Role of transporter-mediated efflux in the placental biodisposition of bupropion and its metabolite, OH-bupropion

Abstract

Cigarette smoking during pregnancy is a preventable risk factor associated with maternal and fetal complications. Bupropion is an antidepressant used successfully for smoking cessation in non-pregnant patients. Our goal is to determine whether it could benefit the pregnant patient seeking smoking cessation. The aim of this investigation was to determine the role of human placenta in the disposition of bupropion and its major hepatic metabolite, OH-bupropion. The expression of efflux transporters P-gp and BCRP was determined in placental brush border membrane (n=200) and revealed a positive correlation (p<0.05). Bupropion was transported by BCRP (K(t) 3 microM, V(max) 30 pmol/mg protein/min) and P-gp (K(t) 0.5 microM, V(max) 6 pmol/mg protein min) in placental inside-out vesicles (IOVs). OH-bupropion crossed the dually-perfused human placental lobule without undergoing further metabolism, nor was it an efflux substrate of P-gp or BCRP. In conclusion, our data indicate that human placenta actively regulates the disposition of bupropion (via metabolism, active transport), but not its major hepatic metabolite, OH-bupropion.

Figure 1

P-gp and BCRP protein expression in human placentas as determined by western blot. The immunoblot (10 μg/lane) was probed with the monoclonal antibody C219 for P-gp, and monoclonal antibody BXP-21 for BCRP. Immunoreactive protein bands detected by immunoblotting were analyzed by densitometry. Expression was determined as a proportion of the total amount of β-actin present per lane. There was a positive correlation between protein expression of P-gp and BCRP (*, t = p<0.05).

Figure 2

Bupropion transport by P-gp and BCRP. Bupropion displayed ATP-dependent transport into placental IOVs by both P-gp and BCRP. (A) P-gp displayed apparent K_t of 0.5 ± 0.2 μM and V_max = 6 ± 0.7 pmol/mg protein*min for bupropion transport (B) BCRP displayed apparent K_t of 3 ± 2 μM and V_max = 30 ± 13 pmol/mg protein*min for bupropion transport, with Lineweaver-Burk plot (inset). Data are presented as mean ± SEM of 4–8 experiments in a pool of 60 placentas.

Figure 2

Bupropion transport by P-gp and BCRP. Bupropion displayed ATP-dependent transport into placental IOVs by both P-gp and BCRP. (A) P-gp displayed apparent K_t of 0.5 ± 0.2 μM and V_max = 6 ± 0.7 pmol/mg protein*min for bupropion transport (B) BCRP displayed apparent K_t of 3 ± 2 μM and V_max = 30 ± 13 pmol/mg protein*min for bupropion transport, with Lineweaver-Burk plot (inset). Data are presented as mean ± SEM of 4–8 experiments in a pool of 60 placentas.

Figure 3

Bupropion stimulation of ATP hydrolysis by P-gp and BCRP. Stimulation of ATPase activity, as measured by inorganic phosphate release in the presence and absence of ATPase inhibitor sodium orthovanadate (100 μM), was detected in baculovirus-transfected insect cells expressing human P-gp (A) and BCRP (B). Bupropion stimulated ATP hydrolysis by both P-gp and BCRP in a concentration-dependent manner, confirming that bupropion interacts with P-gp and BCRP.

Figure 3

Bupropion stimulation of ATP hydrolysis by P-gp and BCRP. Stimulation of ATPase activity, as measured by inorganic phosphate release in the presence and absence of ATPase inhibitor sodium orthovanadate (100 μM), was detected in baculovirus-transfected insect cells expressing human P-gp (A) and BCRP (B). Bupropion stimulated ATP hydrolysis by both P-gp and BCRP in a concentration-dependent manner, confirming that bupropion interacts with P-gp and BCRP.

Figure 4

OH-bupropion transport by placental IOVs. ATP-dependent transport of OH-Bupropion was determined by its accumulation in placental IOVs in the presence and absence of an ATP-regenerating system. The accumulation was similar in the presence and absence of ATP, indicating OH-bupropion is not a transported substrate of P-gp or BCRP in human placenta brush border membrane. The role of non-ATP-dependent transporters in OH-bupropion distribution cannot be ruled out at this time. Data are presented as mean ± SEM of 4 experiments in a pool of 60 placentas.

Figure 5

Placental transfer of OH-bupropion. [¹⁴C]-OH-bupropion added to the maternal circuit at initial concentration of 470 ± 17 ng/ml was perfused using closed-closed configuration for 4 hours. OH-bupropion appeared in the fetal circuit within 5 minutes of perfusion. The concentration of OH-bupropion in the fetal circuit at the end of experiment reached 151 ± 13 ng/ml, which represents 32 ± 3% of its initial concentration in maternal circuit.

Figure 6

OH-bupropion (OH-BUP) metabolism by placental tissue during perfusion. OH-bupropion was not metabolized by the placental tissue during 4 hours of perfusion. HPLC-Radioactive chromatograms of [C¹⁴]-OH-bupropion perfused in human placenta displaying A) [C¹⁴]-OH-bupropion (OH-BUP) standard, B) maternal perfusate at 240 minutes, C) fetal perfusate at 240 minutes, and D) sample of perfused placental tissue at 240 minutes.

Figure 6

OH-bupropion (OH-BUP) metabolism by placental tissue during perfusion. OH-bupropion was not metabolized by the placental tissue during 4 hours of perfusion. HPLC-Radioactive chromatograms of [C¹⁴]-OH-bupropion perfused in human placenta displaying A) [C¹⁴]-OH-bupropion (OH-BUP) standard, B) maternal perfusate at 240 minutes, C) fetal perfusate at 240 minutes, and D) sample of perfused placental tissue at 240 minutes.

Figure 6

OH-bupropion (OH-BUP) metabolism by placental tissue during perfusion. OH-bupropion was not metabolized by the placental tissue during 4 hours of perfusion. HPLC-Radioactive chromatograms of [C¹⁴]-OH-bupropion perfused in human placenta displaying A) [C¹⁴]-OH-bupropion (OH-BUP) standard, B) maternal perfusate at 240 minutes, C) fetal perfusate at 240 minutes, and D) sample of perfused placental tissue at 240 minutes.

Figure 6

OH-bupropion (OH-BUP) metabolism by placental tissue during perfusion. OH-bupropion was not metabolized by the placental tissue during 4 hours of perfusion. HPLC-Radioactive chromatograms of [C¹⁴]-OH-bupropion perfused in human placenta displaying A) [C¹⁴]-OH-bupropion (OH-BUP) standard, B) maternal perfusate at 240 minutes, C) fetal perfusate at 240 minutes, and D) sample of perfused placental tissue at 240 minutes.