The Role of Placental Carbonyl Reducing Enzymes in Biotransformation of Bupropion and 4-methylnitrosamino-1-(3-pyridyl)-1-butanone.

BACKGROUND: Bupropion (BUP) has a potential to be an effective pharmacotherapy for smoking cessation during pregnancy. Smoking during pregnancy stimulates placental carbonyl reductases that catalyze the biotransformation of BUP. 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen of cigarette smoke. Carbonyl reduction of NNK into 4- methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) constitutes a major step in NNK detoxification. Thus, placentas of pregnant smokers on BUP therapy can become a site of drug-drug interaction. Therefore, we investigated the effect of continuous exposure to BUP and cigarette smoke on the activity of placental carbonyl reductases in the formation of NNAL from NNK. METHODS: The reductive metabolism of NNK was determined using microsomal and cytosolic subcellular fractions of placentas obtained from non-smoking women treated with BUP for depression, and women not exposed to BUP: non-smokers (control) and smokers. The effect of BUP and its metabolites on the reductive metabolism of NNK was investigated using subcellular fractions of control placentas. RESULTS: The formation of NNAL from NNK by placental cytosolic fractions of heavy smokers (≥20 cigarettes per day) was lower than that of control (12.1±3.5 nmol.mgP-1 vs 16.5±6.0 nmol.mgP-1, P<0.05). While being exposed to BUP, the activity of placental carbonyl reductases remained unaffected, the formation of NNAL in the placental cytosolic fraction decreased only in the presence of high concentrations of BUP metabolites. CONCLUSION: Smoking during pregnancy decreases the detoxifying capacity of soluble carbonyl reductases towards NNK. Given the experimental conditions, exposure to BUP and its metabolites should not impede the reductive metabolism of NNK by placenta in vivo.

Transplacental transfer and distribution of pravastatin.

OBJECTIVE: The objective of the study was to determine the bidirectional transfer of pravastatin across the dually perfused term human placental lobule and its distribution between the tissue and maternal and fetal circuits. STUDY DESIGN: The transfer of pravastatin was determined in the maternal-to-fetal (n = 11) and fetal-to-maternal (n = 10) directions. Pravastatin was coperfused with its [(3)H]-isotope and the marker compound antipyrine (20 µg/mL) and its [(14)C]-isotope. The concentration of pravastatin in the perfused tissue and the maternal and fetal circuits was determined using liquid scintillation spectrometry. Inside-out vesicles prepared from placental brush border membranes were utilized to investigate the role of efflux transporters in the transplacental transfer of pravastatin. RESULTS: Pravastatin was transferred from the maternal to the fetal circuit and vice versa. In the maternal-to-fetal direction, the distribution of pravastatin at the end of experiment was as follows: 14 ± 5% of the drug was retained by the tissue, 68 ± 5% remained in the maternal circuit, and 18 ± 4% was transferred to the fetal circuit. The normalized transfer of pravastatin (clearance index) to antipyrine in the fetal-to-maternal direction (0.48 ± 0.07) was higher than its transfer in the maternal-to-fetal direction (0.36 ± 0.07, P < .01). Furthermore, pravastatin inhibited the adenosine triphosphate (ATP)-dependent uptake of the paclitaxel and estrone sulfate. CONCLUSION: The transfer of pravastatin across the dually perfused placental lobule suggests that fetal exposure to pravastatin is plausible. The higher transfer of pravastatin in the fetal-to-maternal direction than the reverse as well as its inhibition of the ATP-dependent uptake of [(3)H]-paclitaxel and [(3)H]-estrone sulfate strongly suggest the involvement of efflux transporters in decreasing its transfer across the placenta and support pravastatin's favorable pharmacokinetic profile in pregnancy.

Transplacental transfer and metabolism of buprenorphine in preterm human placenta.

We sought to determine whether gestational age affects the transplacental transfer and metabolism of buprenorphine (BUP). Transfer of BUP (10 ng/mL) and its [ (3)H]-isotope was determined across placentas of 30 to 34 weeks of gestation utilizing the technique of dual perfusion of placental lobule. Concentration of the drug in trophoblast tissue and in maternal and fetal circuits was determined by liquid scintillation spectrometry. Microsomes prepared from placentas of 17 to 37 weeks of gestation were divided into three groups: late second, early third, and late third trimesters. Antibodies raised against human cytochrome P450 (CYP) isoforms were utilized to identify the enzyme(s) catalyzing BUP biotransformation by preterm placental microsomes. The amount of norbuprenorphine formed was determined by liquid chromatography-mass spectrometry (LC-MS). BUP transfer across the placentas of 30 to 34 weeks of gestation was similar to those at term. CYP19 antibodies caused 60% inhibition of BUP metabolism by microsomes of late second and early third trimesters and 85% by microsomes of late third trimester. The developmental changes occurring in human placenta between 30 weeks of gestation through term do not affect the transfer of BUP across human placenta. CYP19 is the major enzyme responsible for biotransformation of BUP beginning at 17 weeks of gestation until term.

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

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.

Role of human placental apical membrane transporters in the efflux of glyburide, rosiglitazone, and metformin.

OBJECTIVE: Substrates of placental efflux transporters could compete for a single transporter, which could result in an increase in the transfer of each substrate to the fetal circulation. Our aim was to determine the role of placental transporters in the biodisposition of oral hypoglycemic drugs that could be used as monotherapy or in combination therapy for gestational diabetes. STUDY DESIGN: Inside-out brush border membrane vesicles from term placentas were used to determine the efflux of glyburide, rosiglitazone, and metformin by P-glycoprotein, breast cancer resistance protein, and multidrug resistance protein. RESULTS: Glyburide was transported by multidrug resistance protein (43 +/- 4%); breast cancer resistance protein (25 +/- 5%); and P-glycoprotein (9 +/- 5%). Rosiglitazone was transported predominantly by P-glycoprotein (71 +/- 26%). Metformin was transported by P-glycoprotein (58 +/- 20%) and breast cancer resistance protein (25 +/- 14%). CONCLUSION: Multiple placental transporters contribute to efflux of glyburide, rosiglitazone, and metformin. Administration of drug combinations could lead to their competition for efflux transporters.

Bupropion metabolism by human placenta.

Smoking during pregnancy is the largest modifiable risk factor for pregnancy-related morbidity and mortality. The success of bupropion for smoking cessation warrants its investigation for the treatment of pregnant patients. Nevertheless, the use of bupropion for the treatment of pregnant smokers requires additional data on its bio-disposition during pregnancy. Therefore, the aim of this investigation was to determine the metabolism of bupropion in placentas obtained from nonsmoking and smoking women, identify metabolites formed and the enzymes catalyzing their formation, as well as the kinetics of the reaction. Data obtained revealed that human placentas metabolized bupropion to hydroxybupropion, erythro- and threohydrobupropion. The rates for formation of erythro- and threohydrobupropion exceeded that for hydroxybupropion by several folds, were dependent on the concentration of bupropion and exhibited saturation kinetics with an apparent K(m) value of 40microM. Human placental 11beta-hydroxysteroid dehydrogenases were identified as the major carbonyl-reducing enzymes responsible for the reduction of bupropion to threo- and erythrohydrobupropion in microsomal fractions. On the other hand, CYP2B6 was responsible for the formation of OH-bupropion. These data suggest that both placental microsomal carbonyl-reducing and oxidizing enzymes are involved in the metabolism of bupropion.

Modulation of human placental P-glycoprotein expression and activity by MDR1 gene polymorphisms.

The ABC transporter P-glycoprotein is a product of the MDR1 gene and its function in human placenta is to extrude xenobiotics from the tissue thus decreasing fetal exposure. The goal of this investigation was to examine the effect of three polymorphisms in the MDR1 gene on the expression and activity of placental P-gp. In 199 term placentas examined, the C1236T variant was associated with 11% lower P-gp protein expression than wild-type, while the C3435T and G2677T/A variants each were associated with a 16% reduction (p<0.05). Homozygotes for the C1236T and C3435T variant allele (TT) were associated with 42% and 47% increase in placental P-gp transport activity, respectively (p=0.04 and p=0.02) of the prototypic substrate, [(3)H]-paclitaxel. These findings indicate that the C3435T and G2677T/A SNPs in MDR1 are significantly associated with decreased placental P-gp protein expression, while the C1236T and C3245T homozygous variants are significantly associated with an increase in its efflux activity.

Opiates inhibit paclitaxel uptake by P-glycoprotein in preparations of human placental inside-out vesicles.

The use of either methadone or buprenorphine for treatment of the pregnant opiate-dependent patient improves maternal and neonatal outcome. However, patient outcomes are often complicated by neonatal abstinence syndrome (NAS). The incidence and severity of NAS should depend on opiate concentration in the fetal circulation. Efflux transporters expressed in human placental brush border membranes decrease fetal exposure to medications by their extrusion to the maternal circulation. Accordingly, the concentration of either methadone or buprenorphine in the fetal circulation is, in part, dependent on the activity of the efflux transporters. The objective of this study was to characterize the activity of P-gp and its interaction with opiates in the placental apical membrane. Therefore, brush border membrane vesicles were prepared from human placenta. The vesicles were oriented approximately 75% inside-out, exhibited saturable ATP-dependent uptake of P-gp substrate [(3)H]-paclitaxel with an apparent K(t) of 66+/-38 nM and V(max) of 20+/-3 pmol mg protein (-1)min(-1). Methadone, buprenorphine, and morphine inhibited paclitaxel transport with apparent K(i) of 18, 44, and 90 microM, respectively. Our data indicate that a method has been established to determine the activity of the efflux transporter P-gp, expressed in placental brush border membranes, and the kinetics for the transfer of its prototypic substrate paclitaxel. Furthermore, the method was used to determine the effects of methadone, buprenorphine, and morphine on paclitaxel transfer by placental P-gp and revealed that they have higher affinity to the transporter than its classical inhibitor verapamil (K(i), 300 microM).

Effect of plasma proteins on buprenorphine transfer across dually perfused placental lobule.

OBJECTIVE: The aim of this investigation is to determine the effect of human serum albumin (HSA) and alpha-acid glycoprotein (AAG) on buprenorphine (BUP) transplacental transfer and distribution. METHODS: The technique of dual perfusion of placental lobule (DPPL) was utilised. BUP was co-perfused with the marker compound antipyrine (AP). In each experiment, the radiolabelled isotopes [(3)H]-BUP and [(14)C]-AP were added to enhance their detection limits. Human plasma proteins, HSA and AAG, were added to both the maternal and fetal circuits separately and in combination at their physiological concentrations in maternal and fetal circulations close to term. RESULTS: Transplacental transfer of BUP, in absence of plasma proteins, is a two-step process: the first is its uptake by the syncytiotrophoblast from the maternal circuit, and the second is its transfer/release from the tissue to the fetal circuit. The addition of HSA to the perfusion medium affected only the second step of BUP transfer, but AAG affected both steps. The combined effect of HSA and AAG was not different from that observed in presence of the latter alone. CONCLUSIONS: Binding of BUP to circulating AAG could have an important role in the transfer of the drug from the maternal to fetal circulation.

Transplacental transfer and metabolism of 17-alpha-hydroxyprogesterone caproate.

OBJECTIVE: Determine transplacental transfer and metabolism of 17-alpha-hydroxyprogesterone caproate and its distribution between the tissue and the maternal and fetal circuits of the dually perfused placental lobule. STUDY DESIGN: 17-alpha-Hydroxyprogesterone caproate (21 ng/mL) and its dual-labeled isotope, 17-alpha-hydroxy-[(3)H] progesterone [(14)C] caproate were added to the maternal circuit. The concentrations of the drug and its metabolite in trophoblast tissue and both circuits were determined by high performance liquid chromatography and liquid scintillation spectrometry. RESULTS: 17-alpha-Hydroxyprogesterone caproate was transferred from the maternal to fetal circuit. After a 4-hour perfusion period, a metabolite of 17-alpha-hydroxyprogesterone caproate that retained both progesterone and caproate moieties was identified in the tissue and the maternal and fetal circuits. Neither 17-alpha-hydroxyprogesterone caproate nor its metabolite, at the concentrations tested, had adverse effect on determined viability and functional parameters of placental tissue. CONCLUSION: 17-alpha-Hydroxyprogesterone caproate was metabolized by term placental lobule during its perfusion and both parent compound and its metabolite(s) transferred to the fetal circuit.

Transfer of metformin across the dually perfused human placental lobule.

OBJECTIVE: The purpose of this study was to determine the transfer characteristics of metformin across placentas that were obtained from uncomplicated pregnancies and from patients with gestational diabetes mellitus. STUDY DESIGN: The technique of dual perfusion of placental lobule was used. Metformin, 5 microg/mL and its [14C]-isotope were co-transfused with the marker compound antipyrine, 20 microg/mL and its [3H]-isotope from the maternal to fetal circuit. The drug content in both circulations and placental tissue was determined by liquid scintillation spectrometry. RESULTS: The transfer of metformin across term human placentas that were obtained from uncomplicated pregnancies was rapid and was not different from that in placentas that were obtained from patients with gestational diabetes mellitus. Metformin is distributed between the maternal and fetal circuits, with reliable amounts retained by the tissue. CONCLUSION: In an ex vivo model system, metformin was transferred readily from the maternal to fetal circuit across placentas that were obtained from uncomplicated pregnancies and pregnancies with gestational diabetes mellitus.