of ABCB1 mRNA compared to untreated control, after 24 h treatment, in a dose dependent manner. Aryl hydrocarbon receptor (AhR) is a nuclear receptor highly expressed in human placenta. Treating the cells with CH223191, a potent, highly selective AhR antagonist, prior to drug treatment, decreased ABCG2 mRNA expression by 3–4 fold, but not ABCB1 expression. Transient transfection of JEG3 cells with an AhR luciferase reporter, followed by drug treatment, caused a 2‐fold increase in luciferase activity. Next, we generated JEG3 cells stably expressing AhR. Transfecting these cells with the AhR luciferase reporter and ABCG2 promoter‐luciferase plasmids followed by drug treatment, resulted in a 3–6 fold induction of luciferase activity.

We report, for the first time, that (1) BUP and NBUP are potent AhR ligands; and (2) ABCG2 expression in human placental cells can be regulated via the AhR/ARNT signaling pathway. However, expression of ABCB1 was not inhibited by the AhR antagonist, suggesting involvement of other signaling cascades. Further studies are needed to fully understand the mechanisms by which ABCB1 and ABCG2 in human placenta are regulated by BUP, NBUP, RMET and SMET.

Support or Funding Information

NIH/NIDA: DA032507