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Background: In this study, a prototype of a targeted nanocarrier for drug delivery for prenatal therapy of the developing fetus was developed and examined in vitro and ex vivo. The folate transport mechanism in the human placenta was utilized as a possible pathway for the transplacental delivery of targeted nanoparticles., Methods: Several types of folic acid-decorated polymeric nanoparticles were synthesized and characterized. During transport studies of targeted and non-targeted fluorescent nanoparticles across the placental barrier, the apparent permeability values, uptake, transfer indices, and distribution in placental tissue were determined., Results: The nanoparticles had no effect on BeWo b30 cell viability. In vitro , studies showed significantly higher apparent permeability of the targeted nanoparticles across the cell monolayers as compared to the nontargeted nanoparticles (P e = 5.92 ± 1.44 ×10 -6 cm/s for PLGA-PEG-FA vs . 1.26 ± 0.31 ×10 -6 cm/s for PLGA-PEG, P < 0.05), and the transport of the targeted nanoparticles was significantly inhibited by excess folate. Ex vivo placental perfusion showed significantly greater accumulation of the targeted nanoparticles in the placental tissue (4.31 ± 0.91%/g for PLGA-PEG-FA vs. 2.07 ± 0.26%/g for PLGA-PEG)., Conclusion: The data obtained suggested different mechanisms for the uptake and transplacental transfer of targeted versus nontargeted nanoparticles. This targeted nanoformulation may be a promising strategy for fetal drug therapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Aims: Indomethacin is used for the treatment of preterm labour, short cervices and idiopathic polyhydramnios during pregnancy. Few studies have described the pharmacokinetics (PK) of indomethacin during pregnancy. This study aimed to determine maternal and fetal PK of indomethacin during different trimesters of pregnancy using physiologically based PK (PBPK) modelling and simulations., Methods: Full PBPK simulations were performed in nonpregnant subjects and pregnant subjects from each trimester of pregnancy at steady state using Simcyp's healthy volunteers and pregnancy PBPK model, respectively. The fetal exposures were predicted using a fetoplacental pregnancy PBPK model. The models were verified by comparing PBPK-based predictions with observed PK profiles., Results: Predicted exposure (AUC 0-6h ) and clearance of indomethacin in nonpregnant women and pregnant women are similar to the clinical observations. AUC 0-6h of indomethacin is approximately 14, 24 and 32% lower, consistent with 18, 34 and 52% higher clearance in the first, second and third trimesters of pregnancy, respectively, compared to nonpregnant women. Predicted fetal plasma exposures increased by approximately 30% from the second trimester to the third trimester of pregnancy., Conclusion: A mechanistic PBPK model adequately described the maternal and the fetal PK of indomethacin during pregnancy. As the pregnancy progresses, a modest decrease (≤32%) in systemic exposures in pregnant women and a 33% increase in fetal exposures to indomethacin were predicted. Higher fetal exposures in the third trimester of pregnancy may pose safety risks to the fetus. Additional studies are warranted to understand the exposure-response relationship and provide appropriate dosing recommendations during pregnancy that consider both safety and efficacy., (© 2021 British Pharmacological Society.)

Diagnosis and treatment of breast cancer in pregnancy can result in morbidity and mortality for the mother and fetus. Many new paclitaxel nanoformulations commercially available worldwide for breast cancer treatment are being adopted due to favorable dosing regimens and side effect profiles, but their transplacental transport and resultant fetal exposure remain unknown. Here, we examine three formulations: Taxol (paclitaxel dissolved in Kolliphor EL and ethanol); Abraxane (albumin nanoparticle); and Genexol-PM (polymeric micelle). In the ex vivo dually perfused human placental cotyledon, placental accumulation of Genexol-PM is higher than Taxol, and both nanoformulations have lower maternal concentrations of paclitaxel over time. In vitro studies of these formulations and fluorescent nanoparticle analogs demonstrate that Genexol-PM allows paclitaxel to overcome P-glycoprotein efflux, but Abraxane behaves as a free drug formulation. We anticipate that these findings will impact future development of rational and safe treatment strategies for pregnancy-associated breast cancer and other diseases., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background and Objective: Cytochrome P450 (CYP) 2C9 catalyzes the biotransformation of indomethacin to its inactive metabolite O-desmethylindomethacin (DMI). The aim of this work was to determine the effect of CYP2C9 polymorphisms on indomethacin metabolism in pregnant women., Methods: Plasma concentrations of indomethacin and DMI at steady state were analyzed with a validated LC-MS/MS method. DNA was isolated from subject blood and buccal smear samples. Subjects were grouped by genotype for comparisons of pharmacokinetic parameters., Results: For subjects with the *1/*2 genotype, the mean steady-state apparent oral clearance (CL/F ss ) of indomethacin was 13.5 ± 7.7 L/h (n = 4) and the mean metabolic ratio (AUC DMI /AUC indomethacin ) was 0.291 ± 0.133. For subjects with the *1/*1 genotype, these values were 12.4 ± 2.7 L/h and 0.221 ± 0.078, respectively (n = 14). Of note, we identified one subject who was a carrier of both the *3 and *4 alleles, resulting in an amino acid change (I359P) which has not been reported previously. This subject had a metabolic ratio of 0.390 and a CL/F ss of indomethacin (24.3 L/h) that was nearly double the wild-type clearance., Conclusion: Although our results are limited by sample size and are not statistically significant, these data suggest that certain genetic polymorphisms of CYP2C9 may lead to an increased metabolic ratio and an increase in the clearance of indomethacin. More data are needed to assess the impact of CYP2C9 genotype on the effectiveness of indomethacin as a tocolytic agent.

Ex vivo placental perfusion experiments are important in understanding the quantity and mechanisms of xenobiotic transport to the fetus during pregnancy. Our study demonstrates that paclitaxel and antipyrine concentrations in placental perfusion medium containing physiological concentrations of human serum albumin during pregnancy (30 mg/mL) can be quantified by RP-HPLC and UV detection. A liquid-liquid extraction method was developed for the quantification of paclitaxel and celecoxib (internal standard) from perfusion medium. Antipyrine, which is a necessary marker in placental perfusions for determining the validity of experiments and calculating the clearance index of xenobiotics, was also analyzed by HPLC and UV detection. Antipyrine concentrations were determined by HPLC after precipitating the perfusion medium in acetonitrile and separating the precipitated proteins by centrifugation. Concentrations were fitted to linear regressions with R 2 values approaching 1. Lower limits of detection for paclitaxel and antipyrine were 100 ng/mL and 200 ng/mL, respectively. Both methods demonstrated high intra-day and inter-day precision and trueness. Additionally, the use of these methods was demonstrated in a placental perfusion experiment using Taxol ® (paclitaxel dissolved in Cremophor-EL). The fetal transfer rate of Taxol was 6.6% after 1 hour.

We analyzed more than 40 cytotrophoblast cultures derived from cell islets that grew from trypsinized tissue fragments of placental microvilli. Phenotypic variability of trophoblasts was demonstrated. Changes in trophoblast morphology from epithelium-like or oval cells to bipolar and spindle-shaped or twisted and then to mesenchymal-like cells as well as intensive expression of cytokeratin-7 and vimentin attested to epithelial-mesenchymal transition of trophoblasts during in vitro culturing. Analysis of the expression of specific markers in long-term trophoblast culture (≥7 passages) revealed the possibility of culture contamination with other non-trophoblast cells including fibroblasts. High risk of trophoblast culture contamination with rapidly growing cells necessitates regular control of the cultures used in fundamental studies. Our experiments confirmed the possibility of long-term culturing of cells maintaining trophoblast properties. The identity and purity of 4 trophoblast cultures free from contamination and retaining the properties of pure culture during long-term (>10 passages) culturing in vitro were confirmed.

Approximately 20% of pregnant women smoke despite intentions to quit. Smoking cessation drugs, such as nicotine replacement therapy (NRT) and bupropion, are recommended treatments. Adverse cardiovascular outcomes in offspring have raised concerns about NRT's safety during pregnancy. However, the effect of bupropion is unknown. Using a rat model, we determined whether NRT and bupropion interventions during pregnancy are safer than continued smoking on offspring's cardiovascular function. Male offspring of controls and dams exposed to cigarette smoke (1.6 packs/day, inhalation), nicotine (2 mg/kg/d subcutaneously), and bupropion (13 mg/kg twice daily orally) were assessed for fetoplacental weight, cardiac function, blood pressure, and vascular reactivity. Fetoplacental weights were decreased and spontaneous beating and intracellular calcium in neonatal cardiomyocytes were increased in smoking, nicotine, and bupropion offspring; however, these effects were more accentuated in smoking followed by nicotine and bupropion offspring. Increased heart rate and decreased cardiac output, stroke volume, and left ventricular percent posterior wall thickening were observed in smoking, nicotine, and bupropion offspring. The left ventricular mass was reduced in smoking and nicotine but not in bupropion offspring. Blood pressure was higher with decreased endothelium-dependent relaxation and exaggerated vascular contraction to angiotensin II in smoking and nicotine offspring, with more pronounced dysfunctions in smoking than nicotine offspring. Maternal bupropion did not impact offspring's blood pressure, endothelium-dependent relaxation, and vascular contraction. In conclusion, maternal nicotine intervention adversely affects offspring's cardiovascular outcomes, albeit less severely than continued smoking. However, bupropion causes cardiac derangement in offspring but does not adversely affect blood pressure and vascular function.

Background: Bupropion is used to treat depression during pregnancy. However, its usefulness as a smoking cessation aid for pregnant women is not fully known., Objective: The objective of the study was to evaluate the preliminary efficacy of bupropion sustained release for smoking cessation during pregnancy., Study Design: We conducted a randomized, prospective, double-blind, placebo-controlled, pilot trial. Pregnant women who smoked daily received individualized behavior counseling and were randomly assigned to a 12 week, twice-a-day treatment with 150 mg bupropion sustained release or placebo. The primary study objectives were to determine whether bupropion sustained release reduces nicotine withdrawal symptoms on the quit date and during the treatment period compared with placebo and whether it increases 7 day point prevalence abstinence at the end of the treatment period and at the end of pregnancy., Results: Subjects in the bupropion (n = 30) and placebo (n = 35) groups were comparable in age, smoking history, number of daily smoked cigarettes, and nicotine dependence. After controlling for maternal age and race, bupropion sustained release reduced cigarette cravings (1.5 ± 1.1 vs 2.1 ± 1.2, P = .02) and total nicotine withdrawal symptoms (3.8 ± 4.3 vs 5.4 ± 5.1, P = .028) during the treatment period. Administration of bupropion sustained release reduced tobacco exposure, as determined by levels of carbon monoxide in exhaled air (7.4 ± 6.4 vs 9.1 ± 5.8, P = .053) and concentrations of cotinine in urine (348 ± 384 ng/mL vs 831 ± 727 ng/mL, P = .007) and increased overall abstinence rates during treatment (19% vs 2%, P = .003). However, there was no significant difference in 7 day point prevalence abstinence rates between the 2 groups at the end of medication treatment (17% vs 3%, P = .087) and at the end of pregnancy (10% vs 3%, P = .328)., Conclusion: Individual smoking cessation counseling along with the twice-daily use of 150 mg bupropion sustained release increased smoking cessation rates and reduced cravings and total nicotine withdrawal symptoms during the treatment period. However, there was no significant difference in abstinence rates between groups at the end of medication treatment and at the end of pregnancy, likely because of the small sample size. A larger study is needed to confirm these findings and to examine the potential benefit/ risk ratio of bupropion sustained release for smoking cessation during pregnancy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Bupropion sustained release is used to promote smoking cessation in males and nonpregnant females. However, its efficacy as a smoking cessation aid during pregnancy is not reported. The pregnancy-associated changes in maternal physiology may alter the pharmacokinetics and pharmacodynamics of bupropion and consequently its efficacy in pregnant smokers. Therefore, the aims of this study were to determine the steady-state pharmacokinetics of bupropion during pregnancy and the effect of functional genetic variants of CYP2B6 and CYP2C19 on bupropion pharmacokinetics in pregnant women. Plasma and urine concentrations of bupropion and its metabolites hydroxybupropion (OHBUP), threohydrobupropion, and erythrohydrobupropion were determined by liquid chromatography-mass spectrometry. Subjects were genotyped for five nonsynonymous single-nucleotide polymorphisms that result in seven CYP2B6 alleles, namely *2, *3, *4, *5, *6, *7, and *9, and for CYP2C19 variants *2, *3, and *17 The present study reports that the isoform-specific effect of pregnancy on bupropion-metabolizing enzymes along with the increase of renal elimination of the drug could collectively result in a slight decrease in exposure to bupropion in pregnancy. In contrast, pregnancy-induced increase in CYP2B6-catalyzed bupropion hydroxylation did not impact the plasma levels of OHBUP, probably due to a higher rate of OHBUP glucuronidation, and renal elimination associated with pregnancy. Therefore, exposure to OHBUP, a pharmacologically active metabolite of the bupropion, appears to be similar to that of the nonpregnant state. The predicted metabolic phenotypes of CYP2B6*6 and variant alleles of CYP2C19 in pregnancy are similar to those in the nonpregnant state., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Bupropion is used for treatment of depression during pregnancy. However, its use as a smoking cessation aid for pregnant women is currently under evaluation., Objective: The aim of this opportunistic study was to investigate the transfer of bupropion and its major pharmacologically active metabolites, hydroxybupropion and threohydrobupropion, across the placenta in vivo. In addition, the concentrations of the drug and its metabolites were determined in the amniotic fluid., Study Design: The following samples were collected at deliveries from 22 women taking bupropion: maternal blood (n = 22), umbilical cord venous blood (n = 22), and amniotic fluid (n = 9). The concentrations of the drug and its metabolites in blood plasma and amniotic fluid were determined by means of liquid chromatography-mass spectrometry. Placental passage was calculated as a ratio of umbilical cord venous plasma to maternal plasma concentrations., Results: The levels of hydroxybupropion and threohydrobupropion in umbilical cord venous plasma were invariably lower than their corresponding concentrations in maternal plasma. The concentrations of bupropion in umbilical cord plasma were lower than in maternal plasma in the majority of the maternal-cord blood pairs. The median values of the umbilical cord venous plasma to maternal plasma ratios were: bupropion, 0.53 (interquartile range 0.35, n = 18), hydroxybupropion, 0.21 (interquartile range 0.12, n = 18), and threohydrobupropion, 0.61 (interquartile range 0.11, n = 21). In umbilical cord venous plasma, the median concentration of bupropion was 5.3 ng/mL; hydroxybupropion, 103.6 ng/mL; and threohydrobupropion, 59.6 ng/mL. Bupropion and its metabolites were detectable in the amniotic fluid but the concentrations of threohydrobupropion were higher than those in the corresponding umbilical cord venous plasma., Conclusion: Bupropion and its active metabolites cross the placenta to the fetal circulation. The concentrations of hydroxybupropion and threohydrobupropion in umbilical cord venous plasma were higher than bupropion concentrations suggesting a higher fetal exposure to the metabolites than the parent drug. The higher levels of threohydrobupropion in the amniotic fluid than those in umbilical cord venous plasma suggest that enzymes involved in the metabolism of bupropion to threohydrobupropion are most likely active in the fetus. The biological consequences of fetal exposure to maternally administered bupropion and/or its active metabolites via placental transfer and recirculation of the amniotic fluid are yet to be determined., (Copyright © 2016. Published by Elsevier Inc.)

This report describes the development and validation of a chromatography/tandem mass spectrometry method for the quantitative determination of pravastatin and its metabolite (3α-hydroxy pravastatin) in plasma and urine of pregnant patients under treatment with pravastatin, as part of a clinical trial. The method includes a one-step sample preparation by liquid-liquid extraction. The extraction recovery of the analytes ranged between 93.8 and 99.5% in plasma. The lower limits of quantitation of the analytes in plasma samples were 0.106 ng/mL for pravastatin and 0.105 ng/mL for 3α-hydroxy pravastatin, while in urine samples they were 19.7 ng/mL for pravastatin and 2.00 ng/mL for 3α-hydroxy pravastatin. The relative deviation of this method was <10% for intra- and interday assays in plasma and urine samples, and the accuracy ranged between 97.2 and 106% in plasma, and between 98.2 and 105% in urine. The method described in this report was successfully utilized for determining the pharmacokinetics of pravastatin in pregnant patients enrolled in a pilot clinical trial for prevention of preeclampsia., (Copyright © 2015 John Wiley & Sons, Ltd.)

This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87-99%, and that from urine samples was 85-95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100-0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984-1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy was 86-114% in plasma, and 94-105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine., (Copyright © 2014 John Wiley & Sons, Ltd.)

Recent in vitro data obtained in our laboratory revealed similarities between baboons and humans in the biotransformation of bupropion (BUP) by both hepatic and placental microsomes. These data supported the use of baboons to study BUP biotransformation during pregnancy. The aim of this investigation was to determine the pharmacokinetics of BUP in baboons during pregnancy and postpartum, as well as fetal exposure to the drug after intravenous administration. Pregnant baboons (n = 5) received a single intravenous bolus dose of bupropion hydrochloride (1 mg/kg) at gestational ages 94-108 days (midpregnancy), 142-156 days (late pregnancy), and 6 weeks postpartum. Blood and urine samples were collected for 12 and 24 hours, respectively. The concentrations of BUP, hydroxybupropion (OH-BUP), threohydrobupropion, and erythrohydrobupropion in plasma were determined by liquid chromatography-tandem mass spectrometry. Relative to the postpartum period, the average midpregnancy clearance of BUP trended higher (3.6 ± 0.15 versus 2.7 ± 0.28 l/h per kg) and the average C(max) (294 ± 91 versus 361 ± 64 ng/ml) and the area under the curve (AUC) of BUP values (288 ± 22 versus 382 ± 42 h·ng/ml) trended lower. AUC(OH-BUP) also tended to be lower midpregnancy compared with postpartum (194 ± 76 versus 353 ± 165 h·ng/ml). Whereas the observed trend toward increased clearance of BUP during baboon pregnancy could be associated with a pregnancy-induced increase in its biotransformation, the trend toward increased renal elimination of OH-BUP may overshadow any corresponding change in the hydroxylation activity of CYP2B., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

The increasing incidence and severity of methicillin- and vancomycin-resistant infections during pregnancy prompted further development of telavancin. The understanding of the pharmacokinetics of telavancin during pregnancy is critical to optimize dosing. Due to ethical and safety concerns the study is conducted on the pregnant baboons. A method using solid-phase extraction coupled with liquid chromatography-single quadrupole mass spectrometry for the quantitative determination of telavancin in baboon plasma samples was developed and validated. Teicoplanin was used as an internal standard. Telavancin was extracted from baboon plasma samples by using Waters Oasis(®) MAX 96-Well SPE plate and achieved extraction recovery was >66% with variation <12%. Telavancin was separated on Waters Symmetry C18 column with gradient elution. Two SIM channels were monitored at m/z 823 and m/z 586 to achieve quantification with simultaneous confirmation of telavancin identification in baboon plasma samples. The linearity was assessed in the range of 0.188μg/mL to75.0μg/mL, with a correlation coefficient of 0.998. The relative standard deviation of this method was <11% for within- and between-run assays, and the accuracy ranged between 96% and 114%., (Published by Elsevier B.V.)

Background and Objectives: Although indomethacin has been widely used for the treatment of preterm labor over the past 40 years, there are few reports regarding its pharmacokinetics in pregnant women., Methods: This opportunistic study assessed the steady-state pharmacokinetics of indomethacin in pregnant subjects to whom an oral dose of 25 mg every 6 h was prescribed. Indomethacin concentrations in plasma and urine were analyzed by a validated high-performance liquid chromatography method with mass spectrometric detection., Results: The mean area under the plasma concentration versus time curve at steady state (AUCss) was 1.91 ± 0.53 μg·h/mL, mean peak plasma concentration (C max) was 1.02 ± 0.49 μg/mL, and mean time to reach C max (t max) was 1.3 ± 0.7 h. The mean apparent clearance at steady state was 14.5 ± 5.5 L/h, which is higher than the apparent clearance reported in the literature for non-pregnant subjects. Indomethacin crosses the placenta; the mean fetal/maternal ratio from five sets of cord blood samples collected at delivery was 4.0 ± 1.1., Conclusions: Further studies are needed to determine whether any dose adjustments are necessary as a result of the increased clearance of indomethacin during pregnancy.

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., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Liquid chromatography with electrospray ionization mass spectrometry for the quantitative determination of famotidine in human urine, maternal and umbilical cord plasma was developed and validated. The plasma samples were alkalized with ammonium hydroxide and extracted twice with ethyl acetate. The extraction recovery of famotidine in maternal and umbilical cord plasma ranged from 53 to 64% and 72 to 79%, respectively. Urine samples were directly diluted with the initial mobile phase then injected into the HPLC system. Chromatographic separation of famotidine was achieved by using a Phenomenex Synergi™ Hydro-RP™ column with a gradient elution of acetonitrile and 10 mm ammonium acetate aqueous solution (pH 8.3, adjusted with ammonium hydroxide). Mass spectrometric detection of famotidine was set in the positive mode and used a selected ion monitoring method. Carbon-13-labeled famotidine was used as internal standard. The calibration curves were linear (r(2) > 0.99) in the concentration ranges of 0.631-252 ng/mL for umbilical and maternal plasma samples and 0.075-30.0 µg/mL for urine samples. The relative deviation of method was <14% for intra- and inter-day assays, and the accuracy ranged between 93 and 110%. The matrix effect of famotidine in human urine, maternal and umbilical cord plasma was less than 17%., (Copyright © 2013 John Wiley & Sons, Ltd.)

A liquid chromatography with single quadrupole mass spectrometry method was developed for the quantitative determination of indomethacin in the maternal plasma and urine of pregnant patients under treatment. A deuterium-labeled isotope of indomethacin (d4-indomethacin) was used as an internal standard. The maternal plasma and urine samples were acidified with 1.0M HCl then extracted with chloroform to achieve the extraction recovery range of 94-104% with variation less than 11%. Chromatographic separation was achieved by a Waters Symmetry C₁₈ column with isocratic elution of 0.05% (v/v) formic acid aqueous solution and acetonitrile (47:53, v/v). An in-source fragmentation was applied on the single quadrupole mass spectrometer equipped with an electrospray ionization source at positive mode. The LC-ESI-MS quantification was performed in the selected ion monitoring mode targeting ions at m/z 139 for indomethacin and m/z 143 for its internal standard. The calibration curves were linear in the concentration ranges between 14.8 and 2.97 × 10(3) ng/mL for plasma samples and between 10.5 and 4.21 × 10(3) ng/mL for urine samples. The relative standard deviation of this method was less than 8% for intra- and inter-day assays, and the accuracy ranged between 90% and 108%., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objectives: Determine the bidirectional transfer of oseltamivir carboxylate (OC) across term human placenta and its distribution between the tissue, maternal and fetal circuits., Methods: The technique of dual perfusion of placental lobule (DPPL) in its recirculating mode was utilized to determine the transfer of the drug. OC (350 ng/mL) was co-perfused with its [(3)H]-isotope and the marker compound antipyrine (AP, 20 µg/mL) together with its [(14)C]-isotope. The concentrations of OC and any of its metabolite(s) formed during perfusion were determined in the tissue, maternal and fetal circuits by liquid scintillation spectrometry following their separation by High Performance Liquid Chromatography (HPLC)., Results: The distribution of OC following its perfusion in the Maternal-to-Fetal direction for 4 h was as follows: 21 ± 4% of the drug was transferred to the fetal circuit, 13 ± 5% was retained by the perfused lobule, and 66 ± 4% remained in the maternal circuit. The normalized transfer of OC to that of AP (Clearance index) in the maternal-to-fetal direction was (0.47 ± 0.11) and was not different from its transfer from the fetal-to-maternal direction (0.47 ± 0.06) suggesting that involvement of placental efflux transporters is unlikely., Conclusions: OC crosses human placenta. As the transfer rate of OC is 47% of the freely diffusible AP, it is likely that fetus could be exposed to OC during pregnancy.

A liquid chromatography in tandem with electro-spray ionization mass spectrometry method has been developed and validated for the quantitative determination of bupropion and its major metabolites (hydroxybupropion, threo- and erythrohydrobupropion) in human umbilical cord plasma and placental tissue. The samples were acidified with trichloroacetic acid, and protein precipitated by adding acetonitrile. Chromatographic separation of drug and metabolites was achieved by using a Waters Symmetry C(18) column, with an isocratic elution of 31% methanol and 69% formic acid (0.04%, v/v) aqueous solution at a flow rate of 1.0 mL/min. Detection was carried out by mass spectrometry using positive electro-spray ionization mode, and the compounds were monitored using multiple reactions monitoring method. Deuterium-labeled isotopes of the compounds were used as internal standards. Calibration curves were linear (r(2)>0.99) in the tested ranges. The lower limit of quantification of analytes in umbilical cord plasma samples is <0.72 ng/mL and 0.92 ng/g in placental tissue samples. The relative deviation of this method was <15% for intra- and inter-day assays, and the accuracy ranged between 88% and 105%. The extraction recovery of the four analytes ranged between 89% and 96% in umbilical cord plasma, and 64% and 80% in placental tissue. No significant matrix effect was observed in the presented method., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Perfusion of 17-alpha-hydroxyprogesterone caproate (17HPC) via the maternal circuit of a dually perfused human placental lobule resulted in the extensive formation of 2 metabolites. On the other hand, human placental microsomes biotransformed 17HPC into 5 monohydroxylated metabolites, which did not correspond to those formed during perfusion. The goal of this investigation was to determine the subcellular localization of the enzymes responsible for the biotransformation of 17HPC during its perfusion in human placenta. Crude subcellular fractions of the human placental tissue were utilized. Six 17HPC metabolites were formed by the placental mitochondrial fraction, of which 4 were identical to those formed by the microsomes; whereas the other 2, namely MM and M₁₉, were formed by the mitochondrial fraction only. The latter metabolites were identical to those formed during 17HPC perfusion, as determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Therefore, these data strongly suggest that the enzymes responsible for the biotransformation of 17HPC during its perfusion are predominantly localized in human placental mitochondria.

The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (Km 36±6 μM, Vmax 258±32 pmol mg protein(-1) min(-1)), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent Km values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: The present study was undertaken to investigate the pharmacokinetics of metronidazole in pregnant patients with bacterial vaginosis., Methods: Twenty patients received metronidazole (Flagyl ®, Pfizer, 235 East 42nd Street, NY, NY 10017) oral dose 500 mg twice a day for 3 consecutive days. Pharmacokinetic analyses of metronidazole were performed after a single oral dose on the morning of day 4., Results: Although absolute estimates of metronidazole total body exposure were highest in women during early term pregnancy, weight-corrected estimates of exposure maximum plasma drug concentration (C(max)) and the area under the plasma concentration-versus-time curve (AUC(0-12)), along with apparent oral clearance and distribution volume, were not significantly different between women at early, middle, and late stages of pregnancy and were in the range of reported values for nonpregnant patients receiving a similar dose., Conclusions: The pharmacokinetic profile of metronidazole did not change at the different time points assessed during pregnancy.

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., (Thieme Medical Publishers.)

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., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

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., (2010 Elsevier Inc. All rights reserved.)

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., (Copyright 2010 Mosby, Inc. All rights reserved.)

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., (Copyright 2009 Elsevier Inc. All rights reserved.)

One of the factors affecting the pharmacokinetics (PK) of a drug during pregnancy is the activity of hepatic and placental metabolizing enzymes. Recently, we reported on the biotransformation of glyburide by human hepatic and placental microsomes to six metabolites that are structurally identical between the two tissues. Two of the metabolites, 4-trans-(M1) and 3-cis-hydroxycyclohexyl glyburide (M2b), were previously identified in plasma and urine of patients treated with glyburide and are pharmacologically active. The aim of this investigation was to identify the major human hepatic and placental CYP450 isozymes responsible for the formation of each metabolite of glyburide. This was achieved by the use of chemical inhibitors selective for individual CYP isozymes and antibodies raised against them. The identification was confirmed by the kinetic constants for the biotransformation of glyburide by cDNA-expressed enzymes. The data revealed that the major hepatic isozymes responsible for the formation of each metabolite are as follows: CYP3A4 (ethylene-hydroxylated glyburide (M5), 3-trans-(M3) and 2-trans-(M4) cyclohexyl glyburide); CYP2C9 (M1, M2a (4-cis-) and M2b); CYP2C8 (M1 and M2b); and CYP2C19 (M2a). Human placental microsomal CYP19/aromatase was the major isozyme responsible for the biotransformation of glyburide to predominantly M5. The formation of significant amounts of M5 by CYP19 in the placenta could render this metabolite more accessible to the fetal circulation. The multiplicity of enzymes biotransforming glyburide and the metabolites formed underscores the potential for its drug interactions in vivo.

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).

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.

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.

Recent data from our laboratory revealed the formation of an unknown metabolite of 17 hydroxyprogesterone caproate (17-HPC), used for treatment of preterm deliveries, during its perfusion across the dually perfused human placental lobule. Previously, we demonstrated that the drug is not hydrolyzed, neither in vivo nor in vitro, to progesterone and caproate. Therefore, the hypothesis for this investigation is that 17-HPC is actively metabolized by human and baboon (Papio cynocephalus) hepatic and placental microsomes. Baboon hepatic and placental microsomes were investigated to validate the nonhuman primate as an animal model for drug use during pregnancy. Data presented here indicate that human and baboon hepatic microsomes formed several mono-, di-, and tri-hydroxylated derivatives of 17-HPC. However, microsomes of human and baboon placentas metabolized 17-HPC to its mono-hydroxylated derivatives only in quantities that were a fraction of those formed by their respective livers, except for two metabolites (M16' and M17') that are unique for placenta and contributed to 25% and 75% of the total metabolites formed by human and baboon, respectively. The amounts of metabolites formed, relative to each other, by human and baboon microsomes were different suggesting that the affinity of 17-HPC to CYP enzymes and their activity could be species-dependent.

Objective: The aims of this investigation were (i) to determine the rate and extent of rosiglitazone transfer across term human placenta, and (ii) to determine the effect of human serum albumin (HSA) on rosiglitazone and glyburide transfer and distribution., Methods: These aims were achieved by utilizing the technique of dual perfusion of placental lobule (DPPL). Each hypoglycemic drug was coperfused with the marker compound antipyrine (AP). In each experiment, the [3H]-isotope of the hypoglycemic drug and the [14C]-isotope of AP were added to enhance the detection limits of each drug. Human serum albumin (HSA) was added to both the maternal and fetal circuits in the experiments in which it was investigated., Results: Transplacental transfer of rosiglitazone and glyburide from the maternal to fetal circuits in media devoid of HSA was similar. However, the addition of HSA to the maternal and fetal circuits had different effects on the transfer and distribution of the two drugs, though their binding to HSA (99.8%) was almost identical. HSA increased the maternal (M) to fetal (F) transfer of rosiglitazone, as revealed by an increase in its F/M concentration ratio from 0.17 +/- 0.01 (in the absence of albumin) to 0.33 +/- 0.07 (p < 0.001). Moreover, the addition of albumin decreased the amount of rosiglitazone retained by placental tissue from 539 +/- 148 to 60 +/- 8 ng/g (p < 0.001). Conversely, the addition of HSA to the perfusion media resulted in a decrease in glyburide transfer, as revealed by the change of its F/M concentration ratio from 0.09 +/- 0.02 (in the absence of albumin) to 0.03 +/- 0.01 (p < 0.01). However, similar to rosiglitazone, glyburide retention by the tissue decreased from 103 +/- 26 to 19 +/- 6 ng/g (p < 0.001)., Conclusions: These data indicate that the binding of the two drugs to albumin, though similar, is only one of the factors that could affect their placental transfer and distribution.

Objective: The aim of this investigation is to determine whether 17alpha-hydroxyprogesterone caproate is hydrolyzed, in vitro, to 17alpha-hydroxyprogesterone and caproate., Study Design: The in vitro hydrolysis of dual radioactively labeled 17alpha-hydroxy-[3H] progesterone [14C] caproate by human plasma, hepatic and placental S9 fractions as well as recombinant esterases was investigated. The formation of [3H]-17alpha-hydroxyprogesterone and [14C]-caproate were determined with the use of high-performance liquid chromatography equipped with an online radioactivity detector. The presence and activity of carboxylesterase and butyrylcholinesterase in the human-derived preparations was confirmed by the hydrolysis of their prototypic substrates p-nitrophenyl acetate, p-nitrophenyl butyrate, and butyrylthiocholine, respectively., Results: The aforementioned human-derived preparations hydrolyzed p-nitrophenyl acetate, p-nitrophenyl butyrate, and butyrylthiocholine. However, when 17alpha-hydroxyprogesterone caproate was incubated with the human-derived preparations under identical experimental conditions neither [3H]-17alpha-hydroxyprogesterone nor [14C]-caproate was detected., Conclusion: 17Alpha-hydroxyprogesterone caproate is not hydrolyzed in vitro by the esterase enzymes present in human plasma, liver, preterm, or term placenta.

Objective: The objective of the study was to determine the effect of gestational age and P-glycoprotein expression on transplacental transfer of methadone., Study Design: Dual perfusion of placental lobule was utilized. Methadone (200 ng/mL) and its [3H]-isotope were cotransfused from the maternal-to-fetal circuit with the marker compound antipyrine (20 microg/mL) and its [14C]-isotope. Concentration of the drugs in trophoblast tissue and both circuits was determined by liquid scintillation spectrometry., Results: Fetal transfer rate of methadone in preterm placentas was 19 +/- 5.8%, and in term placentas it was 31 +/- 9.7% (P < .01). Clearance index of methadone in preterm placentas (0.57 +/- 0.2) was lower than in term placentas (0.95 +/- 0.3, P < .01). P-glycoprotein expression was higher in preterm than term placentas., Conclusion: The ex vivo transfer of methadone across preterm placentas is 30% lower than in term placentas.

Glyburide (glibenclamide) is under investigation for treatment of gestational diabetes. Two metabolites of glyburide have been previously identified in patients, namely, 4-trans-(M1) and 3-cis-(M2) hydroxycyclohexyl glyburide. Recently, the metabolism of glyburide by microsomes of liver and placenta from humans and baboons revealed the formation of four additional metabolites: 4-cis-(M2a), 3-trans-(M3), and 2-trans-(M4) hydroxycyclohexyl glyburide, and ethyl-hydroxy glyburide (M5). The aim of this investigation was to determine the kinetics for the metabolism of glyburide by cytochrome P450 (CYP) isozymes of human and baboon placental and hepatic microsomes. The metabolism of glyburide by microsomes from the four organs revealed saturation kinetics and apparent K(m) values between 4 and 12 microM. However, the rates for formation of the metabolites varied between organs and species. M1 was the major metabolite (36% of total), formed by human hepatic microsomes with V(max) of 80+/-13 pmol mg protein(-1)min(-1), and together with M2, accounted for only 51% of the total. M5 was the major metabolite (87%) formed by human placental microsomes with V(max) of 11 pmol mg protein(-1)min(-1). In baboon liver, M5 had the highest rate of formation (V(max) 135+/-32 pmol mg protein(-1)min(-1), 39% of total), and in its placenta, was M4 (V(max) 0.7+/-0.1 pmol mg protein(-1)min(-1), 65%). The activity of human and baboon hepatic microsomes in metabolizing glyburide was similar, but the activity of human and baboon placental microsomes was 7% and 0.3% of their respective hepatic microsomes. The data obtained suggest that more than 1 CYP isozyme is responsible for catalyzing the hydroxylation of glyburide.

Aromatase, cytochrome P450 19, is a key enzyme in the biosynthesis of estrogens by the human placenta. It is also the major placental enzyme that metabolizes the opiates L-acetylmethadol (LAAM), methadone, and buprenorphine (BUP). Methadone and BUP are used in treatment of the opiate addict and are competitive inhibitors of testosterone conversion to estradiol (E(2)) and 16alpha-hydroxytestosterone (16-OHT) to estriol (E(3)) by aromatase. The aim of this investigation is to determine the effect of 20 opiates, which can be administered to pregnant patients for therapeutic indications or abused, on E(2) and E(3) formation by placental aromatase. Data obtained indicated that the opiates increased, inhibited, or had no effect on aromatase activity. Their effect on E(3) formation was more pronounced than that on E(2) due to the lower affinity of 16-OHT than testosterone to aromatase. The K(i) values for the opiates that inhibited E(3) formation were sufentanil, 7 +/- 1 microM; LAAM, 13 +/- 8 microM; fentanyl, 25 +/- 5 microM; oxycodone, 92 +/- 22 microM; codeine, 218 +/- 69 microM; (+)-pentazocine, 225 +/- 73 microM. The agonists morphine, heroin, hydromorphone, oxymorphone, hydrocodone, propoxyphene, meperidine, levorphanol, dextrorphan, and (-)-pentazocine and the antagonists naloxone and naltrexone caused an increase in E(3) formation by 124-160% of control but had no effect on E(2) formation. Moreover, oxycodone and codeine did not inhibit E(2) formation and the IC(50) values for fentanyl, sufentanil, and (+)-pentazocine were >1000 microM. It is unlikely that the acute administration of the opiates that inhibit estrogen formation would affect maternal and/or neonatal outcome. However, the effects of abusing any of them during the entire pregnancy are unclear at this time.

Glyburide (glibenclamide) is a second-generation sulfonylurea used for treatment of type-2 and gestational diabetes mellitus. To date, two glyburide metabolites have been identified in maternal urine: namely, 4-trans-hydroxycyclohexyl glyburide and 3-cis-hydroxycyclohexyl glyburide. The use of glyburide to treat gestational diabetes prompted us to investigate its metabolism by the placenta. The metabolism of glyburide by microsomal preparations from human and baboon placenta was compared with metabolism by their livers. The metabolites formed by the microsomes of the four tissues were identified by high-performance liquid chromatography-mass spectrometry using retention times, ion current (extracted at m/z 510), and selected-ion monitoring. The data obtained revealed the formation of six distinct hydroxylated derivatives of glyburide by each of the four microsomal preparations. However, the amounts of the six metabolites formed by the placentas were a fraction of that formed by the livers. Moreover, the relative quantities of each metabolite formed differed between species as well as between the two tissues. Also, the structure of the unidentified metabolites was determined by comparison with synthesized standards. These metabolites were identified as the 4-cis-hydroxycyclohexyl glyburide, 3-trans-hydroxycyclohexyl glyburide, and 2-trans-hydroxycyclohexyl glyburide. Therefore, one glyburide metabolite remains to be identified, but the data we obtained allowed us to suggest its structure.

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.

This study examines the role of placental P-glycoprotein (P-gp) in the transfer of buprenorphine (BUP) and L-alpha-acetylmethadol (LAAM) across the dually perfused human placental lobule. BUP (10 ng/mL) and LAAM (35 ng/mL) were perfused in the maternal-to-fetal direction. The following kinetic parameters were determined: fetal transfer rate (TR (f)), maternal clearance (Cl (m)), and clearance index (Cl (index)). The opiates were perfused in the presence of P-gp inhibitor GF120918 (experimental group) and in its absence (control group). The kinetic parameters for the control group were set at 100% and were as follows for LAAM in the experimental group: TR (f), 123 +/- 20%, Cl (m) 116 +/- 23%, and Cl (index) 123 +/- 22% ( P < 0.05). The corresponding parameters for BUP were not different from controls. The data indicate that LAAM, but not BUP, is extruded by the efflux transporter P-gp. Therefore, it is reasonable to assume that the activity of P-gp could be one of the factors affecting the extent of fetal exposure to LAAM during pregnancy.

Glyburide is a second-generation sulfonylurea hypoglycemic drug used for the treatment of select women with pregestational and gestational diabetes mellitus (GDM). In vitro and in vivo investigations demonstrated its very low transplacental transfer to the fetal circulation. However, the factors influencing its low transfer across the human placenta remain unclear. Therefore, the goal of the current investigation was to determine the effect of human serum albumin (HSA) on the transfer and distribution of glyburide across the human placenta. To achieve this goal, the technique of dual perfusion of the placental lobule was utilized. The effect of HSA on the transfer of glyburide was determined at the range of glyburide to HSA molar ratios of 1:2-1:100. The transfer rate of free/unbound glyburide to the fetal circuit was 73+/-10% of the freely diffusible marker compound antipyrine (AP). Data obtained indicates the dependence of glyburide transfer and its retention by the placental tissue on the concentration of HSA.

The objective of this study was to identify the enzyme that metabolizes methadone in preterm placentas. Microsomal fractions were obtained from preterm (17 to 34 weeks) placentas (36 total; 12 per each gestational age group) and their activity in metabolizing methadone to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was determined. The enzyme catalyzing the reaction was identified by using chemical inhibitors selective for various cytochrome P450 isozymes and monoclonal antibodies raised against them. The metabolism of methadone by microsomes revealed saturation kinetics. Methadone was N-demethylated to EDDP by aromatase. The affinity of methadone to aromatase (apparent Km) did not change with gestation, but the activity of the enzyme (Vmax) increased and varied widely between individual placentas. Aromatase/CYP19 is the placental enzyme metabolizing methadone during pregnancy. The variability in enzyme activity among individuals should be reflected by the concentration of methadone in the fetal circulation and might be one of the factors affecting the incidence and intensity of neonatal abstinence syndrome.

Methadone and buprenorphine (BUP) are used for treatment of the pregnant opiate addict. CYP19/aromatase is the major placental enzyme responsible for the metabolism of methadone to 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and BUP to norbuprenorphine (norBUP). The aim of this investigation was to determine the effects of methadone and BUP on the activity of placental microsomal aromatase in the conversion of its endogenous substrates testosterone to 17beta-estradiol (E(2)) and 16alpha-hydroxytestosterone (16-OHT) to estriol (E(3)). The conversion of testosterone and 16-OHT by human placental microsomes exhibited saturation kinetics, and the apparent K(m) values were 0.2 +/- 1 and 6 +/- 3 microM, respectively. V(max) values for E(2) and E(3) formation were 70 +/- 16 and 28 +/- 10 pmol/mg proteinmin, respectively. Also, data obtained revealed that methadone and BUP are competitive inhibitors of testosterone conversion to E(2) and 16-OHT to E(3). The K(i) for methadone inhibition of E(2) and E(3) formation were 393 +/-144 and 53 +/- 28 microM, respectively, and for BUP the K(i) was 36 +/- 9 and 6 +/- 1 microM. The higher potency of the two opiates and their metabolites in inhibiting E(3) formation is in agreement with the lower affinity of 16-OHT than testosterone to aromatase. Moreover, the metabolites EDDP and norBUP were weaker inhibitors of aromatase than their parent compounds. The determined inhibition constants of methadone and BUP for E(3) formation by a cDNA-expressed CYP19 preparation were similar to those for placental microsomes. Therefore, data reported here suggest that methadone, BUP, and their metabolites are inhibitors of androgen aromatization in the placental biosynthesis of estrogens.

The adverse effects of smoking during pregnancy on fetal development are, in part, due to nicotine. These effects may be due to the actions of nicotine in fetal circulation or on placental functions. In pregnant rats, vaccination with a nicotine immunogen reduces the transfer of nicotine from the maternal to fetal circulation. However, extrapolation of these results to pregnant women might not be valid due to the well-recognized differences between human and rat placentas. In the current investigation, the effects of nicotine-specific antibodies on the transfer of nicotine from the maternal to fetal circuit of the dually perfused human placental lobule were determined. Two types of nicotine-specific antibodies were investigated; nicotine-specific mouse monoclonal antibody (Nic311, K(d) for nicotine 60nM) and IgG from rabbits vaccinated with a nicotine immunogen (Nic-IgG, K(d) 1.6nM). Transfer of the antibodies from maternal to fetal circuits was negligible. Both rabbit Nic-IgG and, to a lesser extent, mouse monoclonal Nic311 significantly reduced nicotine transfer from the maternal to fetal circuit as well as the retention of the drug by placental tissue. These effects were mediated by a substantial increase in the protein binding of nicotine and a reduction in the unbound nicotine concentration. Therefore, the data cited in this report suggest that the use of nicotine-specific antibodies might reduce fetal exposure to the drug, and that antibody affinity for nicotine is a key determinant of the extent of nicotine transfer.

Methadone maintenance programs are considered the standard of care for the pregnant opiate addict. However, data on changes in methadone pharmacokinetics (PK) during pregnancy are limited and do not include its disposition by the placenta due to obvious ethical and safety considerations. Accordingly, investigations in our laboratory are focusing on human placental disposition of opiates including methadone. Recently, we reported on methadone metabolism by placental aromatase and provide here data on its bidirectional transfer across the tissue utilizing the technique of dual perfusion of placental lobule. The concentrations of the opiate transfused into the term placental tissue were those reported for its in vivo levels in the maternal serum of women under treatment with the drug. Data obtained indicated that the opiate has no adverse effects on placental viability and functional parameters and that it is retained by the tissue. Also, methadone transfer and its clearance index in the fetal to maternal direction (0.97+/-0.05) was significantly higher (P<0.05) than in the maternal to fetal (0.83+/-0.09). The observed asymmetry in methadone transfer could be explained by the unidirectional activity of the efflux transporter P glycoprotein (P-gp) that is highly expressed in variable amounts in trophoblast tissue. Therefore, placental disposition of methadone might be an important contributor to the regulation of its concentration in the fetal circulation and consequently may affect the incidence and intensity of neonatal abstinence syndrome for women treated with the drug during pregnancy.

Methadone pharmacotherapy is considered the standard for treatment of the pregnant heroin/opioid addict. One of the factors affecting the transfer kinetics of opioids across human placenta and their levels in the fetal circulation is their metabolism by the tissue. The aim of this investigation is to identify the enzyme(s) responsible for the metabolism of methadone, determine the kinetics of the reaction and the metabolites formed utilizing placental tissue obtained from term healthy pregnancies. Microsomal fractions of trophoblast tissue homogenates had the highest activity in catalyzing the metabolism of methadone. The product formed was identified by HPLC-UV as 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). Inhibitors selective for cytochrome P450 (CYP) isozymes were used to identify the enzyme catalyzing the biotransformation of methadone. Aminoglutethimide and 4-hydroxyandrostenedione inhibited EDDP formation by 88 and 70%, respectively, suggesting that CYP19/aromatase is the enzyme catalyzing the reaction. This was confirmed by the effect of monoclonal antibodies raised against CYP19 that caused an 80% inhibition of the reaction. The apparent K(m) and V(max) values for the CYP19 catalyzed metabolism of methadone to EDDP were 424 +/- 92 microM and 420 +/- 89 pmol(mgprotein)(-1)min(-1), respectively. Kinetic analysis of a cDNA-expressed CYP19 for the metabolism of methadone to EDDP was identical to that by placental microsomes. Taken together, these data indicate that CYP19/aromatase is the major enzyme responsible for the metabolism of methadone to EDDP in term human placentas obtained from healthy pregnancies.

Levo-alpa-acetylmethadol (LAAM) is a methadone derivative used to treat the opiate addict. We previously reported on the kinetics for transplacental transfer of LAAM and its levels in the fetal circuit using the technique of dual perfusion of the placental lobule. The aim of this investigation was to identify the enzyme responsible for the biotransformation of LAAM and norLAAM and the metabolites formed in the term human placenta. Placental microsomes exhibited higher activities than the mitochondrial and cytosolic fractions in metabolizing LAAM to norLAAM. None of these subcellular fractions catalyzed the formation of dinorLAAM from either LAAM or norLAAM as determined by HPLC/UV. Evidence obtained from the effects of cytochrome P450 (CYP) inhibitors on the demethylation of LAAM to norLAAM by placental microsomes suggested that CYP 19/aromatase is the major enzyme involved. Out of 10 monoclonal antibodies raised against various CYP isoforms, only that for aromatase caused over 80% inhibition of norLAAM formation. The biotransformation of LAAM to norLAAM exhibited monophasic kinetics with apparent Km and Vmax values of 105 +/- 57 microM and 86.8 +/- 15.6 pmol mg(-1) protein min(-1), respectively. The kinetic profile determined for a cDNA-expressed CYP 19 metabolism of LAAM to norLAAM was similar to that determined for placental microsomes. Taken together, the above data indicate that CYP 19/aromatase is the enzyme responsible for the N-demethylation of LAAM to norLAAM in term human placentas obtained from healthy pregnant women.

Buprenorphine (BUP) is a partial opiate agonist used for treatment of the adult and the pregnant addicted to this class of narcotics. The kinetic parameters for transplacental transfer and the metabolism of BUP during its perfusion in a placental lobule were the subject of an earlier report from our laboratory. The aim of this investigation is to identify and characterize the enzyme catalyzing the metabolism of BUP in term human placenta. Norbuprenorphine (norBUP) is the only metabolite formed as determined by high performance liquid chromatography and mass spectrometry. The activity of the enzyme responsible for BUP metabolism is highest in the microsomal fraction and lowest in the cytosolic, with the mitochondrial in between. Compounds with selective affinity to the enzyme aromatase (CYP 19), namely 4-hydroxyandrostenedione and aminoglutethimide, caused >70% inhibition of norBUP formation. Monoclonal antibodies raised against CYP 19 were the most potent inhibitors of BUP dealkylation. A comparison between the data obtained from the saturation isotherm for BUP dealkylation by placental microsomes and a commercially available system of cDNA-expressed CYP 19 indicated similar kinetic parameters, with apparent Km values of 12 +/- 4.0 and 14 +/- 8.0 microM, respectively. Therefore, aromatase is the major enzyme catalyzing the biotransformation of BUP to norBUP in term human placentas obtained from healthy pregnancies. The minor involvement of other cytochrome P450 isoforms or enzyme(s) in the metabolism of BUP in placental tissue cannot be ruled out.

The agonists buprenorphine and l-alpha-acetylmethadol (LAAM) were introduced as alternatives to methadone for treatment of the adult opiate addict. The direct and indirect effects of these drugs on normal fetal growth and development are currently under investigation in our laboratory. The goal of this report is to provide part of the data necessary to assess the safety of LAAM in treatment of the pregnant opiate addict. To achieve this goal, the technique of dual perfusion of placental lobule was utilized to determine the kinetics for transplacental transfer of LAAM and its effects on the viability and functional parameters of the tissue. LAAM is rapidly metabolized to the pharmacologically active norLAAM that was also included in this investigation. The two opiates were transfused at their plasma levels in patients under treatment, a concentration of 35 ng/ml. The drugs exhibited similar pharmacokinetic profiles, characterized by an initial phase of distribution into placental tissue followed by their low transfer to the fetal circuit. During the 4-h experimental period, the transfused tissue retained significant amounts of LAAM and norLAAM, and neither drug was metabolized. LAAM did not affect placental tissue viability and functional parameters. However, norLAAM caused a significant decrease in the release of human chorionic gonadotropin. At this time, it is unclear whether a similar effect for norLAAM may occur in vivo and, if so, what the consequences would be on its role in implantation and normal fetal growth and development.