Drug transporters in drug disposition - the year 2022 in review.

On behalf of all the authors, I am pleased to share our third annual review on drug transporter science with an emphasis on articles published and deemed influential in signifying drug transporters' role in drug disposition in the year 2022. As the drug transporter field is rapidly evolving several key findings were noted including promising endogenous biomarkers, rhythmic activity, IVIVE approaches in transporter-mediated clearance, new modality interaction, and transporter effect on gut microbiome. As identified previously (Chothe et Cal. 2021, 2022) the goal of this review is to highlight key findings without a comprehensive overview of each article and to this end, each coauthor independently selected 1-3 peer-reviewed articles published or available online in the year 2022 (Table 1). Each article is summarized in synopsis and commentary with unbiased viewpoints by each coauthor. We strongly encourage readers to consult original articles for specifics of the study. Finally, I would like to thank all coauthors for their continued support in writing this annual review on drug transporters and invite anyone interested in contributing to future versions of this review.

Using a competitive counterflow assay to identify novel cationic substrates of OATP1B1 and OATP1B3.

OATP1B1 and OATP1B3 are two drug transporters that mediate the uptake of multiple endo- and xenobiotics, including many drugs, into human hepatocytes. Numerous inhibitors have been identified, and for some of them, it is not clear whether they are also substrates. Historically radiolabeled substrates or LC-MS/MS methods were needed to test for transported substrates, both of which can be limiting in time and money. However, the competitive counterflow (CCF) assay originally described for OCT2 and, more recently, for OCT1, OATP2B1, and OATP1A2 does not require radiolabeled substrates or LC-MS/MS methods and, as a result, is a more cost-effective approach to identifying substrates of multidrug transporters. We used a CCF assay based on the stimulated efflux of the common model substrate estradiol-17ß-glucuronide (E17ßG) and tested 30 compounds for OATP1B1- and OATP1B3-mediated transport. Chinese Hamster Ovary (CHO) cells stably expressing OATP1B1 or OATP1B3 were preloaded with 10 nM [3H]-estradiol-17ß-glucuronide. After the addition of known substrates like unlabeled estradiol-17ß-glucuronide, estrone-3-sulfate, bromosulfophthalein, protoporphyrin X, rifampicin, and taurocholate to the outside of the preloaded CHO cells, we observed efflux of [3H]-estradiol-17ß-glucuronide due to exchange with the added compounds. Of the tested 30 compounds, some organic cation transporter substrates like diphenhydramine, metformin, and salbutamol did not induce [3H]-estradiol-17ß-glucuronide efflux, indicating that the two OATPs do not transport them. However, 22 (for OATP1B1) and 16 (for OATP1B3) of the tested compounds resulted in [3H]-estradiol-17ß-glucuronide efflux, suggesting that they are OATP substrates. Among these compounds, we further tested clarithromycin, indomethacin, reserpine, and verapamil and confirmed that they are substrates of the two OATPs. These results demonstrate that the substrate spectrum of the well-characterized organic anion transporting polypeptides includes several organic cations. Furthermore, as for other drug uptake transporters, the CCF assay is an easy-to-use screening tool to identify novel OATP substrates.

Sinusoidal Organic Anion-Transporting Polypeptide 1B1/1B3 and Bile Canalicular Multidrug Resistance-Associated Protein 2 Play an Essential Role in the Hepatobiliary Disposition of a Synthetic Cyclic Dinucleotide (STING Agonist).

The liver is central to the elimination of many drugs from the body involving multiple processes and understanding of these processes is important to quantitively assess hepatic clearance of drugs. The synthetic STING (STimulator of INterferon Genes protein) agonist is a new class of drugs currently being evaluated in clinical trials as a potential anticancer therapy. In this study, we used ML00960317 (synthetic STING agonist) to investigate the hepatobiliary disposition of this novel molecular entity. A bile-duct cannulated (BDC) rat study indicated that biliary excretion is the major route of elimination for ML00960317 (84% of parent dose in bile). The human biliary clearance using in vitro sandwich cultured human hepatocyte model predicted significant biliary excretion of ML00960317 (biliary excretion index (BEI) of 47%). Moreover, the transport studies using transporter expressing cell lines, hepatocytes, and membrane vesicles indicated that ML00960317 is a robust substrate of OATP1B1, OATP1B3, and MRP2. Using relative expression factor approach, the combined contribution of OATP1B1 (fraction transported (ft) = 0.62) and OATP1B3 (ft = 0.31) was found to be 93% of the active uptake clearance of ML00960317 into the liver. Furthermore, OATP1B1 and OATP1B3-mediated uptake of ML00960317 was inhibited by rifampicin with IC50 of 6.5 and 2.3 µM, respectively indicating an in vivo DDI risk (R value of 1.5 and 2.5 for OATP1B1 and OATP1B3, respectively). These results highlighted an important role of OATP1B1, OATP1B3, and MRP2 in the hepatobiliary disposition of ML00960317. These pathways may act as rate-determining steps in the hepatic clearance of ML00960317 thus presenting clinical DDI risk.

Novel insights in drug transporter sciences: the year 2021 in review.

On behalf of the team I am pleased to present the second annual 'novel insights into drug transporter sciences review' focused on peer-reviewed articles that were published in the year 2021. In compiling the articles for inclusion, preprints available in 2021 but officially published in 2022 were considered to be in scope. To support this review the contributing authors independently selected one or two articles that were thought to be impactful and of interest to the broader research community. A similar approach as published last year was adopted whereby key observations, methods and analysis of each paper is concisely summarized in the synopsis followed by a commentary highlighting the impact of the paper in understanding drug transporters' role in drug disposition. As the goal of this review is not to provide a comprehensive overview of each paper but rather highlight important findings that are well supported by the data, the reader is encouraged to consult the original articles for additional information. Further, and keeping in line with the goals of this review, it should be noted that all authors actively contributed by writing synopsis and commentary for individual papers and no attempt was made to standardize language or writing styles. In this way, the review article is reflective of not only the diversity of the articles but also that of the contributors. I extend my thanks to the authors for their continued support and also welcome Diane Ramsden and Pallabi Mitra as contributing authors for this issue (Table 1).[Table: see text].

Sinusoidal Uptake Determines the Hepatic Clearance of Pevonedistat (TAK-924) as Explained by Extended Clearance Model.

Quantitative assessment of hepatic clearance (CLH) of drugs is critical to accurately predict human dose and drug-drug interaction (DDI) liabilities. This is challenging for drugs that involve complex transporter-enzyme interplay. In this study, we demonstrate this interplay in the CLH and DDI effect in the presence of CYP3A4 perpetrator for pevonedistat using both the conventional clearance model (CCM) and the extended clearance model (ECM). In vitro metabolism and hepatocyte uptake data showed that pevonedistat is actively transported into the liver via multiple uptake transporters and metabolized predominantly by CYP3A4 (88%). The active uptake clearance (CLact,inf) and passive diffusion clearance (CLdiff,inf) were 21 and 8.7 ml/min/kg, respectively. The CLact,inf was underpredicted as Empirical Scaling Factor of 13 was needed to recover the in vivo plasma clearance (CLplasma). Both CCM and ECM predicted CLplasma of pevonedistat reasonably well (predicted CLplasma of 30.8 (CCM) and 32.1 (ECM) versus observed CLplasma of 32.2 ml/min/kg). However, both systemic and liver exposures in the presence of itraconazole were well predicted by ECM but not by CCM (predicted pevonedistat plasma area under the concentration-time curve ratio (AUCR) 2.73 (CCM) and 1.23 (ECM))., The ECM prediction is in accordance with the observed clinical DDI data (observed plasma AUCR of 1.14) that showed CYP3A4 inhibition did not alter pevonedistat exposure systemically, although ECM predicted liver AUCR of 2.85. Collectively, these data indicated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat and are consistent with the lack of systemic clinical DDI with itraconazole. SIGNIFICANCE STATEMENT: In this study, we successfully demonstrated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat. Both the conventional and extended clearance models predict CLplasma of pevonedistat well however, only the ECM accurately predicted DDI effect in the presence of itraconazole, thus providing further evidence for the lack of DDI with CYP3A4 perpetrators for drugs that involve complex transporter-enzyme interplay as there are currently not many examples in the literature except prototypical OATP substrate drugs.

Recent advances in drug transporter sciences: highlights from the year 2020.

Drug Metabolism Reviews has an impressive track record of providing scientific reviews in the area of xenobiotic biotransformation over 47 years. It has consistently proved to be resourceful to many scientists from pharmaceutical industry, academia, regulatory agencies working in diverse areas including enzymology, pharmacology, pharmacokinetics, and toxicology. Over the last 5 years Drug metabolism Reviews has annually published an industry commentary aimed to highlight novel insights and approaches that have made significant impacts on the field of biotransformation (led by Cyrus Khojasteh). We hope to continue this tradition by providing an overview of advances made in the field of drug transporters during 2020. The field of drug transporters is rapidly evolving as they play an essential role in drug absorption, distribution, clearance, and elimination. In this review, we have selected outstanding drug transporter articles that have significantly contributed to moving forward the field of transporter science with respect to translation and improved understanding of diverse aspects including uptake clearance, clinical biomarkers, induction, proteomics, emerging transporters, and tissue targeting. The theme of this review consists of a synopsis that summarizes each article followed by our commentary. The objective of this work is not to provide a comprehensive review but rather to exemplify novel insights and state-of-the-art highlights of recent research that have advanced our understanding of drug transporters in drug disposition. We are hopeful that this effort will prove useful to the scientific community and as such request feedback, and further extend an invitation to anyone interested in contributing to future reviews.

Kinetic basis of metformin-MPP interactions with organic cation transporter OCT2.

Organic cation transporter 2 (OCT2) clears the blood of cationic drugs. Efforts to understand OCT2 selectivity as a means to predict the potential of new molecular entities (NMEs) to produce unwanted drug-drug interactions typically assess the influence of the NMEs on inhibition of transport. However, the identity of the substrate used to assess transport activity can influence the quantitative profile of inhibition. Metformin and 1-methyl-4-phenylpyridinium (MPP), in particular, display markedly different inhibitory profiles, with IC50 values for inhibition of MPP transport often being more than fivefold greater than IC50 values for the inhibition of metformin transport by the same compound, suggesting that interaction of metformin and MPP with OCT2 cannot be restricted to competition for a single binding site. Here, we determined the kinetic basis for the mutual inhibitory interaction of metformin and MPP with OCT2 expressed in Chinese hamster ovary cells. Although metformin did produce simple competitive inhibition of MPP transport, MPP was a mixed-type inhibitor of metformin transport, decreasing the maximum rate of mediated substrate transport and increasing the apparent Michaelis constant (Ktapp) for OCT2-mediated metformin transport. Furthermore, whereas the IC50 value for metformin's inhibition of MPP transport did not differ from the Ktapp value for metformin transport, the IC50 value for MPP's inhibition of metformin transport was less than its Ktapp value for transport. The simplest model to account for these observations required the influence of a distinct inhibitory site for MPP that, when occupied, decreases the translocation of substrate. These observations underscore the complexity of ligand interaction with OCT2 and argue for use of multiple substrates to obtain the needed kinetic assessment of NME interactions with OCT2.

Assessment of Substrate-Dependent Ligand Interactions at the Organic Cation Transporter OCT2 Using Six Model Substrates.

Organic cation transporter (OCT) 2 mediates the entry step for organic cation secretion by renal proximal tubule cells and is a site of unwanted drug-drug interactions (DDIs). But reliance on decision tree-based predictions of DDIs at OCT2 that depend on IC50 values can be suspect because they can be influenced by choice of transported substrate; for example, IC50 values for the inhibition of metformin versus MPP transport can vary by 5- to 10-fold. However, it is not clear whether the substrate dependence of a ligand interaction is common among OCT2 substrates. To address this question, we screened the inhibitory effectiveness of 20 µM concentrations of several hundred compounds against OCT2-mediated uptake of six structurally distinct substrates: MPP, metformin, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA), TEA, cimetidine, and 4-4-dimethylaminostyryl-N-methylpyridinium (ASP). Of these, MPP transport was least sensitive to inhibition. IC50 values for 20 structurally diverse compounds confirmed this profile, with IC50 values for MPP averaging 6-fold larger than those for the other substrates. Bayesian machine-learning models of ligand-induced inhibition displayed generally good statistics after cross-validation and external testing. Applying our ASP model to a previously published large-scale screening study for inhibition of OCT2-mediated ASP transport resulted in comparable statistics, with approximately 75% of "active" inhibitors predicted correctly. The differential sensitivity of MPP transport to inhibition suggests that multiple ligands can interact simultaneously with OCT2 and supports the recommendation that MPP not be used as a test substrate for OCT2 screening. Instead, metformin appears to be a comparatively representative OCT2 substrate for both in vitro and in vivo (clinical) use.

CB1-Dependent Long-Term Depression in Ventral Tegmental Area GABA Neurons: A Novel Target for Marijuana.

The VTA is necessary for reward behavior with dopamine cells critically involved in reward signaling. Dopamine cells in turn are innervated and regulated by neighboring inhibitory GABA cells. Using whole-cell electrophysiology in juvenile-adolescent GAD67-GFP male mice, we examined excitatory plasticity in fluorescent VTA GABA cells. A novel CB1-dependent LTD was induced in GABA cells that was dependent on metabotropic glutamate receptor 5, and cannabinoid receptor 1 (CB1). LTD was absent in CB1 knock-out mice but preserved in heterozygous littermates. Bath applied Δ9-tetrahydrocannabinol depressed GABA cell activity, therefore downstream dopamine cells will be disinhibited; and thus, this could potentially result in increased reward. Chronic injections of Δ9-tetrahydrocannabinol occluded LTD compared with vehicle injections; however, a single exposure was insufficient to do so. As synaptic modifications by drugs of abuse are often tied to addiction, these data suggest a possible mechanism for the addictive effects of Δ9-tetrahydrocannabinol in juvenile-adolescents, by potentially altering reward behavioral outcomes.SIGNIFICANCE STATEMENT The present study identifies a novel form of glutamatergic synaptic plasticity in VTA GABA neurons, a currently understudied cell type that is critical for the brain's reward circuit, and how Δ9-tetrahydrocannabinol occludes this plasticity. This study specifically addresses a potential unifying mechanism whereby marijuana could exert rewarding and addictive/withdrawal effects. Marijuana use and legalization are a pressing issue for many states in the United States. Although marijuana is the most commonly abused illicit drug, the implications of legalized, widespread, or continued usage are speculative. This study in juvenile-adolescent aged mice identifies a novel form of synaptic plasticity in VTA GABA cells, and the synaptic remodeling that can occur after Δ9-tetrahydrocannabinol use.

Correlation between Apparent Substrate Affinity and OCT2 Transport Turnover.

Organic cation (OC) transporter 2 (OCT2) mediates the first step in the renal secretion of many cationic drugs: basolateral uptake from blood into proximal tubule cells. The impact of this process on the pharmacokinetics of drug clearance as estimated using a physiologically-based pharmacokinetic approach relies on an accurate understanding of the kinetics of transport because the ratio of the maximal rate of transport to the Michaelis constant (i.e., Jmax/ Kt) provides an estimate of the intrinsic clearance (Clint) used in in vitro-in vivo extrapolation of experimentally determined transport data. Although the multispecificity of renal OC secretion, including that of the OCT2 transporter, is widely acknowledged, the possible relationship between relative affinity of the transporter for its diverse substrates and the maximal rates of their transport has received little attention. In this study, we determined the Jmax and apparent Michaelis constant (Ktapp) values for six structurally distinct OCT2 substrates and found a strong correlation between Jmax and Ktapp; high-affinity substrates [Ktapp values <50 µM, including 1-methyl-4-phenylpyridinium, or 1-methyl-4-phenylpyridinium (MPP), and cimetidine] displayed systematically lower Jmax values (<50 pmol cm-2 min-1) than did low-affinity substrates (Ktapp >200 µM, including choline and metformin). Similarly, preloading OCT2-expressing cells with low-affinity substrates resulted in systematically larger trans-stimulated rates of MPP uptake than did preloading with high-affinity substrates. The data are quantitatively consistent with the hypothesis that dissociation of bound substrate from the transporter is rate limiting in establishing maximal rates of OCT2-mediated transport. This systematic relationship may provide a means to estimate Clint for drugs for which transport data are lacking.