Plasma and urinary CP I and CP III concentrations in chimeric mice with human hepatocytes after rifampicin administration.

The interest in transporter-mediated drug interactions has been increasing in the field of drug development. In this study, we measured the plasma and urinary concentrations of coproporphyrin (CP) I and CP III as endogenous substrates for organic anion-transporting polypeptide (OATP) using chimeric mice with human hepatocytes (PXB mice) and examined the influence of an OATP inhibitor, rifampicin (RIF). CP I and CP III were actively taken up intracellularly, and RIF inhibited the uptake in a concentration-dependent manner for both CP I and CP III in human hepatocytes (PXB-cells). Single doses of RIF at 10 and 30 mg/kg were orally or intravenously administered to PXB mice and wild-type ICR mice. Plasma concentrations (AUC0-8h) of CP I increased in both mice. However, a marked increase in CP III was only observed in ICR mice, after intravenous administration of RIF at 30 mg/kg. The IC50 values of RIF for intracellular CP I/III uptake and the unbound plasma concentrations of RIF suggested that the increase in plasma CP I is associated with the exposure of RIF to OATPs. The 24-h cumulative urinary excretions of CP I and CP III increased in both mice, but more markedly in PXB mice. Thus, RIF increased the plasma and urinary concentrations of CP I and CP III in the mice, as reported in humans, and CP I may be a more sensitive biomarker of OATP-mediated drug interactions in PXB mice.

18ß-Glycyrrhetinic acid synergizes with enzalutamide to counteract castration-resistant prostate cancer by inhibiting OATP2B1 uptake of dehydroepiandrosterone sulfate.

Androgen dependence is a key feature of prostate cancer, and androgen deprivation is effective in treating prostate cancer. However, the disease often worsens and develops into castration-resistant prostate cancer after short-term control. The current study aimed to explore the mechanism of the synergistic action of 18ß-glycyrrhetinic acid (18ß-GA) and enzalutamide (ENZ) against prostate cancer. Our findings showed that 18ß-GA significantly inhibited the expression of OATP2B1 and the transport of dehydroepiandrosterone sulfate (DHEAS) in LNCap and 22RV1 cells. It also downregulated the expression of androgen receptor (AR) to some extent. ENZ strongly inhibited AR expression, but it did not affect OATP2B1-mediated uptake of DHEAS. Compared to the effects of 18ß-GA and ENZ alone, the combination of 18ß-GA and ENZ significantly enhanced the inhibitory effects on AR, prostate-specific antigen (PSA) expression, tumor cell proliferation, and migration. The results obtained in castrated model mice matched the findings of in vitro experiments. 18ß-GA significantly reduced the uptake of DHEAS mediated by OATP2B1 in mouse tumor tissues and cooperated with ENZ to further inhibit the expression of AR and PSA, combat the growth of tumor cells, and promote the apoptosis of tumor cells. In conclusion, 18ß-GA considerably decreased the uptake of DHEAS and androgen production in cells by inhibiting the transport function of OATP2B1, while ENZ inhibited the nuclear translocation of AR and reduced the expression of AR. The combination of 18ß-GA and ENZ can simultaneously inhibit androgen production and AR expression and exhibit a synergistic effect against castration and prostate cancer progression.

Organic anion transporters in remote sensing and organ crosstalk.

The organic anion transporters, OAT1 and OAT3, regulate the movement of drugs, toxins, and endogenous metabolites. In 2007, we proposed that OATs and other SLC22 transporters are involved in "remote sensing" and organ crosstalk. This is now known as the Remote Sensing and Signaling Theory (RSST). In the proximal tubule of the kidney, OATs regulate signaling molecules such as fatty acids, bile acids, indoxyl sulfate, kynurenine, alpha-ketoglutarate, urate, flavonoids, and antioxidants. OAT1 and OAT3 function as key hubs in a large homeostatic network involving multi-, oligo- and monospecific transporters, enzymes, and nuclear receptors. The Remote Sensing and Signaling Theory emphasizes the functioning of OATs and other "drug" transporters in the network at multiple biological scales (inter-organismal, organism, organ, cell, organelle). This network plays an essential role in the homeostasis of urate, bile acids, prostaglandins, sex steroids, odorants, thyroxine, gut microbiome metabolites, and uremic toxins. The transported metabolites have targets in the kidney and other organs, including nuclear receptors (e.g., HNF4a, AHR), G protein-coupled receptors (GPCRs), and protein kinases. Feed-forward and feedback loops allow OAT1 and OAT3 to mediate organ crosstalk as well as modulate energy metabolism, redox state, and remote sensing. Furthermore, there is intimate inter-organismal communication between renal OATs and the gut microbiome. Extracellular vesicles containing microRNAs and proteins (exosomes) play a key role in the Remote Sensing and Signaling System as does the interplay with the neuroendocrine, hormonal, and immune systems. Perturbation of function with OAT-interacting drugs (e.g., probenecid, diuretics, antivirals, antibiotics, NSAIDs) can lead to drug-metabolite interactions. The RSST has general applicability to other multi-specific SLC and ABC "drug" transporters (e.g., OCT1, OCT2, SLCO1B1, SLCO1B3, ABCG2, P-gp, ABCC2, ABCC3, ABCC4). Recent high-resolution structures of SLC22 and other transporters, together with chemoinformatic and artificial intelligence methods, will aid drug development and also lead to a deeper mechanistic understanding of polymorphisms.

Probing the Chemical Space of Guanidino-Carboxylic Acids to Identify the First Blockers of the Creatine-Transporter-1.

The creatine transporter-1 (CRT-1/SLC6A8) maintains the uphill transport of creatine into cells against a steep concentration gradient. Cellular creatine accumulation is required to support the ATP-buffering by phosphocreatine. More than 60 compounds have been explored in the past for their ability to inhibit cellular creatine uptake, but the number of active compounds is very limited. Here, we show that all currently known inhibitors are full alternative substrates. We analyzed their structure-activity relation for inhibition of CRT-1 to guide a rational approach to the synthesis of novel creatine transporter ligands. Measurements of both, inhibition of [3H]creatine uptake and transport associated currents, allowed for differentiating between full and partial substrates and true inhibitors. This combined approach led to a refined understanding of the structural requirements for binding to CRT-1, which translated into the identification of three novel compounds - i.e. compound 1 (2-(N-benzylcarbamimidamido)acetic acid), and MIPA572 (=carbamimidoylphenylalanine) and MIPA573 (=carbamimidoyltryptophane) that blocked CRT-1 transport, albeit with low affinity. In addition, we found two new alternative full substrates, namely MIP574 (carbamimidoylalanine) and GiDi1257 (1-carbamimidoylazetidine-3-carboxylic acid), which was superior in affinity to all known CTR-1 ligands, and one partial substrate, namely GiDi1254 (1-carbamimidoylpiperidine-4-carboxylic acid). Significance Statement The creatine transporter-1 (CRT-1) is required to maintain intracellular creatine levels. Inhibition of CRT-1 has been recently proposed as a therapeutic strategy for cancer, but pharmacological tools are scarce. In fact, all available inhibitors are alternative substrates. We tested existing and newly synthesized guanidinocarboxylic acids for CRT-1 inhibition and identified three blockers, one partial and two full substrates of CRT-1. Our results support a refined structural understanding of ligand binding to CRT-1 and provide a proof-of-principle for blockage of CRT-1.

Identification of pyrimidine structure-based compounds as allosteric ligands of the dopamine transporter as therapeutic agents for NeuroHIV.

The disruption of dopamine neurotransmission by the HIV-1 Transactivator of transcription (Tat) during HIV-1 infection has been linked to the development of neurocognitive disorders, even under combined antiretroviral therapy (cART) treatment. We have demonstrated that SRI-32742, a novel allosteric modulator of dopamine (DA) transporter (DAT), attenuates cocaine- and Tat-binding to DAT, alleviates Tat-induced cognitive deficits and potentiation of cocaine reward in inducible Tat transgenic mice. The current study determined the in vitro pharmacological profile of SRI-32743 and its optimized second-generation analogs and their effects as allosteric modulators. Through structure-activity relationship studies of SRI-32743, 170 compounds were synthesized and evaluated for their ability to modulate DAT function. We identified 21 analogs as atypical competitors of DAT (Emax {less than or equal to}60%). Four compounds, SRI-46564, SRI-47056, SRI-46286 and SRI-47867, displayed IC50 values for [3H]DA uptake inhibition from 9.33 {plus minus} 0.50 to 0.96 {plus minus} 0.05 µM and from 3.96 {plus minus} 1.36 to 1.29 {plus minus} 0.19 for DAT binding, respectively. The four analogs also displayed high potency at two different concentrations (0.5 nM and 0.05 nM) to attenuate Tat-induced inhibition of [3H]DA uptake and cocaine-mediated dissociation of [3H]WIN35,428 binding in CHO cells expressing hDAT, suggesting that the effects occur through an allosteric mechanism. In further ex vivo studies using Fast-Scan Cyclic Voltammetry, we demonstrated that the analogs do not disrupt the baseline phasic-like DA release. These findings provide a new insight into the potential for development of novel therapeutic agents to attenuate DAT-Tat interactions to normalize DA neurotransmission in NeuroHIV. Significance Statement The allosteric inhibition of the dopamine (DA) transporter by the HIV-1 Transactivator of transcription (Tat) disrupts dopamine homeostasis, leading to HIV-associated neurocognitive disorders (HANDs). Analogs of SRI-32743, a novel allosteric modulator of the Tat-DAT interaction, were evaluated in the current study and characterized as atypical ligands of DA uptake. Four novel lead compounds demonstrated high potency to attenuate Tat-induced inhibition of hDAT-mediated DA uptake in an allosteric modulatory manner with no effects on the dynamics of DA uptake-release in DAT.

Human organic anion transporting polypeptide 1B3 (OATP1B3) is more heavily N-glycosylated than OATP1B1 in extracellular loops 2 and 5.

Human organic anion transporting polypeptide 1B3 (OATP1B3) and 1B1 are two liver-specific and highly homologous uptake transporters, whose structures consist of 12 transmembrane domains. The present study showed that OATP1B3 is more heavily N-glycosylated than OATP1B1 in extracellular loop 2 (EL2) and EL5. OATP1B3 has six N-glycosylation sites, namely N134, N145, N151, N445, N503, and N516, which is twice of that of OATP1B1. Single removal of individual N-glycans seems to have minimal influence on the surface expression and function of OATP1B3. However, simultaneous removal of all N-glycans will lead to OATP1B3's large retention in the endoplasmic reticulum and cellular degradation and thus significantly disrupts its surface expression. While N-glycosylation plays a crucial role in the surface expression of OATP1B3, it also has some effect on the transport function of OATP1B3 per se, which is not due to a decrease of substrate binding affinity but due to a reduced transporter's turnover number. Taken together, N-glycosylation is essential for normal surface expression and function of OATP1B3. Its disruption by some liver diseases such as NASH might alter the pharmacokinetic/pharmacodynamic properties of OATP1B3's substrate drugs.

Redefining Statin Dosage Post-Gastric Bypass: Insights from a Population Pharmacokinetics-Pharmacodynamics Link Approach.

Roux-en-Y gastric bypass (RYGB) involves creating a small stomach pouch, bypassing part of the small intestine, and rerouting the digestive tract. These alterations can potentially change the drug exposure and response. Our primary aim was to assess the impact of RYGB on the pharmacokinetics of simvastatin lactone (SV) and its active metabolite, simvastatin hydroxy acid (SVA). Ultimately, we aimed to optimize dosing for this understudied population by employing a population pharmacokinetic-pharmacodynamic link approach. The study comprised patients who had undergone RYGB surgery and individuals without a previous history of RYGB. All participants received a single oral dose of simvastatin. Plasma concentration data were analyzed with a nonlinear mixed-effect modeling approach. A parent-metabolite model with first-order absorption, 2-compartments for SV and 1-compartment for SVA, linear elimination, and enterohepatic circulation best described the data. The model was linked to the turnover pharmacodynamic model to describe the SVA inhibition on LDL-cholesterol production. Our simulations indicated that following RYGB surgery, the exposure to SV and SVA decreased by 40%. Consequently, for low-intensity statin patients, we recommend increasing the dose from 10 to 20 mg in post-RYGB patients to maintain a comparable response to that of non-operated subjects. Moderate-intensity statin patients should require increasing doses to 40 or 60 mg or the addition of a non-statin medication to achieve similar therapeutic outcomes. In conclusion, individuals post-RYGB exhibit diminished exposure to SV and may benefit from increasing the dose or adjunctive therapy with non-statin drugs to attain equivalent responses and mitigate potential adverse events.

In Vivo Activity of Intestinal P-Glycoprotein and Hepatic Organic Anion Transporters Polypeptide in Pregnancy and Postpartum.

This study investigates the influence of pregnancy on the in vivo activity of the intestinal P-glycoprotein (P-gp) and hepatic organic anion transporters polypeptide (OATP/BCRP) using, respectively, fexofenadine and rosuvastatin as probe drugs. Eleven healthy participants were investigated during the third trimester of pregnancy (Phase 1, 28 to 38 weeks of gestation) and in the postpartum period (Phase 2, 8 to 12 weeks postpartum). In both phases, after administration of a single oral dose of fexofenadine (60 mg) and rosuvastatin (5 mg), serial blood samples were collected for up to 24 h. Rosuvastatin and fexofenadine in plasma were analyzed by LC-MS/MS using previously validated methods. The pharmacokinetic parameters of fexofenadine and rosuvastatin (Phoenix WinNonLin software) with normal distribution (Shapiro-Wilk test) are presented as geometric mean and 90% confidence interval. Phases 1 and 2 were compared using the t test (P < .05). Fexofexadine AUC0-24 values do not differ (P-value: .0715) between Phase 1 (641.9 ng h/mL [500.6-823.1]) and Phase 2 (823.8 ng h/mL [641.5-1057.6]) showing that pregnancy (third trimester) does not alter intestinal P-gp activity. However, rosuvastatin AUC0-24 values are higher (P-value: .00005) in Phase 1 (18.7 ng h/mL [13.3-26.4]) when compared to Phase 2 (9.5 ng h/mL [6.7-13.4]), suggesting inhibition of OATP1B1/OATP1B3 transporters. In conclusion, pregnancy assessed during the third trimester does not alter the intestinal P-gp activity but reduces the activity of hepatic OATP1B1/OATP1B3 transporters. Therefore, adjustments in dosage regimens may be necessary for drugs with low therapeutic index, substrates of the OATP1B1/OATP1B3 transporters, administered during the third trimester of pregnancy.

Transport of aromatic amino acids l-tryptophan, l-tyrosine, and l-phenylalanine by the organic anion transporting polypeptide (OATP) 3A1.

Amino acids are important for cellular metabolism. Their uptake across the plasma membrane is mediated by transport proteins. Despite the fact that the organic anion transporting polypeptide 4C1 (OATP4C1, Uniprot: Q6ZQN7) mediates transport of l-arginine and l-arginine derivatives, other members of the OATP family have not been characterized as amino acid transporters. The OATP family member OATP3A1 (gene symbol SLCO3A1, Uniprot: Q9UIG8) is ubiquitously expressed in human cells and highly expressed in many cancer tissues and cell lines. However, only a few substrates are known for OATP3A1. Accordingly, knowledge about its biological relevance is restricted. Our aim was to identify new substrates of OATP3A1 to gain insights into its (patho-)physiological function. In an LC-MS-based untargeted metabolomics assay using untreated OATP3A1-overexpressing HEK293 cells and control cells, we identified several amino acids as potential substrates of OATP3A1. Subsequent uptake experiments using exogenously added substrates revealed OATP3A1-mediated transport of l-tryptophan, l-tyrosine, and l-phenylalanine with 194.8 ± 28.7% (P < 0.05), 226.2 ± 18.7% (P < 0.001), and 235.2 ± 13.5% (P < 0.001), respectively, in OATP3A1-overexpressing cells compared to control cells. Furthermore, kinetic transport parameters (Km values) were determined (Trp = 61.5 ± 14.2 µm, Tyr = 220.8 ± 54.5 µm, Phe = 234.7 ± 20.6 µm). In summary, we identified the amino acids l-tryptophan, l-tyrosine, and l-phenylalanine as new substrates of OATP3A1. These findings could be used for a better understanding of (patho-)physiological processes involving increased demand of amino acids, where OATP3A1 should be considered as an important uptake transporter of l-tryptophan, l-tyrosine, and l-phenylalanine.

Genetic alteration of SLCO1B3 defines constitutional indocyanine green excretory defect in patients who underwent hepatectomy.

AIM: Constitutional indocyanine green (ICG) excretory defects must be distinguished when assessing liver function. The absence of OATP1B3 expression due to homogenous alterations in the SLCO1B3 gene has been recently reported to induce ICG excretory defects; however, its association with the clinical examinations and the clinical implications of heterogeneous SLCO1B3 gene alteration remain unclear. METHODS: OATP1B3 expression was evaluated in 49 patients who underwent hepatectomy after evaluation of the ICG retention rate at 15 min (ICGR15) and technetium-99 m-galactosyl serum albumin (99mTc-GSA) hepatic scintigraphy. Additionally, alterations in SLCO1B3 were analyzed in patients without OATP1B3 expression. Subsequently, 59 patients who underwent hepatectomy for colorectal liver metastasis (CRLM) were analyzed. RESULTS: Of 49 patients, 6 (12%) had absent OATP1B3 expression. They had significantly higher ICGR15 value (74.7% vs. 23.5%; p < 0.0001), better modified albumin-bilirubin (ALBI) grade (≤grade 2A, 100% vs. 42%; p = 0.010), more normal 99mTc-GSA hepatic scintigraphy (100% vs. 28%; p = 0.0003), and better pathological liver fibrosis (F0-1, 100% vs. 49%; p = 0.027) compared to those with OATP1B3 expression. Three available frozen blocks of cases without OATP1B3 expression showed homozygous alterations in SLCO1B3. Of 59 patients with CRLM in normal liver background, five (8.5%) had heterozygous insertion in SLCO1B3, however they had no difference in ICGR15 values or other clinical findings compared to the other patients. CONCLUSIONS: Constitutional ICG excretory defects may be defined by the complete absence of OATP1B3 expression. The modified ALBI grade and 99mTc-GSA hepatic scintigraphy were useful for detecting constitutional ICG excretory defects.

Innovative Approaches to Optimize Clinical Transporter Drug-Drug Interaction Studies.

Of the 450 cell membrane transporters responsible for shuttling substrates, nutrients, hormones, neurotransmitters, antioxidants, and signaling molecules, approximately nine are associated with clinically relevant drug-drug interactions (DDIs) due to their role in drug and metabolite transport. Therefore, a clinical study evaluating potential transporter DDIs is recommended if an investigational product is intestinally absorbed, undergoes renal or hepatic elimination, or is suspected to either be a transporter substrate or perpetrator. However, many of the transporter substrates and inhibitors administered during a DDI study also affect cytochrome P450 (CYP) activity, which can complicate data interpretation. To overcome these challenges, the assessment of endogenous biomarkers can help elucidate the mechanism of complex DDIs when multiple transporters or CYPs may be involved. This perspective article will highlight how creative study designs are currently being utilized to address complex transporter DDIs and the role of physiology-based -pharmacokinetic (PBPK) models can play.

Identification of reversible OATP1B1 and time-dependent CYP3A4 inhibition as the major risk factors for drug-induced cholestasis (DIC).

Hepatic bile acid regulation is a multifaceted process modulated by several hepatic transporters and enzymes. Drug-induced cholestasis (DIC), a main type of drug-induced liver injury (DILI), denotes any drug-mediated condition in which hepatic bile flow is impaired. Our ability in translating preclinical toxicological findings to human DIC risk is currently very limited, mainly due to important interspecies differences. Accordingly, the anticipation of clinical DIC with available in vitro or in silico models is also challenging, due to the complexity of the bile acid homeostasis. Herein, we assessed the in vitro inhibition potential of 47 marketed drugs with various degrees of reported DILI severity towards all metabolic and transport mechanisms currently known to be involved in the hepatic regulation of bile acids. The reported DILI concern and/or cholestatic annotation correlated with the number of investigated processes being inhibited. Furthermore, we employed univariate and multivariate statistical methods to determine the important processes for DILI discrimination. We identified time-dependent inhibition (TDI) of cytochrome P450 (CYP) 3A4 and reversible inhibition of the organic anion transporting polypeptide (OATP) 1B1 as the major risk factors for DIC among the tested mechanisms related to bile acid transport and metabolism. These results were consistent across multiple statistical methods and DILI classification systems applied in our dataset. We anticipate that our assessment of the two most important processes in the development of cholestasis will enable a risk assessment for DIC to be efficiently integrated into the preclinical development process.

Functional knockout of the Oatp1d1 membrane transporter affects toxicity of diclofenac in zebrafish embryos.

Organic anion transporting polypeptides (OATPs) facilitate the cellular uptake of a large number of compounds. Zebrafish Oatp1d1 matches the functional capabilities of human OATP orthologs, particularly in hormone and drug transport. It is highly expressed in the liver and later stages of embryonic development, indicating its critical role in zebrafish physiology and development. Data from previous in vitro analyses have shown a high affinity of zebrafish Oatp1d1 for pharmaceuticals and xenobiotics, providing the basis for further in vivo studies on its defence and developmental functions. Using CRISPR-Cas9 technology, we have generated an Oatp1d1 zebrafish mutant that has highly reduced Oatp1d1 expression in embryos and adult tissues compared to wild type (WT). The absence of Oatp1d1 was confirmed using custom-made antibodies. To evaluate its ecotoxicological relevance, mutant and WT embryos were exposed to increasing concentrations of diclofenac, an NSAID known for its wide and frequent use, environmental pseudo-persistence and ecological implications. WT embryos showed developmental delays and malformations such as spinal curvature, cardiac edema and blood pooling at higher diclofenac concentrations, whereas the Oatp1d1 mutant embryos showed marked resilience, with milder developmental defects and delayed toxic effects. These observations suggest that the absence of Oatp1d1 impedes the efficient entry of diclofenac into hepatocytes, thereby slowing its biotransformation into potentially more toxic metabolites. In addition, the changes in transcript expression of other uptake transporters revealed a highly probable and complex network of compensatory mechanisms. Therefore, the results of this study point to the importance of Oatp1d1-mediated transport of diclofenac, as demonstrated for the first time in vivo using an Oatp1 deficient zebrafish line. Finally, our data indicates that the compensatory role of other transporters with overlapping substrate preferences needs to be considered for a reliable understanding of the physiological and/or defensive role(s) of membrane transporters.

A pedigree analysis of Rotor hyperbilirubinemia combined with hepatitis B virus infection in a SLCO1B1 and SLCO1B3 gene mutations patient.

Background: Rotor syndrome (RS, OMIM#237450) is an extremely rare autosomal digenic recessive disorder characterized by mild non-hemolytic hereditary conjugated hyperbilirubinemia, caused by biallelic variation of SLCO1B1 and SLCO1B3 genes that resulted in OATP1B1/B3 dysfunction in the sinusoidal membrane leading to impaired bilirubin reuptake ability of hepatocytes. Methods: One RS pedigree was recruited and clinical features were documented. Whole genome second-generation sequencing was used to screen candidate genes and mutations, Sanger sequencing confirmed predicted mutations. Results: This study detected a homozygous nonsense variant c.1738C > T (p.R580*) in the coding region of the SLCO1B1 (NM006446) gene in a family with RS and hepatitis B virus infection by Variants analysis and Sanger sequencing, and confirmed by Copy Number Variation (CNV) analysis and Long Range PCR that there was a homozygous insertion of intron 5 of the SLCO1B3 gene into intron 5 of long-interspersed element 1 (LINE1). A few cases of such haplotypes have been reported in East Asian populations. A hepatitis B virus infection with fatty liver disease was indicated by pathology, which revealed mild-moderate lobular inflammation, moderate lobular inflammation, moderate hepatocellular steatosis, and fibrosis stage 1-2 (NAS score: 4 points/S1-2) alterations. Heterozygotes carrying p.R580* and LINE1 insertions were also detected in family members (I1, I2, III2, III3), but they did not develop conjugated hyperbilirubinemia. Conclusion: The mutations may be the molecular genetic foundation for the presence of SLCO1B1 c.1738C > T(p.R580*) and SLCO1B3 (LINE1) in this RS pedigree.

The role of drug efflux and uptake transporters in the plasma pharmacokinetics and tissue disposition of morphine and its main metabolites.

Morphine is a widely used opioid for the treatment of pain. Differences in drug transporter expression and activity may contribute to variability in morphine pharmacokinetics and response. Using appropriate mouse models, we investigated the impact of the efflux transporters ABCB1 and ABCG2 and the OATP uptake transporters on the pharmacokinetics of morphine, morphine-3-glucuronide (M3G), and M6G. Upon subcutaneous administration of morphine, its plasma exposure in Abcb1a/1b-/-;Abcg2-/--, Abcb1a/1b-/-;Abcg2-/-;Oatp1a/1b-/-;Oatp2b1-/- (Bab12), and Oatp1a/1b-/-;Oatp2b1-/- mice was similar to that found in wild-type mice. Forty minutes after dosing, morphine brain accumulation increased by 2-fold when mouse (m)Abcb1 and mAbcg2 were ablated. Relative recovery of morphine in small intestinal content was significantly reduced in all the knockout strains. In the absence of mOatp1a/1b and mOatp2b1, plasma levels of M3G were markedly increased, suggesting a lower elimination rate. Moreover, Oatp-deficient mice displayed reduced hepatic and intestinal M3G accumulation. Mouse Oatps similarly affected plasma and tissue disposition of subcutaneously administered M6G. Human OATP1B1/1B3 transporters modestly contribute to the liver accumulation of M6G. In summary, mAbcb1, in combination with mAbcg2, limits morphine brain penetration and its net intestinal absorption. Variation in ABCB1 activity due to genetic polymorphisms/mutations and/or environmental factors might, therefore, partially affect morphine tissue exposure in patients. The ablation of mOatp1a/1b increases plasma exposure and decreases the liver and small intestinal disposition of M3G and M6G. Since the contribution of human OATP1B1/1B3 to M6G liver uptake was quite modest, the risks of undesirable drug interactions or interindividual variation related to OATP activity are likely negligible.

Evaluation of In Vitro Metabolism- and Transporter-Based Drug Interactions with Sunscreen Active Ingredients.

PURPOSE: The aim of this study was to examine the ability of sunscreen active ingredients to inhibit in vitro drug metabolism via cytochrome P450 (CYP) enzymes and drug uptake transporters. METHODS: Metabolism assays with human liver microsomes were conducted for CYP2C9, CYP2D6 and CYP3A4 using probe substrates warfarin, bufuralol and midazolam, respectively. Uptake transporter assays with transfected cell lines were conducted for OAT3, OCT2 and OATP1B1 with probe substrates estrone-3-sulfate, metformin and rosuvastatin, respectively. Six sunscreen active ingredients, avobenzone, enzacamene, oxybenzone, octinoxate, trolamine, and homosalate, were evaluated up to their aqueous solubility limits in the assays. RESULTS: None of the sunscreen active ingredients inhibited CYP2D6 or CYP3A4 activities in the microsomes at concentration ranges up to tenfold higher than their known clinical total plasma levels. Only enzacamene, oxybenzone and trolamine were found to be inhibitory to CYP2C9 activity with IC50 values of 14.76, 22.46 and 154.7 µM, respectively. Avobenzone, enzacamene, homosalate and octinoxate were not inhibitory to the uptake transporters at the evaluated concentrations. Oxybenzone was inhibitory to OAT3 and OCT2 with IC50 values of 39.93 and 42.77 µM, respectively. Trolamine also inhibited uptake in OAT3 and OCT2 transfected cells with IC50 values of 448.1 and 1376 µM, respectively. CONCLUSIONS: Although enzacamene, oxybenzone and trolamine inhibited CYP2C9 and the renal transporters OAT3 and OCT2 in vitro, their IC50 values exceeded total plasma levels found in clinical studies. Therefore, it is unlikely that these sunscreen active ingredients in sunscreen products will inhibit the metabolism or transport of co-administered drugs in consumers.

3,3',5-Triiodothyroacetic Acid Transporters.

Introduction: Thyroid hormone transporters are essential for thyroid hormones to enter target cells. Monocarboxylate transporter (MCT) 8 is a key transporter and is expressed at the blood-brain barrier (BBB), in neural cells and many other tissues. Patients with MCT8 deficiency have severe neurodevelopmental delays because of cerebral hypothyroidism and chronic sequelae of peripheral thyrotoxicosis. The T3 analog 3,3',5-triiodothyroacetic acid (TRIAC) rescued neurodevelopmental features in animal models mimicking MCT8 deficiency and improved key metabolic features in patients with MCT8 deficiency. However, the identity of the transporter(s) that facilitate TRIAC transport are unknown. Here, we screened candidate transporters that are expressed at the human BBB and/or brain-cerebrospinal fluid barrier and known thyroid hormone transporters for TRIAC transport. Materials and Methods: Plasma membrane expression was determined by cell surface biotinylation assays. Intracellular accumulation of 1 nM TRIAC was assessed in COS-1 cells expressing candidate transporters in Dulbecco's phosphate-buffered saline (DPBS)/0.1% glucose or Dulbecco's modified Eagle's medium (DMEM) with or without 0.1% bovine serum albumin (BSA). Expression of Slc22a8 was determined by fluorescent in situ hybridization in brain sections from wild-type and Mct8/Oatp1c1 knockout mice at postnatal days 12, 21, and 120. Results: In total, 59 plasma membrane transporters were selected for screening of TRIAC accumulation (n = 40 based on expression at the human BBB and/or brain-cerebrospinal fluid barrier and having small organic molecules as substrates; n = 19 known thyroid hormone transporters). Screening of the selected transporter panel showed that 18 transporters facilitated significant intracellular accumulation of TRIAC in DPBS/0.1% glucose or DMEM in the absence of BSA. In the presence of BSA, substantial transport was noted for SLCO1B1 and SLC22A8 (in DPBS/0.1% glucose and DMEM) and SLC10A1, SLC22A6, and SLC22A24 (in DMEM). The zebrafish and mouse orthologs of these transporters similarly facilitated intracellular accumulation of TRIAC. Highest Slc22a8 mRNA expression was detected in mouse brain capillary endothelial cells and choroid plexus epithelial cells at early postnatal time points, but was reduced at P120. Conclusions: Human SLC10A1, SLCO1B1, SLC22A6, SLC22A8, and SLC22A24 as well as their mouse and zebrafish orthologs are efficient TRIAC transporters. These findings contribute to the understanding of TRIAC treatment in patients with MCT8 deficiency and animal models thereof.

Downregulation of OATP2B1 by proinflammatory cytokines leads to 5-ASA hyposensitivity in Ulcerative colitis.

5-Aminosalicylic acid (5-ASA) is a first-line agent in both remission and maintenance therapy for ulcerative colitis (UC). However, the mucosal concentration of 5-ASA was significantly lower in patients with severe histological inflammation, which further led to a poor response to 5-ASA treatment. Our study aimed to clarify the mechanism of 5-ASA uptake into colonic epithelial cells and to further explore the reason for the decreased colonic mucosal 5-ASA concentration in UC patients. Our results demonstrated that the colonic 5-ASA concentration was notably reduced in DSS-induced colitis mice and inversely correlated with colonic inflammation. 5-ASA was not a substrate of carnitine/organic cation transporter 1/2 (OCTN1/2) or multidrug resistance protein 1 (MDR1), whereas organic anion transporting polypeptide 2B1 (OATP2B1) and sodium-coupled monocarboxylate transporter 1 (SMCT1) mediated the uptake of 5-ASA, with a greater contribution from OATP2B1 than SMCT1. Inhibitors and siRNAs targeting OATP2B1 significantly reduced 5-ASA absorption in colonic cell lines. Moreover, OATP2B1 expression was dramatically downregulated in colon tissues from UC patients and dextran sodium sulfate (DSS)-induced colitis mice, and was also negatively correlated with colonic inflammation. Mechanistically, mixed proinflammatory cytokines downregulated the expression of OATP2B1 in a time- and concentration-dependent manner through the hepatocyte nuclear factor 4 α (HNF4α) pathway. In conclusion, OATP2B1 was the pivotal transporter involved in colonic 5-ASA uptake, which indicated that inducing OATP2B1 expression may be a strategy to promote 5-ASA uptake and further improve the concentration and anti-inflammatory efficacy of 5-ASA in UC.

Interactions between grapefruit juice and psychotropic medications: an update of the literature and an original case series.

INTRODUCTION: There is a large body of preclinical data implicating that grapefruit juice (GJ) inhibits many CYP 450 isoforms. The potential of GJ-to-drug is of high relevance to clinical psychiatry, because a wide range of psychotropic medicines undergo CYP 450 metabolism and P-gp transport. AREAS COVERED: Relevant data were identified by searching the electronic databases up to February 2024. This work constitutes a summary of preclinical and clinical data on GJ impact on CYP 450 metabolism, P-glycoprotein, and organic anion-transporting polypeptides (OATPs), with focus on studies that assessed GJ-to-psychotropic drug interactions. Additionally, an unpublished case series of nine patients is provided. EXPERT OPINION: The impact of GJ on CYP 3A4 appears to be the critical mechanism for the majority of GJ-to-psychopharmacotherapy interactions described in human studies or case reports. However, there are studies and cases of patients clearly showing that this is not the only route explaining the GJ effect, and at times, this particular is of no relevance and that other CYP 450 isoforms as well as drug transporting proteins might be involved. The risk of GJ-to-psychotropic drugs needs to be further evaluated in a 'real-world' setting and apply not only measures of pharmacokinetics but also treatment effectiveness and safety.

Effect of Antioxidants in Medicinal Products on Intestinal Drug Transporters.

The presence of mutagenic and carcinogenic N-nitrosamine impurities in medicinal products poses a safety risk. While incorporating antioxidants in formulations is a potential mitigation strategy, concerns arise regarding their interference with drug absorption by inhibiting intestinal drug transporters. Our study screened thirty antioxidants for inhibitory effects on key intestinal transporters-OATP2B1, P-gp, and BCRP in HEK-293 cells (OATP2B1) or membrane vesicles (P-gp, BCRP) using 3H-estrone sulfate, 3H-N-methyl quinidine, and 3H-CCK8 as substrates, respectively. The screen identified that butylated hydroxyanisole (BHA) and carnosic acid inhibited all three transporters (OATP2B1, P-gp, and BCRP), while ascorbyl palmitate (AP) inhibited OATP2B1 by more than 50%. BHA had IC50 values of 71 ± 20 µM, 206 ± 14 µM, and 182 ± 49 µM for OATP2B1, BCRP, and P-gp, respectively. AP exhibited IC50 values of 23 ± 10 µM for OATP2B1. The potency of AP and BHA was tested with valsartan, an OATP2B1 substrate, and revealed IC50 values of 26 ± 17 µM and 19 ± 11 µM, respectively, in HEK-293-OATP2B1 cells. Comparing IC50 values of AP and BHA with estimated intestinal concentrations suggests an unlikely inhibition of intestinal transporters at clinical concentrations of drugs formulated with antioxidants.