Qingcheng Mao

Background/Objectives: Pregnancy is associated with increased renal secretory clearance of drugs mediated by organic anion transporters (OATs) and organic cation transporter 2 (OCT2). Circulating concentrations of pregnancy-related hormones (PRHs) increase with gestational age, providing a plausible mechanism for renal OAT and OCT2 regulation. Methods: Using primary human proximal tubular epithelial cells (PTECs), we quantified the effects of PRHs, at trimester-specific concentrations, on the mRNA expression of renal drug transporters (apical and basal) and metabolizing enzymes (DMETs), as well as endocytic receptors. PTECs from three female, premenopausal donors were cultured in an optimized Transwell system that maintains measurable OAT activity. PTECs were then exposed for 72 h to trimester-matched PRH cocktails at physiologic (1×) or supraphysiologic (10×) concentrations, with medium replaced every 24 h. DMET and endocytic receptor mRNA were quantified by RT-qPCR, and uptake activities of OAT1/2/3, OCT2, OAT4, and OCTN1 were measured with selective substrates or substrate–inhibitor pairs. Results: At 1× PRHs, renal DMET and endocytic receptor mRNA expression was unchanged across trimester-related PRH concentration except for consistent downregulation of PEPT2. Uptake activity for all measured transporters was unchanged. At 10× PRHs, selective changes in mRNA expression of transporters were observed (e.g., induction of OAT1), but these changes did not translate into changes in activity. Conclusions: Our data argue against PRHs as the main driver of the increase in OAT-mediated drug secretion during pregnancy. Alternative mechanisms (e.g., flow-dependent mechanotransduction and untested hormones [e.g., prolactin, hCG]) should be evaluated to explain gestation-dependent changes in renal secretory clearance of drugs.

Introduction: Infection and inflammation elevate circulating pro-inflammatory cytokines that can affect renal drug clearance. Accordingly, we sought to (i) quantify the extent of modulation of renal drug-metabolizing enzymes and transporters (DMETs) by cytokines and (ii) identify the mechanism(s) underlying these effects. Methods: Fresh primary human proximal tubular epithelial cells (PTECs) were cultured on extracellular matrix-coated Transwells. PTECs were exposed every 24 h, for 48 h, to IL-6, IL-1β, TNF-α, IFN-γ, IL-4, or IL-10 (0.1 or 1 ng/mL), individually or as a cocktail. mRNA expression of 25 renal DMETs was quantified by RT-qPCR. Individual activity of OAT1–4, OCT2, and OCTN1 was measured. To determine mechanisms of these effects, selective MAPK/NF-κB inhibitors (ERK [PD98059], p38 ^MAPK [SB203580], JNK [SP600125], and NF-κB [PDTC]), individually or as a cocktail, were used. IL-6, soluble IL-6 receptor (sIL-6Rα), and IL-6 + sIL-6Rα were used to probe endogenous/exogenous IL-6 classic versus trans-signaling. Results: IL-1β was the predominant modulator, downregulating mRNA expression of OAT1–3, OCT2, OAT4, MATE2-K, MRP2, and OATP4C1, and upregulating mRNA expression of OCTN1 and MRP3. TNF-α downregulated OAT1–3 mRNA expression to an extent similar to IL-1β, but did not affect other transporters. Activity changes for the major uptake transporters mirrored mRNA directionality. MAPK/NF-κB blockade by the inhibitor cocktail reduced IL-6 secretion while completely reversing the IL-1β-driven downregulation of OAT1–3 mRNA. JNK inhibition alone restored OAT1/3 mRNA. Inhibition of p38 ^MAPK blunted OAT2 mRNA downregulation. OCTN1 mRNA induction required NF-κB. Downregulation of OAT4/OCT2 mRNA was largely MAPK/NF-κB-independent. IL-6 alone, sIL-6Rα alone, or IL-6 + sIL-6Rα did not reproduce IL-1β-driven changes in transporter mRNA. Conclusions: IL-1β is the principal driver of cytokine-mediated regulation of human renal transporters in PTECs via JNK/p38 ^MAPK /NF-κB nodes. These mechanistic, exposure-verified data provide inputs for physiologically based pharmacokinetic predictions of renal secretory clearance and pathway-mediated drug interactions during inflammation. Visual Translational Statement: Systemic inflammation increases cytokine concentrations and alters drug pharmacokinetics. Yet, cytokine regulation of renal drug transporters remains poorly defined, even though the kidney clears many anti-infective drugs via active secretion. Using an optimized primary human proximal tubular epithelial cell model that preserves expression and function of major renal transporters, we found that IL-1β is the predominant cytokine that downregulates the mRNA and activity of OAT1–3, OCT2, and OAT4, while upregulating the mRNA and activity of OCTN1. We further showed that IL-1β-driven downregulation of OAT1/3 occurs through JNK signaling, OAT2 through p38 ^MAPK , and OCTN1 through NF-κB. These data provide quantitative inputs for physiologically based pharmacokinetic models to predict how inflammation alters renal transporter-mediated drug clearance, informing dose adjustment and risk assessment for disease-drug and drug-drug interactions in patients with inflammatory kidney disease or systemic infections. They also highlight signaling nodes where anti-inflammatory therapies might inadvertently modify renal drug transport.

Δ⁹-Tetrahydrocannabinol (THC) is the primary psychoactive component of cannabis which is being increasingly consumed by pregnant people. In humans, THC is sequentially metabolized in the liver to its circulating metabolites 11-hydroxy-THC (11-OH-THC, psychoactive) and 11-nor-9-carboxy-THC (THC-COOH, non-psychoactive). Human and macaque data show that fetal exposure to THC is considerably lower than the corresponding maternal exposure. Through perfused human placenta studies, we showed that this is due to the active efflux of THC (fetal-to-maternal) by a placental transporter(s) other than P-glycoprotein or breast cancer resistance protein. The identity of this placental transporter(s) as well as whether THC or its metabolites are substrates or inhibitors of hepatic solute carrier transporters is unknown. Therefore, we investigated whether 5 μM THC, 0.3 μM 11-OH-THC, and 2.5 μM THC-COOH are substrates and/or inhibitors of placental or hepatic solute carrier transporters at their pharmacologically relevant concentrations. Using HEK cells overexpressing human OATP1B1, OATP1B3, OATP2B1, OCT1, OCT3, OAT2, OAT4, or NTCP, and prototypic substrates/inhibitors of these transporters, we found that THC and THC-COOH were substrates but not inhibitors of OCT1. THC-COOH was a weak substrate of OCT3 and a weak inhibitor of OAT4. THC, 11-OH-THC, and THC-COOH were found not to be substrates/inhibitors of the remaining transporters investigated.

Δ9-Tetrahydrocannabinol (THC) is the primary psychoactive component of cannabis which is in-creasingly consumed by pregnant people. In humans, THC is sequentially metabolized in the liver to its circulating metabolites 11-hydroxy-THC (11-OH-THC, psychoactive) and 11-nor-9-carboxy-THC (THC-COOH, non-psychoactive). Human and macaque data show that fetal exposure to THC is considerably lower than its corresponding maternal exposure. Through perfused human placenta studies, we have shown that this is due to active efflux of THC (fe-tal-to-maternal) by a placental transporter(s) other than P-glycoprotein or breast cancer resistance protein. The identity of this placental transporter(s) as well as whether THC or its metabolites are substrates or inhibitors of hepatic solute carrier transporters is unknown. Therefore, we investigated whether 5 μM THC, 0.3 μM 11-OH-THC, and 2.5 μM THC-COOH are substrates and/or inhibitors of placental or hepatic solute carrier transporters at their pharmacological-relevant concentrations. Using HEK cells overexpressing human OATP1B1, OATP1B3, OATP2B1, OCT1, OCT3, OAT2, OAT4, or NTCP, and prototypic substrates/inhibitors of these transporters, we found that THC and THC-COOH were substrates but not inhibitors of OCT1. THC-COOH was a weak substrate of OCT3 and a weak inhibitor of OAT4. THC, 11-OH-THC, and THC-COOH were found not to be substrates/inhibitors of the remaining transporters investigated.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3–5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

ID 20942 Poster Board 388 In vivo studies have shown that the clearance of drugs primarily cleared by cytochrome P450 (CYP) metabolism is reduced during inflammation or infections, potentially resulting in drug toxicity. Inflammation or infectious diseases (e.g. HIV, COVID-19) are associated with increased production of pro-inflammatory cytokines which are known to down-regulate the expression or activity of CYP enzymes. However, their effect on human hepatic drug transporters has been poorly studied, primarily with individual cytokines at supraphysiological concentrations. This makes it difficult to translate these findings to in vivo. Therefore, the goal of this study was to systematically investigate the effect of major cytokines, individually or in combination, on the mRNA expression of hepatic drug transporters in plated primary human hepatocytes. Primary human hepatocytes (lot ZKF, YND, ADR, BioIVT) were treated with interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN- γ), either in combination (each at 0.1 and 1 ng/mL) or individually (0.01, 0.1, 1 and 10 ng/mL) for 72 hours with daily replenishment of media. The cytokine concentrations used encompass the in vivo observed plasma concentrations of cytokines in infectious diseases (e.g. HIV) and autoimmune diseases (rheumatoid arthritis). Then, the total RNA was then isolated, and the mRNA levels of 11 hepatic drug transporters were quantified by real time q-PCR, including organic anion transporting polypeptide 1B1 (OATP1B1), organic anion transporting polypeptide 1B3 (OATP1B3), organic anion transporting polypeptide 2B1 (OATP2B1), organic anion transporter (OAT2), organic cation transporter (OCT1), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), multidrug and toxin extrusion protein 1 (MATE1), multi-drug resistance protein 2,3, and 4 (MRP2/3/4). OATP1B1, OATP1B3, OAT2, P-gp, BCRP, and MATE1 mRNA expression was significantly down-regulated by the combination of cytokines in a concentration-dependent manner. OAT2 mRNA was most affected. It was down-regulated by 82% at 0.1 ng/mL and 95% at 1 ng/mL. Conversely, OCT1 mRNA expression was induced by 2-fold and 3-fold at 0.1 ng/mL and 1 ng/mL, respectively. The individual cytokines also modulated transporter mRNA expression in a concentration-dependent manner. The greatest repression (at 10 ng/ml) was of OATP1B1 by TNF-α (by 60%) and IFN- γ (by 60%), OATP1B3 by IL-6 (by 75%) and IFN- γ (by 60%), and OAT2 by TNF-α (by 90%) and IL-1β (by 90%), P-gp by IL-6 (by 50%) and TNF-α (by 50%). While OATP2B1 was down-regulated by IL-1β (by 70%) and induced by IL-6 (by 1.5-fold) at 10 g/ml, this down-regulation was abolished by the combination cytokine treatment. Our data suggested that there is an additive effect of the cytokines on the mRNA expression of most of the hepatic transporters. Similar results were obtained with plated human hepatocytes from three donors. Also, cytokines generally exhibited a greater impact on uptake transporters vs. efflux transporters. Next, we intend to determine if these observed changes in transporter mRNA expression correspond to changes in transporter activity (by transport assays) and/or abundance (by quantitative proteomics). Then, these data will be used to predict changes in transporter-mediated drug PK caused by inflammation or infectious diseases using physiologically based pharmacokinetic modeling and simulations. This work was supported by the NIH Grant R01HD102786.

(-)-Δ⁹-tetrahydrocannabinol (THC) is the primary pharmacological active constituent of cannabis. 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) are respectively the active and nonactive circulating metabolites of THC in humans. While previous animal studies reported that THC could be a substrate of mouse P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), we have shown, in vitro, that only THC-COOH is a weak substrate of human BCRP, but not of P-gp. To confirm these findings and to investigate the role of P-gp and/or Bcrp in the maternal-fetal disposition of THC and its metabolites, we administrated 3 mg/kg of THC retro-orbitally to FVB wild-type (WT), P-gp^−/−, Bcrp^−/−, or P-gp^−/−/Bcrp^−/− pregnant mice on gestation day 18 and estimated the area under the concentration-time curve (AUC) of the cannabinoids in the maternal plasma, maternal brain, placenta, and fetus, as well as the tissue/maternal plasma AUC geometric mean ratios (GMRs) using a pooled data bootstrap approach. We found that the dose-normalized maternal plasma AUCs of THC in P-gp^−/− and P-gp^−/−/Bcrp^−/− mice, and the placenta-to-maternal plasma AUC GMR of THC in Bcrp^−/− mice were 279%, 271%, and 167% of those in WT mice, respectively. Surprisingly, the tissue-to-maternal plasma AUC GMRs of THC and its major metabolites in the maternal brain, placenta, or fetus in P-gp^−/−, Bcrp^−/− or P-gp^−/−/Bcrp^−/− mice were 28–78% of those in WT mice. This study revealed that P-gp and Bcrp do not play a role in limiting maternal brain and fetal exposure to THC and its major metabolites in pregnant mice. SIGNIFICANCE STATEMENT This study systematically investigated whether P-gp and/or Bcrp in pregnant mice can alter the disposition of THC, 11-OH-THC, and THC-COOH. Surprisingly, except for Bcrp, which limits placental (but not fetal) exposure to THC, we found that P-gp^−/−, Bcrp^−/−, and/or P-gp^−/−/Bcrp^−/− significantly decreased exposure to THC and/or its metabolites in maternal brain, placenta, or fetus. The mechanistic basis for this decrease is unclear and needs further investigation. If replicated in humans, P-gp- or BCRP-based drug–cannabinoid interactions are not of concern.

Pregnant women are often complicated with diseases that require treatment with medication. Most drugs administered to pregnant women are off-label without the necessary dose, efficacy, and safety information. Knowledge concerning drug transfer across the placental barrier is essential for understanding fetal drug exposure and hence drug safety and efficacy to the fetus. Transporters expressed in the placenta, including adenosine triphosphate (ATP)-binding cassette efflux transporters and solute carrier uptake transporters, play important roles in determining drug transfer across the placental barrier, leading to fetal exposure to the drugs. In this review, we provide an update on placental drug transport, including in vitro cell/tissue, ex vivo human placenta perfusion, and in vivo animal studies that can be used to determine the expression and function of drug transporters in the placenta as well as placental drug transfer and fetal drug exposure. We also describe how the knowledge of placental drug transfer through passive diffusion or active transport can be combined with physiologically based pharmacokinetic modeling and simulation to predict systemic fetal drug exposure. Finally, we highlight knowledge gaps in studying placental drug transport and predicting fetal drug exposure and discuss future research directions to fill these gaps.

(-)-Δ⁹-tetrahydrocannabinol (THC) is the primary psychoactive constituent of cannabis. In humans, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) are psychoactive and non-psychoactive circulating metabolites of THC, respectively. Whether these cannabinoids are substrates or inhibitors of human P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) is unknown. Previous animal studies suggest that THC and its metabolites could be substrates of these transporters. Therefore, we performed Transwell, cellular accumulation, and vesicular transport assays, at pharmacologically relevant concentrations of these cannabinoids, using Madin-Darby canine kidney II (MDCKII) cells or plasma membrane vesicles overexpressing human P-gp or BCRP. Neither THC nor 11-OH-THC was found to be a substrate or inhibitor of P-gp or BCRP. The efflux ratio of THC-COOH in MDCKII-BCRP cells was 1.6, which was significantly decreased to 1.0 by the BCRP inhibitor Ko143. Likewise, cellular accumulation of THC-COOH was significantly increased 1.6-fold in the presence vs. absence of Ko143. THC-COOH also significantly inhibited BCRP-mediated transport of lucifer yellow, a BCRP substrate; however, THC-COOH was neither a substrate nor an inhibitor of P-gp. Collectively, these results indicate that THC and 11-OH-THC are not substrates or inhibitors (at pharmacologically relevant concentrations) of either P-gp or BCRP. THC-COOH is a weak substrate and inhibitor of BCRP, but not of P-gp. Accordingly, we predict that P-gp/BCRP will not modulate the disposition of these cannabinoids in humans. In addition, use of these cannabinoids will not result in P-gp- or BCRP-based drug interactions. Significance Statement In this study, we systematically investigated whether THC and its major metabolites 11-OH-THC and THC-COOH are substrates and/or inhibitors of human P-gp and BCRP at pharmacologically relevant concentrations. The results obtained are highly valuable for mechanistic understanding and prediction of the roles of P-gp and BCRP in determining the human pharmacokinetics, tissue distribution, and drug interactions of cannabinoids.