[177Lu]Lu-PSMA-617 has recently been successfully approved by the FDA, the MHRA, Health Canada and the EMA as Pluvicto®. However, salivary gland (SG) and kidney toxicities account for its main dose-limiting side-effects, while its corresponding uptake and retention mechanisms still remain elusive. Recently, the presence of different ATP-binding cassette (ABC) transporters, such as human breast cancer resistance proteins (BCRP), multidrug resistance proteins (MDR1), multidrug-resistance-related proteins (MRP1, MRP4) and solute cassette (SLC) transporters, such as multidrug and toxin extrusion proteins (MATE1, MATE2-K), organic anion transporters (OAT1, OAT2v1, OAT3, OAT4) and peptide transporters (PEPT2), has been verified at different abundances in human SGs and kidneys. Therefore, our aim was to assess whether [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 are substrates of these ABC and SLC transporters. For in vitro studies, the novel isotopologue ([α,β-3H]Nal)Lu-PSMA-617 was used in cell lines or vesicles expressing the aforementioned human ABC and SLC transporters for inhibition and uptake studies, respectively. The corresponding probe substrates and reference inhibitors were used as controls. Our results indicate that [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 are neither inhibitors nor substrates of the examined transporters. Therefore, our results show that human ABC and SLC transporters play no central role in the uptake and retention of [177Lu]Lu-PSMA-617 and [225Ac]Ac-PSMA-617 in the SGs and kidneys nor in the observed toxicities.

Uptake drug transporters play a significant role in the pharmacokinetic of drugs within the brain, facilitating their entry into the central nervous system (CNS). Understanding brain drug disposition is always challenging, especially with respect to preclinical to clinical translation. These transporters are members of the solute carrier (SLC) superfamily, which includes organic anion transporter polypeptides (OATPs), organic anion transporters (OATs), organic cation transporters (OCTs), and amino acid transporters. In this systematic review, we provide an overview of the current knowledge of uptake drug transporters in the brain and their contribution to drug disposition. Here, we also assemble currently available proteomics-based expression levels of uptake transporters in the human brain and their application in translational drug development. Proteomics data suggest that in association with efflux transporters, uptake drug transporters present at the BBB play a significant role in brain drug disposition. It is noteworthy that a significant level of species differences in uptake drug transporters activity exists, and this may contribute toward a disconnect in inter-species scaling. Taken together, uptake drug transporters at the BBB could play a significant role in pharmacokinetics (PK) and pharmacodynamics (PD). Continuous research is crucial for advancing our understanding of active uptake across the BBB.

Assessing the interactions of a new drug candidate with transporters, either as a substrate, inhibitor, or inducer, is no simple matter. There are many clinically relevant transporters, as many as nine to be evaluated for an FDA submission and up to 11 for the EMA as of 2020. Additionally, it is likely that if a compound is a substrate or inhibitor of one transporter, it will be so for other transporters as well. There are practically no specific substrates or inhibitors, presumably because the specificities of drug transporters are so broad and overlapping, and even fewer clinically relevant probes that can be used to evaluate transporter function in humans. In the case of some transporters, it is advisable to evaluate an NCE with more than one test system and/or more than one probe substrate in order to convince oneself (and regulatory authorities) that a clinical drug interaction study is not warranted. Finally, each test system has its own unique set of advantages and disadvantages. One has to appreciate the nuances of the available tools (test systems, probe substrates, etc.) to select the most relevant tools for the study and design the optimal in vitro experiment. In this chapter, several examples are used to illustrate the successful interpretation of in vitro data for both efflux and uptake transporters. Some data presented in this chapter are unpublished at the time of the compilation of this book. It has been included in this chapter to provide a sense of the complexities in transporter kinetics to the reader.

Organic anion and cation transporting proteins (OATs, OATPs, and OCTs), as well as the Multidrug and Toxin Extrusion (MATE) transporters of the Solute Carrier (SLC) family are playing a pivotal role in the discovery and development of new drugs due to their involvement in drug disposition, drug-drug interactions, adverse drug effects and related toxicity. Computational methods to understand and predict clinically relevant transporter interactions can provide useful guidance at early stages in drug discovery and design, especially if they include contemporary data science approaches. In this review, we summarize the current state-of-the-art of computational approaches for exploring ligand interactions and selectivity for these drug (uptake) transporters. The computational methods discussed here by highlighting interesting examples from the current literature are ranging from semiautomatic data mining and integration, to ligand-based methods (such as quantitative structure-activity relationships, and combinatorial pharmacophore modeling), and finally structure-based methods (such as comparative modeling, molecular docking, and molecular dynamics simulations). We are focusing on promising computational techniques such as fold-recognition methods, proteochemometric modeling or techniques for enhanced sampling of protein conformations used in the context of these ADMET-relevant SLC transporters with a special focus on methods useful for studying ligand selectivity.

A thorough understanding of species-dependent differences in hepatic uptake transporters is critical for predicting human pharmacokinetics (PKs) from preclinical data. In this study, the activities of organic anion transporting polypeptide (OATP/Oatp), organic cation transporter 1 (OCT1/Oct1), and sodium-taurocholate cotransporting polypeptide (NTCP/Ntcp) in cultured rat, dog, monkey and human hepatocytes were compared. The activities of hepatic uptake transporters were evaluated with respect to culture duration, substrate and species-dependent differences in hepatocytes. Longer culture duration reduced hepatic uptake transporter activities across species except for Oatp and Ntcp in rats. Comparable apparent Michaelis-Menten constant (Km,app) values in hepatocytes were observed across species for atorvastatin, estradiol-17β-glucuronide and metformin. The Km,app values for rosuvastatin and taurocholate were significantly different across species. Rat hepatocytes exhibited the highest Oatp percentage of uptake transporter-mediated permeation clearance (PSinf,act) while no difference in %PSinf,act of probe substrates were observed across species. The in vitro hepatocyte inhibition data in rats, monkeys and humans provided reasonable predictions of in vivo drug-drug interaction (DDIs) between atorvastatin/rosuvastatin and rifampin. These findings suggested that using human hepatocytes with a short culture time is the most robust preclinical model for predicting DDIs for compounds exhibiting active hepatic uptake in humans.

The blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) separate the brain and cerebrospinal fluid (CSF) from the systemic circulation and represent a barrier to the uptake of both endogenous compounds and xenobiotics into the brain. For compounds whose passive diffusion is limited due to their ionization or hydrophilicity, membrane transporters can facilitate their uptake across the BBB or BCSFB. Members of the solute carrier (SLC) and ATP-binding case (ABC) families are present on these barriers. Differences exist in the localization and expression of transport proteins between the BBB and BCSFB, resulting in functional differences in transport properties. This review focuses on the expression, membrane localization, and different isoforms present at each barrier. Diseases that affect the central nervous system including brain tumors, HIV, Alzheimer\'s disease, Parkinson\'s disease, and stroke affect the integrity and expression of transporters at the BBB and BCSFB and will be briefly reviewed.

To date, little is known about the transporter-mediated drug-drug interaction (DDI) potential of evogliptin, a novel DPP-4 inhibitor. The objective of this study was to evaluate the DDI potential of evogliptin using various in vitro assays in transporter-expressing cell lines. After incubating evogliptin with cells overexpressing OAT1, OAT3, OCT2, OATP1B1 and OATP1B3, there was no notable cellular accumulation of evogliptin (fold accumulation, 0.41-1.86). In bidirectional transport assays using a Caco-2 cell monolayer, a high efflux ratio (ER, 522) of evogliptin was observed, which was significantly decreased (97.96%) in the presence of a potent P-gp inhibitor. In assays using MDCKII-BCRP cell monolayers, by contrast, a low net ER (1.16-1.26) was found. In similar cellular uptake and bidirectional studies with probe substrates of P-gp, BCRP, OAT1, OAT3, OCT2, OATP1B1 and OATP1B3, the active transport of the substrates was not significantly suppressed by evogliptin. These results suggest that evogliptin may be a substrate of P-gp, but not a substrate of BCRP, OAT1B1, OAT1B3, OAT1, OAT3 or OCT2, and not an inhibitor of any of these transporters. Therefore, it could be concluded that evogliptin has some DDI potential involving P-gp, but it has low potential of DDI mediated by the other transporters.

Glucuronidation is a well-recognized phase II metabolic pathway for a variety of chemicals including drugs and endogenous substances. Although it is usually the secondary metabolic pathway for a compound preceded by phase I hydroxylation, glucuronidation alone could serve as the dominant metabolic pathway for many compounds, including some with high aqueous solubility. Glucuronidation involves the metabolism of parent compound by UDP-glucuronosyltransferases (UGTs) into hydrophilic and negatively charged glucuronides that cannot exit the cell without the aid of efflux transporters. Therefore, elimination of parent compound via glucuronidation in a metabolic active cell is controlled by two driving forces: the formation of glucuronides by UGT enzymes and the (polarized) excretion of these glucuronides by efflux transporters located on the cell surfaces in various drug disposition organs. Contrary to the common assumption that the glucuronides reaching the systemic circulation were destined for urinary excretion, recent evidences suggest that hepatocytes are capable of highly efficient biliary clearance of the gut-generated glucuronides. Furthermore, the biliary- and enteric-eliminated glucuronides participate into recycling schemes involving intestinal microbes, which often prolong their local and systemic exposure, albeit at low systemic concentrations. Taken together, these recent research advances indicate that although UGT determines the rate and extent of glucuronide generation, the efflux and uptake transporters determine the distribution of these glucuronides into blood and then to various organs for elimination. Recycling schemes impact the apparent plasma half-life of parent compounds and their glucuronides that reach intestinal lumen, in addition to prolonging their gut and colon exposure.

Background: Perivascular adipose tissue (PVAT) can decrease vascular contraction to NE. We tested the hypothesis that metabolism and/or uptake of vasoactive amines by mesenteric PVAT (MPVAT) could affect NE-induced contraction of the mesenteric resistance arteries. Methods: Mesenteric resistance vessels (MRV) and MPVAT from male Sprague-Dawley rats were used. RT-PCR and Western blots were performed to detect amine metabolizing enzymes. The Amplex® Red Assay was used to quantify oxidase activity by detecting the oxidase reaction product H2O2 and the contribution of PVAT on the mesenteric arteries\' contraction to NE was measured by myography. Results: Semicarbazide sensitive amine oxidase (SSAO) and monoamine oxidase A (MAO-A) were detected in MRV and MPVAT by Western blot. Addition of the amine oxidase substrates tyramine or benzylamine (1 mM) resulted in higher amine oxidase activity in the MRV, MPVAT, MPVAT\'s adipocyte fraction (AF), and the stromal vascular fraction (SVF). Inhibiting SSAO with semicarbazide (1 mM) decreased amine oxidase activity in the MPVAT and AF. Benzylamine-driven, but not tyramine-driven, oxidase activity in the MRV was reduced by semicarbazide. By contrast, no reduction in oxidase activity in all sample types was observed with use of the monoamine oxidase inhibitors clorgyline (1 μM) or pargyline (1 μM). Inhibition of MAO-A/B or SSAO individually did not alter contraction to NE. However, inhibition of both MAO and SSAO increased the potency of NE at mesenteric arteries with PVAT. Addition of MAO and SSAO inhibitors along with the H2O2 scavenger catalase reduced PVAT\'s anti-contractile effect to NE. Inhibition of the norepinephrine transporter (NET) with nisoxetine also reduced PVAT\'s anti-contractile effect to NE. Conclusions: PVAT\'s uptake and metabolism of NE may contribute to the anti-contractile effect of PVAT. MPVAT and adipocytes within MPVAT are a source of SSAO.

Microcystin-LR (MC-LR) and nodularin are hepatotoxins produced by several cyanobacterial species. Their toxicity is based on active cellular uptake and subsequent inhibition of protein phosphatases PP1/2A, leading to hyperphosphorylation and cell death. To date, uptake of MC-LR and nodularin in fish is poorly understood. Here, we investigated the role of the organic anion transporting polypeptide Oatp1d1 in zebrafish (drOatp1d1, Slco1d1) in cellular uptake in zebrafish. We stably transfected CHO and HEK293 cell lines expressing drOatp1d1. In both transfectants, uptake of MC-LR and nodularin was demonstrated by competitive inhibition of uptake with fluorescent substrate lucifer yellow. Direct uptake of MC-LR was demonstrated by immunostaining, and indirectly by the high cytotoxicity in stable transfectants. By means of a synthesized fluorescent labeled MC-LR derivative, direct uptake was further confirmed in HEK293 cells expressing drOatp1d1. Additionally, uptake and toxicity was investigated in the permanent zebrafish liver cell line ZFL. These cells had only a low relative abundance of drOatp1d1, drOatp2b1 and drOatp1f transcripts, which correlated with the lack of MC-LR induced cytotoxicity and transcriptional changes of genes indicative of endoplasmic reticulum stress, a known effect of this toxin. Our study demonstrates that drOatp1d1 functions as an uptake transporter for both MC-LR and nodularin in zebrafish.