Placental Efflux Research Articles

Quantification of sertraline maternal/fetal ratio and amniotic fluid concentration using a pregnancy physiologically based pharmacokinetic model.

Selective serotonin reuptake inhibitors (SSRIs) are indicated for a variety of psychiatric conditions which may require treatment during pregnancy. Knowledge of appropriate SSRI dosages that maintain maternal therapeutic benefit and minimize fetal risk are needed. Assessment of fetal exposure to drugs is challenging since sampling is often limited to a single concentration from the umbilical cord at delivery. Physiologically based pharmacokinetic (PBPK) modelling provides a non-invasive approach to quantify exposure in pregnancy. We incorporated sertraline clearances mediated by passive diffusion, placental efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) into our previously published pregnancy PBPK model for sertraline. Simulations were performed for various sertraline doses (25-200 mg) at 40 weeks gestational age to predict the minimum (Cmin ), maximum (Cmax ) and average (Cavg ) sertraline maternal and fetal plasma concentrations and evaluated them against observed maternal and cord concentrations obtained at delivery from five clinical studies. The accuracy of the PBPK predictions as indicated by the average fold error (AFE) value for Cmax , Cmin and Cavg for maternal plasma sertraline concentrations at delivery was 1.7, 1.2 and 1.4, respectively. The AFE for the Cmax , Cmin and Cavg for cord blood sertraline concentration at delivery was 1.2, 1 and 1.1, respectively. The AFE for cord-maternal sertraline concentration ratio at delivery for Cmax , Cmin and Cavg was 0.7, 0.9 and 0.8, respectively. The PBPK model we developed may serve as a guide for maternal sertraline dose adjustment during pregnancy considering changes in exposures for both mother and fetus.

Protective role of the placental efflux transporter BCRP/ABCG2 in the relationship between prenatal cadmium exposure, placenta weight, and size at birth

Background and aimPlacental efflux transporter proteins, such as BCRP, reduce the placental and fetal toxicity of environmental contaminants but have received little attention in perinatal environmental epidemiology. Here, we evaluate the potential protective role of BCRP following prenatal exposure to cadmium, a metal that preferentially accumulates in the placenta and adversely impacts fetal growth. We hypothesized that individuals with a reduced function polymorphism in ABCG2, the gene encoding BCRP, would be most vulnerable to the adverse impacts of prenatal cadmium exposure, notably, smaller placental and fetal size. MethodsWe measured cadmium in maternal urine samples at each trimester and in term placentas from UPSIDE-ECHO study participants (NY, USA; n = 269). We fit adjusted multivariable linear regression and generalized estimating equation models to examine log-transformed urinary and placental cadmium concentrations in relation to birthweight, birth length, placental weight, and fetoplacental weight ratio (FPR) and stratified models by ABCG2 Q141K (C421A) genotype. ResultsOverall 17% of participants expressed the reduced-function ABCG2 C421A variant (AA or AC). Placental cadmium concentrations were inversely associated with placental weight (β = −19.55; 95%CI: −37.06, −2.04) and trended towards higher FPR (β = 0.25; 95%CI: −0.01, 0.52) with stronger associations in 421A variant infants. Notably, higher placental cadmium concentrations in 421A variant infants were associated with reduced placental weight (β = −49.42; 95%CI: 98.87, 0.03), and higher FPR (β = 0.85, 95%CI: 0.18, 1.52), while higher urinary cadmium concentration was associated with longer birth length (β = 0.98; 95%CI: 0.37, 1.59), lower ponderal index (β = −0.09; 95%CI: 0.15, −0.03), and higher FPR (β = 0.42; 95%CI: 0.14, 0.71). ConclusionsInfants with reduced function ABCG2 polymorphisms may be particularly vulnerable to the developmental toxicity of cadmium as well as other xenobiotics that are BCRP substrates. Additional work examining the influence of placental transporters in environmental epidemiology cohorts is warranted.

Mid-pregnancy poly(I:C) viral mimic disrupts placental ABC transporter expression and leads to long-term offspring motor and cognitive dysfunction

Limited information is available about the effect of mid-pregnancy viral infections on the placental expression of efflux transporters and offspring behavior. We hypothesized that maternal exposure to polyinosinic-polycytidylic acid [poly(I:C)], a synthetic double-stranded RNA viral mimic, would impair placental cell turnover, the expression of selected ABC transporters and adult offspring behavior. C57BL/6 mice were administered poly(I:C) (10 mg/Kg;ip) or vehicle at gestational day (GD) 13.5 (mid-pregnancy). Dams were euthanized for blood collection 4 h after injection, fetal and placental collection at GD18.5 or allowed to deliver spontaneously at term. At GD 13.5, poly(I:C) induced an acute pro-inflammatory response characterized by an increase in maternal plasma levels of IL-6, CXCL-1 and CCL-2/MCP-1. At GD 18.5, poly(I:C) decreased cell proliferation/death in the labyrinthine and increased cell death in the junctional zones, characterizing a disruption of placental cell turnover. Abca1 and Abcg1 immunolabelling was decreased in the labyrinthine zone, whereas Abca1, Abcg1 and breast cancer resistance transporter (Bcrp) expression increased in the junctional zone. Moreover, adult offspring showed motor and cognitive impairments in the Rotarod and T-water maze tests. These results indicate that viral infection during mid-pregnancy may disrupt relevant placental efflux transporters, as well as placental cell turnover and offspring behavior in adult life.

A High-Throughput Toxicity Screen of 42 Per- and Polyfluoroalkyl Substances (PFAS) and Functional Assessment of Migration and Gene Expression in Human Placental Trophoblast Cells.

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants that have been associated with adverse pregnancy outcomes in women and experimental research models. Adverse developmental and reproductive outcomes have been investigated for relatively few PFAS, and such studies are not scalable to address the thousands of unique chemical structures. As the placenta has been reported as a PFAS target tissue, the human placental trophoblast JEG-3 cell line was employed in a high-throughput toxicity screen (HTTS) to evaluate the effects of 42 unique PFAS on viability, proliferation, and mitochondrial membrane potential (MMP). HTTS concentration-response curve fitting determined EC50 values for 79% of tested compounds for at least one of the three endpoints. Trophoblast migratory potential was evaluated for a subset of six prioritized PFAS using a scratch wound assay. Migration, measured as the percent of wound closure after 72 h, was most severely inhibited by exposure to 100 µM perfluorooctanoic acid (PFOA; 72% closure), perfluorooctanesulfonic acid (PFOS; 57% closure), or ammonium perfluoro-2-methyl-3-oxahexanoate (GenX; 79% closure). PFOA and GenX were subsequently evaluated for disrupted expression of 46 genes reported to be vital to trophoblast health. Disrupted regulation of oxidative stress was suggested by altered expression of GPEX1 (300 µM GenX and 3 µM GenX), GPER1 (300 µM GenX), and SOD1 and altered cellular response to xenobiotic stress was indicated by upregulation of the placental efflux transporter, ABCG2 (300 µM GenX, 3 µM GenX, and 100 µM PFOA). These findings suggest the placenta is potentially a direct target of PFAS exposure and indicate that trophoblast cell gene expression and function are disrupted at PFAS levels well below the calculated cytotoxicity threshold (EC50). Future work is needed to determine the mechanism(s) of action of PFAS towards placental trophoblasts.

Estimation of Fetal-to-Maternal Unbound Steady-State Plasma Concentration Ratio of P-Glycoprotein and/or Breast Cancer Resistance Protein Substrate Drugs Using a Maternal-Fetal Physiologically Based Pharmacokinetic Model.

Pregnant women are frequently prescribed drugs to treat chronic diseases such as human immunodeficiency virus infection, but little is known about the benefits and risks of these drugs to the fetus that are driven by fetal drug exposure. The latter can be estimated by fetal-to-maternal unbound plasma concentration at steady state (Kp,uu,fetal). For drugs that are substrates of placental efflux transporters [i.e., P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP)], Kp,uu,fetal is expected to be <1. Here, we estimated the in vivo Kp,uu,fetal of selective P-gp and BCRP substrate drugs by maternal-fetal physiologically based pharmacokinetic (m-f-PBPK) modeling of umbilical vein (UV) plasma and maternal plasma (MP) concentrations obtained simultaneously at term from multiple maternal-fetal dyads. To do so, three drugs were selected: nelfinavir (P-gp substrate), efavirenz (BCRP substrate), and imatinib (P-gp/BCRP substrate). An m-f-PBPK model for each drug was developed and validated for the nonpregnant population and pregnant women using the Simcyp simulator (v20). Then, after incorporating placental passive diffusion clearance, the in vivo Kp,uu,fetal of the drug was estimated by adjusting the placental efflux clearance until the predicted UV/MP values best matched the observed data (Kp,uu,fetal) of nelfinavir = 0.41, efavirenz = 0.39, and imatinib = 0.35. Furthermore, Kp,uu,fetal of nelfinavir and efavirenz at gestational weeks (GWs) 25 and 15 were predicted to be 0.34 and 0.23 (GW25) and 0.33 and 0.27 (GW15). These Kp,uu,fetal values can be used to adjust dosing regimens of these drugs to optimize maternal-fetal drug therapy throughout pregnancy, to assess fetal benefits and risks of these dosing regimens, and to determine if these estimated in vivo Kp,uu,fetal values can be predicted from in vitro studies.SIGNIFICANCE STATEMENTThe in vivo fetal-to-maternal unbound steady-state plasma concentration ratio (Kp,uu,fetal) of nelfinavir [P-glycoprotein (P-gp) substrate], efavirenz [breast cancer resistance protein (BCRP) substrate], and imatinib (P-gp and BCRP substrate) was successfully estimated using maternal-fetal physiologically based pharmacokinetic (m-f-PBPK) modeling. These Kp,uu,fetal values can be used to adjust dosing regimens of these drugs to optimize maternal-fetal drug therapy throughout pregnancy, to assess fetal benefits and risks of these dosing regimens, and to determine if these estimated in vivo Kp,uu,fetal values can be predicted from in vitro studies.

Regulation of Placental Efflux Transporters during Pregnancy Complications.

The placenta is essential for regulating the exchange of solutes between the maternal and fetal circulations. As a result, the placenta offers support and protection to the developing fetus by delivering crucial nutrients and removing waste and xenobiotics. ATP-binding cassette transporters, including multidrug resistance protein 1, multidrug resistance-associated proteins, and breast cancer resistance protein, remove chemicals through active efflux and are considered the primary transporters within the placental barrier. Altered transporter expression at the barrier could result in fetal exposure to chemicals and/or accumulation of xenobiotics within trophoblasts. Emerging data demonstrate that expression of these transporters is changed in women with pregnancy complications, suggesting potentially compromised integrity of placental barrier function. The purpose of this review is to summarize the regulation of placental efflux transporters during medical complications of pregnancy, including 1) placental inflammation/infection and chorioamnionitis, 2) hypertensive disorders of pregnancy, 3) metabolic disorders including gestational diabetes and obesity, and 4) fetal growth restriction/altered fetal size for gestational age. For each disorder, we review the basic pathophysiology and consider impacts on the expression and function of placental efflux transporters. Mechanisms of transporter dysregulation and implications for fetal drug and toxicant exposure are discussed. Understanding how transporters are up- or downregulated during pathology is important in assessing possible exposures of the fetus to potentially harmful chemicals in the environment as well as the disposition of novel therapeutics intended to treat placental and fetal diseases. SIGNIFICANCE STATEMENT: Diseases of pregnancy are associated with reduced expression of placental barrier transporters that may impact fetal pharmacotherapy and exposure to dietary and environmental toxicants.

Preterm Birth Associates With Increased Placental Expression of MDR Transporters Irrespective of Prepregnancy BMI.

Preterm birth (PTB) and suboptimal prepregnancy body mass index (BMI) operate through inflammatory pathways to impair fetoplacental development. Placental efflux transporters mediate fetal protection and nutrition; however, few studies consider the effect of both PTB and BMI on fetal protection. We hypothesized that PTB would alter the expression of placental multidrug resistance (MDR) transporters and selected proinflammatory cytokines, and that maternal underweight and obesity would further impair placental phenotype. To determine whether placental MDR transporters P-glycoprotein (P-gp, encoded by ABCB1) and breast cancer resistance protein (BCRP/ABCG2), and proinflammatory cytokine levels are altered by PTB and maternal BMI. A cross-sectional study was conducted to assess the effect of PTB (with/without chorioamnionitis), or the effect of maternal prepregnancy BMI on placental MDR transporter and interleukin (IL)-6 and -8 expression in 60 preterm and 36 term pregnancies. ABCB1 expression was increased in preterm compared to term placentae (P = .04). P-gp (P = .008) and BCRP (P = .01) immunolabeling was increased among all preterm compared to term placentae, with P-gp expression further increased in preterm pregnancies with chorioamnionitis (PTC, P = .007). Placental IL-6 mRNA expression was decreased in PTC compared to term placentae (P = .0005) and PTC associated with the greatest proportion of anti-inflammatory medications administered during pregnancy. Maternal BMI group did not influence placental outcomes. PTB and infection, but not prepregnancy BMI, alter placental expression of MDR transporters and IL-6. This may have implications for fetal exposure to xenobiotics that may be present in the maternal circulation in pregnancies complicated by PTB.

Effect of Selected Antidepressants on Placental Homeostasis of Serotonin: Maternal and Fetal Perspectives.

Depression is a prevalent condition affecting up to 20% of pregnant women. Hence, more than 10% are prescribed antidepressant drugs, mainly serotonin reuptake inhibitors (SSRIs) and selective serotonin and noradrenaline reuptake inhibitors (SNRIs). We hypothesize that antidepressants disturb serotonin homeostasis in the fetoplacental unit by inhibiting serotonin transporter (SERT) and organic cation transporter 3 (OCT3) in the maternal- and fetal-facing placental membranes, respectively. Paroxetine, citalopram, fluoxetine, fluvoxamine, sertraline, and venlafaxine were tested in situ (rat term placenta perfusion) and ex vivo (uptake studies in membrane vesicles isolated from healthy human term placenta). All tested antidepressants significantly inhibited SERT- and OCT3-mediated serotonin uptake in a dose-dependent manner. Calculated half-maximal inhibitory concentrations (IC50) were in the range of therapeutic plasma concentrations. Using in vitro and in situ models, we further showed that the placental efflux transporters did not compromise mother-to-fetus transport of antidepressants. Collectively, we suggest that antidepressants have the potential to affect serotonin levels in the placenta or fetus when administered at therapeutic doses. Interestingly, the effect of antidepressants on serotonin homeostasis in rat placenta was sex dependent. As accurate fetal programming requires optimal serotonin levels in the fetoplacental unit throughout gestation, inhibition of SERT-/OCT3-mediated serotonin uptake may help explain the poor outcomes of antidepressant use in pregnancy.

Development and Verification of a Linked Δ 9-THC/11-OH-THC Physiologically Based Pharmacokinetic Model in Healthy, Nonpregnant Population and Extrapolation to Pregnant Women.

Conducting clinical trials to understand the exposure risk/benefit relationship of cannabis use is not always feasible. Alternatively, physiologically based pharmacokinetic (PBPK) models can be used to predict exposure of the psychoactive cannabinoid (-)-Δ9-tetrahydrocannabinol (THC) and its active metabolite 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC). Here, we first extrapolated in vitro mechanistic pharmacokinetic information previously quantified to build a linked THC/11-OH-THC PBPK model and verified the model with observed data after intravenous and inhalation administration of THC in a healthy, nonpregnant population. The in vitro to in vivo extrapolation of both THC and 11-OH-THC disposition was successful. The inhalation bioavailability (Finh) of THC after inhalation was higher in chronic versus casual cannabis users (Finh = 0.35 and 0.19, respectively). Sensitivity analysis demonstrated that 11-OH-THC but not THC exposure was sensitive to alterations in hepatic intrinsic clearance of the respective compound. Next, we extrapolated the linked THC/11-OH-THC PBPK model to pregnant women. Simulations showed that THC plasma area under the curve (AUC) does not change during pregnancy, but 11-OH-THC plasma AUC decreases by up to 41%. Using a maternal-fetal PBPK model, maternal and fetal THC serum concentrations were simulated and compared with the observed THC serum concentrations in pregnant women at term. To recapitulate the observed THC fetal serum concentrations, active placental efflux of THC needed to be invoked. In conclusion, we built and verified a linked THC/11-OH-THC PBPK model in healthy nonpregnant population and demonstrated how this mechanistic physiologic and pharmacokinetic platform can be extrapolated to a special population, such as pregnant women. SIGNIFICANCE STATEMENT: Although the pharmacokinetics of cannabinoids have been extensively studied clinically, limited mechanistic pharmacokinetic models exist. Here, we developed and verified a physiologically based pharmacokinetic (PBPK) model for (-)-Δ9-tetrahydrocannabinol (THC) and its active metabolite, 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC). The PBPK model was verified in healthy, nonpregnant population after intravenous and inhalation administration of THC, and then extrapolated to pregnant women. The THC/11-OH-THC PBPK model can be used to predict exposure in special populations, predict drug-drug interactions, or impact of genetic polymorphism.

Pregnancy Has No Clinically Significant Effect on the Pharmacokinetics of Bupropion or Its Metabolites.

Bupropion (BUP) is a chiral antidepressant and smoking cessation aide with benefits and side effects correlated with parent and active metabolite concentrations. BUP is metabolized by CYP2B6, CYP2C19, and CYP3A4 to hydroxy-BUP (OH-BUP) as well as by 11β-hydroxysteroid dehydrogenase-1 and aldo-keto reductases to threohydrobupropion (Threo) and erythrohydrobupropion (Erythro), respectively. As pregnancy alters the activity of drug-metabolizing enzymes, the authors hypothesized that BUP metabolism and BUP metabolite concentrations would be altered during pregnancy, potentially affecting the efficacy and safety of BUP in pregnant women. Pregnant women (n = 8) taking BUP chronically were enrolled, and steady-state plasma samples and dosing interval urine samples were collected during pregnancy and postpartum. Maternal and umbilical cord venous blood samples were collected at delivery from 3 subjects, and cord blood/maternal plasma concentration ratios were calculated. The concentrations of BUP stereoisomers and their metabolites were measured. Paired t tests were used to compare pharmacokinetic parameters during pregnancy and postpartum. No significant changes were observed in the steady-state plasma concentrations, metabolite to parent ratios, formation clearances, or renal clearance of any of the compounds during pregnancy when compared with postpartum. The umbilical cord venous plasma concentrations of BUP and its metabolites were 30%-60% lower than maternal plasma concentrations. This study showed that there are no clinically meaningful differences in the stereoselective disposition of BUP or its metabolites during pregnancy, indicating that dose adjustment during pregnancy may not be necessary. The results also showed that the placenta provides a partial barrier for bupropion and its metabolite distribution to the fetus, with possible placental efflux transport of bupropion and its metabolites.

697 Exploring sex differences in syncytiotrophoblast aromatase and placental efflux transporter expression in opioid exposed pregnancies

Male sex is associated with the development of severe neonatal opioid withdrawal syndrome (NOWS) in opioid exposed pregnancies. Placental efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), are critical for transfer of drugs from the placenta to maternal circulation. Aromatase is responsible for metabolizing Methadone and Buprenorphine in the human placenta. We sought to determine whether sex differences exist in placental aromatase and efflux transporter density within the apical syncytiotrophoblast. This was a retrospective cohort study from 2014-2019 at a single tertiary site. Pregnant women ≥ age 18 with non-anomalous fetuses on maintenance Methadone or Buprenorphine with gestational age ≥33 weeks were included. De-paraffinized placental sections, including the apical syncytiotrophoblast membrane, were labeled with monoclonal antibodies for aromatase, P-gp and BCRP. Placentas were scored for presence and intensity of staining using the Allred scoring schema. Severe NOWS requiring opioid treatment was defined by 3 consecutive Finnegan scores ≥8 or the sum of 3 consecutive Finnegan scores ≥24 within 72 hours of birth. Data was analyzed using descriptive, parametric and non-parametric statistics. A p-value of <0.05 was considered significant. There were 110 opioid exposed neonates included in this analysis with a mean gestational age of 37 weeks. Of the 46% (51/110) of infants with severe NOWS, 69% (35/51) were Methadone exposed and 31% (16/51) were Buprenorphine exposed (p =0.01). There were significantly more males in the group with severe NOWS (non-severe 24 (41%) vs severe 34 (67%), p<0.01). There were no differences in median placental Allred scores for aromatase, P-gp or BCRP between groups (p>0.05). Aromatase and placental efflux transporters had weak, but significant correlation (rho=0.22). P-gp and BCRP exhibited strong correlation (rho=0.59). Male infants within our cohort were significantly more likely to develop severe NOWS. We found no sex differences in aromatase, P-gp and BCRP within apical syncytiotrophoblasts.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

696 Severe neonatal opioid withdrawal syndrome is associated with reduced aromatase in the apical syncytiotrophoblast

Placental cytochrome p450 (CYP) enzymes and efflux transporters, P-glycoprotein (p-gp) and breast cancer resistance protein (BCRP), are critical for transfer of drugs from the placenta to maternal circulation. CYP19A1 (aromatase) is the enzyme responsible for metabolizing Methadone and Buprenorphine in the human placenta. We sought to determine how CYP and efflux transporter density within the syncytiotrophoblast affects neonatal outcomes in opioid exposed pregnancies. This was a retrospective cohort study from 2014-2019 at a single tertiary care center. Pregnant women ≥ age 18 with non-anomalous fetuses on maintenance Methadone or Buprenorphine with gestational age ≥33 weeks were included. De-paraffinized placental sections, inclusive of the apical syncytiotrophoblast membrane, were labeled with monoclonal antibodies for aromatase, P-gp and BCRP. Placentas were scored for the presence and intensity of staining using the Allred scoring schema. Severe neonatal opioid withdrawal syndrome (NOWS) requiring opioid treatment was defined by 3 consecutive Finnegan scores ≥8 or the sum of 3 consecutive Finnegan scores ≥24 within 72 hours of birth. Data was analyzed using descriptive, parametric and non-parametric statistics. A p-value of <0.05 was considered significant. There were 110 opioid exposed neonates included in this analysis with a mean gestational age of 37 weeks. Of the 46% (51/110) of infants with severe NOWS, 69% (35/51) were Methadone exposed and 31% (16/51) were Buprenorphine exposed (p =0.01). The median placental Allred scores for aromatase were significantly different in the group with severe NOWS (p=.03). There were no differences in P-gp and BCRP scores between groups. Aromatase and the placental efflux transporters had weak, but significant correlation (rho=0.22). P-gp and BCRP exhibited strong correlation (rho=0.59). Infants with severe NOWS had significantly lower placental aromatase in the apical syncytiotrophoblast compared with those without severe NOWS. Further investigation into the role of aromatase and fetal opioid exposure is needed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

703 Impact of opioid exposure on apical syncytiotrophoblast aromatase and placental efflux transporters in term pregnancies

Placental cytochrome p450 (CYP) enzymes and efflux transporters, P-glycoprotein (p-gp) and breast cancer resistance protein (BCRP), are critical for transfer of drugs from the placenta to maternal circulation. CYP19A1 (aromatase) is the enzyme responsible for metabolizing Methadone and Buprenorphine in the human placenta. We set out to assess placental aromatase, P-gp and BCRP density in placental syncytiotrophoblasts of opioid exposed and unexposed term pregnancies. This was a retrospective case-control study from 2014-2019 at a single tertiary care center. Cases included pregnant women ≥ age 18 with non-anomalous fetuses on maintenance Methadone or Buprenorphine with gestational age ≥37 weeks. Controls included pregnant women with no medication exposure and no comorbid conditions delivering with gestational age ≥37 weeks. De-paraffinized placental sections, inclusive of the apical syncytiotrophoblast membrane, were labeled with monoclonal antibodies for aromatase, P-gp and BCRP. Placentas were scored for the presence and intensity of staining using the Allred scoring schema. Data was analyzed using descriptive and parametric statistics. A p-value of <0.05 was considered significant. There were 69 opioid exposed and 10 unexposed pregnancies included in this analysis. Maternal and neonatal characteristics of the opioid exposed cases and unexposed controls are noted in Table 1. The median placental Allred scores for aromatase were significantly lower in the opioid exposed group compared with the unexposed controls (exposed 6.8± 1.4 vs unexposed 7.5± 0.7, p=.03). There were no differences in P-gp and BCRP scores between groups. Aromatase and the placental efflux transporters had weak, but significant correlation (rho=0.22). P-gp and BCRP exhibited strong correlation (rho=0.59). Apical syncytiotrophoblast aromatase density was reduced in opioid exposed cases compared to unexposed controls. There were no observed differences in P-gp or BCRP between groups. Further exploration into the role of aromatase in fetal opioid exposure is warranted.

Maternal-fetal disposition of domoic acid following repeated oral dosing during pregnancy in nonhuman primate

Domoic acid (DA) is a marine algal toxin that causes acute and chronic neurotoxicity in animals and humans. Prenatal exposure to DA has been associated with neuronal damage and cognitive and behavioral deficits in juvenile California sea lions, cynomolgus monkeys and rodents. Yet, the toxicokinetics (TK) of DA during pregnancy and the maternal-fetal disposition of DA have not been fully elucidated. In this study, we investigated the TK before, during, and after pregnancy and the maternal-fetal disposition of DA in 22 cynomolgus monkeys following daily oral doses of 0.075 or 0.15mg/kg/day of DA. The AUC0-τ of DA was not changed while the renal clearance of DA was increased by 30-90% during and after pregnancy when compared to the pre-pregnancy values. DA was detected in the infant plasma and in the amniotic fluid at delivery. The infant plasma concentrations correlated positively with both the maternal plasma and the amniotic fluid concentrations. The paired infant-to-maternal plasma DA concentration ratios ranged from 0.3 to 0.6 and increased as a function of time which suggests placental efflux and longer apparent fetal half-life than the maternal half-life. The paired amniotic fluid-to-infant plasma DA concentration ratios ranged from 4.5 to 7.5 which indicates significant accumulation of DA in the amniotic fluid. A maternal-fetal TK model was developed to explore the processes that give the observed maternal-fetal disposition of DA. The final model suggests that placental transport and recirculation of DA between the fetus and amniotic fluid are major determining factors of the maternal-fetal TK of DA.

Quantitative Prediction of the Role of Placental Efflux to Optimize Dosing Regimens of Dexamethasone and Betamethasone to Prevent Respiratory Distress Syndrome

IntroductionRespiratory distress syndrome is one of the major causes of newborn deaths. Dexamethasone (DEX) and Betamethasone (BET) may promote fetal lung maturation when administered to pregnant women at risk of preterm birth. Usually, DEX‐phosphate (6 mg) is administered IM q 12 hrs over 48 hrs while BET is administered IM as a 50:50 phosphate and acetate combination (12 mg total) q 24 hrs over 48 hrs. Recent non‐clinical trials suggest lower concentration would result in effective treatment for these drugs. In sheep, fetal BET plasma concentrations &gt;1 ng/mL appears to adequately promote fetal lung maturation. Therefore, there is a need to optimize the dosing regimen of these drugs. DEX and BET are substrates of P‐gp, a placental efflux transporter, that can reduce their fetal plasma concentrations. We have recently developed a maternal‐fetal PBPK model to predict maternal‐fetal plasma concentrations of drugs throughout pregnancy. The model has been populated with physiological changes observed in pregnancy including that in hepatic activity of CYP enzymes. Using this model, our aims were to 1) predict maternal plasma concentration‐time profiles of DEX and BET, after IM administration, and compare them with those observed; 2) determine the extent of placental P‐gp activity necessary to match the predicted and observed fetal DEX/BET plasma concentrations; 3) use these data to design maternal dosing regimens of DEX and BET that minimize maternal plasma concentrations while maintaining fetal plasma concentrations &gt; 1 ng/ml.MethodNon‐pregnant PBPK model for both drugs was developed in SimCYP where hepatic intrinsic clearance by CYP3A (CLhep,int), renal clearance and absorption rate constants of IM DEX and BET, were optimized based on literature data. A single first‐order absorption rate was used for IM DEX‐phosphate. Distinct absorption rate constants were used for IM BET‐phosphate and IM BET‐acetate. The maternal PBPK model was developed in MATLAB Simulink and populated with maternal gestational age‐dependent changes as well as placental passive diffusion and fetal hepatic clearance of the drugs. Theoretical DEX and BET fetal plasma concentration‐time profiles were simulated with varying magnitudes of P‐gp efflux until their predicted fetal plasma concentration‐time profiles adequately encompassed the observed data and the fetal PK parameters (AUC and Cmax) fell within 0.8 to 1.25‐fold of observed values.Results and DiscussionBoth non‐pregnant and pregnancy PBPK models for DEX were verified with the observed data. The pregnancy model for BET (i.e CLhep, int) needed to be optimized to describe the observed maternal plasma concentration data. Sensitivity analysis showed that the fraction of DEX and BET transported by placental P‐gp needed to be 0.6 and 0.5, respectively, to best describe the observed fetal plasma concentration data. Using these data, we are devising dosing regimens of DEX and BET for pregnant women that have the potential to maximize their efficacy and minimize their toxicity. These dosing regimens will be presented.Support or Funding InformationThis work is Supported by Bill &amp; Melinda Gates Foundation grant OPP1190468

The Effects of P‐glycoprotein Inhibition on Norbuprenorphine‐induced Neonatal Abstinence Syndrome (NAS)

Treating pregnant, opioid‐addicted women with the opioid buprenorphine improves mother‐child outcomes, but can cause neonatal abstinence syndrome (NAS), a potentially life‐threatening withdrawal syndrome newborns often develop following chronic prenatal exposure to opioids. NAS severity is independent of maternal buprenorphine dose, suggesting that inter‐individual variance in the pharmacokinetics of buprenorphine may influence NAS. We previously showed that prenatal exposure to the major active metabolite of buprenorphine, norbuprenorphine (NorBUP), can induce NAS. NorBUP is a substrate of the highly polymorphic placental efflux transporter P‐glycoprotein, which transports its substrates from fetal to maternal circulation. We tested the hypothesis that inhibiting P‐glycoprotein increases NorBUP‐induced NAS severity. We administered a subthreshold dose of NorBUP or vehicle to pregnant rats from gestation day (GD) 9 until post‐delivery and treated them twice daily with the p‐glycoprotein inhibitor Elacridar or vehicle on GD18‐21. After delivery, pups were observed for precipitated withdrawal signs following administration of naltrexone or saline. NorBUP and Elacridar together, but not individually, increased naltrexone‐precipitated withdrawal signs, providing the first evidence that P‐glycoprotein activity can substantially modulate the effects of NorBUP on the fetus and may therefore contribute to the uncoupling of maternal buprenorphine dose and NAS severity.Support or Funding InformationThe project described was supported by a Summer Undergraduate Research Fellowship from the American Society for Pharmacology and Experimental Therapeutics, the UAMS Fellow‐to‐Faculty Award (Arkansas Biomedical Institute, Tobacco Settlement Funds), a UAMS Research Scholar Pilot Grant Award in Child Health, and the Translational Research Institute (TRI), grants 1U54TR001629‐01A1 and KL2TR000063 through the National Center for Advancing Translational Sciences of the National Institutes of Health (NIH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Promoter Haplotypes of the ABCB1 Gene Encoding the P-Glycoprotein Differentially Affect Its Promoter Activity by Altering Transcription Factor Binding.

Promoter single nucleotide polymorphisms (SNPs) of the ABCB1 gene, encoding the placental efflux transporter P-glycoprotein, can alter its expression and affect fetal exposure to therapeutics and environmental xenobiotics. SNPs are not arrayed as independent variants but as combinations forming defined haplotypes. Recently, we defined the haplotypes encompassing ABCB1 promoter SNPs and found that ABCB1 haplotypes differentially affect its promoter activity. The mechanism(s) by which ABCB1 haplotypes alter its promoter activity are not known. We hypothesize that the haplotype-dependent differences in ABCB1 promoter activity are due to haplotype-specific alterations in transcription factor (TF) binding. To test our hypothesis, we used a TF binding profile array and determined whether differences in TF binding exist across different ABCB1 haplotypes. TFs showing significant haplotype binding differences were mechanistically evaluated using small interfering RNA (siRNA) in cultured human placental cells. Our data indicate significant haplotype-dependent differences in TF binding. Our siRNA studies showed that the regulatory effects of TFs on promoter activity are also haplotype dependent. Our data provide a mechanistic explanation for the differential effects of ABCB1 haplotypes on its promoter activity and underscore the importance of evaluating genetic variants in the context of haplotypes rather than individual SNPs when investigating their effects on gene/protein expression and disease risk.

Role of the efflux transporters BCRP and MRP1 in human placental bio-disposition of pravastatin

The expression and activity of human placental transporters during pregnancy could be altered by several factors including pathological changes associated with preeclampsia. The aims of this study were to identify the placental efflux transporters involved in the bio-disposition of pravastatin, determine the protein expression of these transporters and their encoding genes as well as the activity of pravastatin uptake in placentas obtained from patients with preeclampsia. ATP-dependent uptake of [3H]-pravastatin by trophoblast tissue apical and basal membrane vesicles exhibited sigmoidal kinetics. The curved shapes of Eadie-Hofstee plots indicate that more than one placental transporter are involved in the uptake of pravastatin. ATP-dependent uptake of [3H]-pravastatin into vesicles expressing MRP1-5, BCRP, and P-gp, as well as the results of inhibition studies suggest that BCRP and MRP1 are the major placental efflux transporters responsible for the in vitro uptake of pravastatin. Compared to placentas from healthy pregnancies, preeclamptic placentas had increased number of syncytial knots with increased expression of BCRP in their apical membrane and increased expression of MRP1 in the cytoplasm of the syncytiotrophoblast and in cytoplasm of syncytial knots. There was a concomitant increase in ABCC1 but not in ABCG2 gene expressions in preeclamptic placentas. ATP-dependent uptake of [3H]-pravastatin by vesicles prepared from apical membranes of preeclamptic placentas was similar to the uptake by vesicles prepared from placentas obtained after uncomplicated pregnancies (13.9 ± 6.5 vs 14.1 ± 5.8 pmol·mg protein−1 min−1). The transporter-specific changes in the expression of BCRP and MRP1 in preeclamptic placentas did not affect the efflux activity of transporters localized on the apical membrane of the syncytiotrophoblast.

Differential effect of ABCB1 haplotypes on promoter activity.

Promoter single-nucleotide polymorphisms (SNPs) of the ABCB1 gene, encoding the placental efflux transporter P-glycoprotein, can affect its expression and alter xenobiotic transfer from the maternal to the fetal circulation. Because SNPs are arranged in specific combinations as defined haplotypes, the aims of this study were to: (i) determine the placental haplotype structure of the ABCB1 promoter and (ii) determine the differential effect of these haplotypes on placental ABCB1 promoter activity. DNA samples from 100 healthy placentas were PCR-amplified and sequenced to identify existing SNPs in the proximal ABCB1 promoter. The haplotype structure encompassing these SNPs was inferred by PHASE analysis. Luciferase reporter constructs representing these haplotypes were generated and transfected into human placental 3A cells and their effect on ABCB1 promoter activity was determined using a dual-luciferase assay. We identified 12 ABCB1 promoter SNPs. These SNPs were predicted by PHASE to segregate into 28 haplotypes with frequencies ranging between 0.019 and 0.88. We found 12 of these haplotypes in our population in addition to two haplotypes not predicted by PHASE. We also generated two haplotypes to determine individual SNP effects for a total of 16 studied. Compared with the ancestral haplotype, three haplotypes significantly up-regulated (107-266% increase; P<0.05), one significantly down-regulated (95.4% decrease; P<0.01), and 12 had no statistically significant effect on ABCB1 promoter activity. Our data show that the effect of SNPs on promoter activity depends on their presence in a specific haplotype. This indicates that haplotypes, rather than individual SNPs, could play a significant role in regulating placental P-glycoprotein expression and affect placental transfer and fetal exposure to xenobiotics.

Genetic and Dietary Regulation of Glyburide Efflux by the Human Placental Breast Cancer Resistance Protein Transporter.

Glyburide is frequently used to treat gestational diabetes owing to its low fetal accumulation resulting from placental efflux by the breast cancer resistance protein (BCRP)/ABCG2 transporter. Here we sought to determine how exposure to the dietary phytoestrogen genistein and expression of a loss-of-function polymorphism in the ABCG2 gene (C421A) impacted the transport of glyburide by BCRP using stably transfected human embryonic kidney 293 (HEK) cells, human placental choriocarcinoma BeWo cells, and human placental explants. Genistein competitively inhibited the BCRP-mediated transport of (3)H-glyburide in both wild-type (WT) and C421A-BCRP HEK-expressing cells, with greater accumulation of (3)H-glyburide in cells expressing the C421A variant. In BeWo cells, exposure to genistein for 60 minutes increased the accumulation of (3)H-glyburide 30%-70% at concentrations relevant to dietary exposure (IC50 ∼180 nM). Continuous exposure of BeWo cells to genistein for 48 hours reduced the expression of BCRP mRNA and protein by up to 40%, which impaired BCRP transport activity. Pharmacologic antagonism of the estrogen receptor attenuated the genistein-mediated downregulation of BCRP expression, suggesting that phytoestrogens may reduce BCRP levels through this hormone receptor pathway in BeWo cells. Interestingly, genistein treatment for 48 hours did not alter BCRP protein expression in explants dissected from healthy term placentas. These data suggest that whereas genistein can act as a competitive inhibitor of BCRP-mediated transport, its ability to downregulate placental BCRP expression may only occur in choriocarcinoma cells. Overall, this research provides important mechanistic data regarding how the environment (dietary genistein) and a frequent genetic variant (ABCG2, C421A) may alter the maternal-fetal disposition of glyburide.