SLCO1B3 and SLCO2B1 genotypes, androgen deprivation therapy, and prostate cancer outcomes: a prospective cohort study and meta-analysis.

Solute carrier organic anion (SLCO) transporters (OATP transporters) are involved in cellular uptake of drugs and hormones. Germline variants in SLCO1B3 and SLCO2B1 have been implicated in prostate cancer progression and therapy response, including to androgen deprivation and statin medications, but results have appeared heterogeneous. We conducted a cohort study of five single-nucleotide polymorphisms (SNPs) in SLCO1B3 and SLCO2B1 with prior evidence among 3208 men with prostate cancer who participated in the Health Professionals Follow-up Study or the Physicians' Health Study, following participants prospectively after diagnosis over 32 years (median, 14 years) for development of metastases and cancer-specific death (lethal disease, 382 events). Results were suggestive of, but not conclusive for, associations between some SNPs and lethal disease and differences by androgen deprivation and statin use. All candidate SNPs were associated with SLCO mRNA expression in tumor-adjacent prostate tissue. We also conducted a systematic review and harmonized estimates for a dose-response meta-analysis of all available data, including 9 further studies, for a total of 5598 patients and 1473 clinical events. The A allele of the exonic SNP rs12422149 (14% prevalence), which leads to lower cellular testosterone precursor uptake via SLCO2B1, was associated with lower rates of prostate cancer progression (hazard ratio per A allele, 0.80; 95% confidence interval, 0.69-0.93), with little heterogeneity between studies (I2, 0.27). Collectively, the totality of evidence suggests a strong association between inherited genetic variation in SLCO2B1 and prostate cancer prognosis, with potential clinical use in risk stratification related to androgen deprivation therapy.

A Dual-Gene Reporter-Amplifier Architecture for Enhancing the Sensitivity of Molecular MRI by Water Exchange.

The development of genetic reporters for magnetic resonance imaging (MRI) is essential for investigating biological functions in vivo. However, current MRI reporters have low sensitivity, making it challenging to create significant contrast against the tissue background, especially when only a small fraction of cells express the reporter. To overcome this limitation, we developed an approach for amplifying the sensitivity of molecular MRI by combining a chemogenetic contrast mechanism with a biophysical approach to increase water diffusion through the co-expression of a dual-gene construct comprising an organic anion transporting polypeptide, Oatp1b3, and a water channel, Aqp1. We first show that the expression of Aqp1 amplifies MRI contrast in cultured cells engineered to express Oatp1b3. We demonstrate that the contrast amplification is caused by Aqp1-driven increase in water exchange, which provides the gadolinium ions internalized by Oatp1b3-expressing cells with access to a larger water pool compared with exchange-limited conditions. We further show that our methodology allows cells to be detected using approximately 10-fold lower concentrations of gadolinium than that in the Aqp1-free scenario. Finally, we show that our approach enables the imaging of mixed-cell cultures containing a low fraction of Oatp1b3-labeled cells that are undetectable on the basis of Oatp1b3 expression alone.

Rifampin- and Silymarin-Mediated Pharmacokinetic Interactions of Exogenous and Endogenous Substrates in a Transgenic OATP1B Mouse Model.

Organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3, encoded by the SLCO gene family of the solute carrier superfamily, are involved in the disposition of many exogenous and endogenous compounds. Preclinical rodent models help assess risks of pharmacokinetic interactions, but interspecies differences in transporter orthologs and expression limit direct clinical translation. An OATP1B transgenic mouse model comprising a rodent Slco1a/1b gene cluster knockout and human SLCO1B1 and SLCO1B3 gene insertions provides a potential physiologically relevant preclinical tool to predict pharmacokinetic interactions. Pharmacokinetics of exogenous probe substrates, pitavastatin and pravastatin, and endogenous OATP1B biomarkers, coproporphyrin-I and coproporphyrin-III, were determined in the presence and absence of known OATP/Oatp inhibitors, rifampin or silymarin (an extract of milk thistle [Silybum marianum]), in wild-type FVB mice and humanized OATP1B mice. Rifampin increased exposure of pitavastatin (4.6- and 2.8-fold), pravastatin (3.6- and 2.2-fold), and coproporphyrin-III (1.6- and 2.1-fold) in FVB and OATP1B mice, respectively, but increased coproporphyrin-I AUC0-24h only (1.8-fold) in the OATP1B mice. Silymarin did not significantly affect substrate AUC, likely because the silymarin flavonolignan concentrations were at or below their reported IC50 values for the relevant OATPs/Oatps. Silymarin increased the Cmax of pitavastatin 2.7-fold and pravastatin 1.9-fold in the OATP1B mice. The data of the OATP1B mice were similar to those of the pitavastatin and pravastatin clinical data; however, the FVB mice data more closely recapitulated pitavastatin clinical data than the data of the OATP1B mice, suggesting that the OATP1B mice are a reasonable, though costly, preclinical strain for predicting pharmacokinetic interactions when doses are optimized to achieve clinically relevant plasma concentrations.

Associations between UGT1A1, SLCO1B1, SLCO1B3, BLVRA and HMOX1 polymorphisms and susceptibility to neonatal severe hyperbilirubinemia in Chinese Han population.

BACKGROUND: Severe neonatal hyperbilirubinemia could lead to kernicterus and neonatal death. This study aimed to analyze the association between single nucleotide polymorphisms in genes involved in bilirubin metabolism and the incidence of severe hyperbilirubinemia. METHODS: A total of 144 neonates with severe hyperbilirubinemia and 50 neonates without or mild hyperbilirubinemia were enrolled in 3 institutions between 2019 and 2020. Twelve polymorphisms of 5 genes (UGT1A1, SLCO1B1, SLCO1B3, BLVRA, and HMOX1) were analyzed by PCR amplification of genomic DNA. Genotyping was performed using an improved multiplex ligation detection reaction technique based on ligase detection reaction. RESULTS: The frequencies of the A allele in UGT1A1-rs4148323 and the C allele in SLCO1B3-rs2417940 in the severe hyperbilirubinemia group (30.2% and 90.6%, respectively) were significantly higher than those in the controls (30.2% vs.13.0%, 90.6% vs. 78.0%, respectively, both p < 0.05). Haplotype analysis showed the ACG haplotype of UGT1A1 were associated with an increased hyperbilirubinemia risk (OR 3.122, p = 0.001), whereas the GCG haplotype was related to a reduced risk (OR 0.523, p = 0.018). CONCLUSION: The frequencies of the A allele in rs4148323 and the C allele in rs2417940 are highly associated with the incidence of severe hyperbilirubinemia in Chinese Han neonates. TRIAL REGISTRATION: Trial registration number:ChiCTR1800020424; Date of registration:2018-12-29.

[Clinical characteristics and genetic analysis of four children with Rotor syndrome].

OBJECTIVE: To explore the characteristics of SLCO1B1/SLCO1B3 gene variants among children with Rotor syndrome (RS). METHODS: Four children who were admitted to the Department of Hepatology of Hunan Children's Hospital between January 2019 and January 2022 were selected as the study subjects. Trio-whole exome sequencing was carried out for the four families, and gel electrophoresis was used to verify an insertional variant of long-interspersed element-1 (LINE-1). RESULTS: Genetic testing has identified three variants of the SLCO1B1 gene, including c.1738C>T (p.R580*), c.757C>T (p.R253*) and c.1622A>C (p.Q541P), and two variants of the SLCO1B3 gene, including c.481+22insLINE-1 and c.1747+1G>A among the children. Three of them were found to harbor homozygous variants of the SLCO1B1/SLCO1B3 genes, and one has harbored compound heterozygous variants. Sanger sequencing confirmed the existence of all variants, and gel electrophoresis has confirmed the existence of the LINE-1 insertional variant of about 6 kb within intron 6 of the SLCO1B3 gene in all children. CONCLUSION: The pathogenesis of the RS among the four children may be attributed to the variants of the SLCO1B1/SLCO1B3 genes. The LINE-1 insertion variant of the SLCO1B3 gene may be common among Chinese RS patients.

SLCO1B1 and SLCO1B3 genetic mutations in Taiwanese patients with Rotor syndrome.

Rotor syndrome is a rare, benign, inherited disorder that is commonly associated with mild hyperbilirubinemia. It is caused by bi-allelic pathological variants in both SLCO1B1 and SLCO1B3 genes, causing defective OATP1B1 and OATP1B3 in the sinusoidal membrane and interrupted bilirubin uptake of the hepatocytes. We report five Taiwanese pediatric and adult patients aged 5-32 years presenting with conjugated hyperbilirubinemia, and were found to have genetic variants of SLCO1B1 and SLCO1B3. Two also had history of prolonged neonatal jaundice. Genetic analysis using panel-based next generation sequencing revealed three patients with homozygous mutations c.1738C>T (p.R580∗) in SLCO1B1 and a transposon LINE-1 insertion in SLCO1B3, one patient with homozygous mutations for another haplotype, c.757C>T (p.R253∗) in SLCO1B1 and c.1747+1G>A in SLCO1B3. Another patient had heterozygous c.1738C>T (p.R580∗) in SLCO1B1 linked with a LINE-1 insertion in SLCO1B3, and heterozygous c.757C>T (p.R253∗) in SLCO1B1 linked with c.1747+1G>A in SLCO1B3. In conclusion, we present the first time of genetic diagnosis of Rotor syndrome in Taiwan. Advanced genetic testing has enhanced the diagnosis of rare diseases with mild symptoms.

Intronic LINE-1 insertion in SLCO1B3 as a highly prevalent cause of rotor syndrome in East Asian population.

Rotor syndrome is caused by digenic loss-of-function variants in SLCO1B1 and SLCO1B3 but only a few studies have reported co-occurring inactivating variants from both genes. A rotor syndrome-causing long interspersed element-1 (LINE-1) insertion in SLCO1B3 had been reported to be highly prevalent in the Japanese population but there has been no additional report. In spite of its known association with various human diseases, LINE-1 is hard to detect with current sequencing technologies. In this study, we aimed to devise a method to screen the LINE-1 insertion variant and investigate the frequency of this variant in various populations. A chimeric sequence, that was generated by concatenating the reference sequence at the junction and a part of inserted LINE-1 sequence, was searched from 725 raw sequencing data files. In cases containing the chimeric sequence, confirmatory long-range PCR and gap-PCR were performed. In total, 95 (13.1%) of 725 patients were positive for the chimeric sequence, and all were confirmed to have the SLCO1B3 LINE-1 insertion by PCR-based tests. The same chimeric sequence was searched from the 1000 Genomes Project data repository and the carrier frequency was remarkably high in the East Asian populations (10.1%), especially in Southern Han Chinese (18.5%), but almost absent in other populations. This SLCO1B3 LINE-1 insertion should be screened in a population-specific manner under suspicion of Rotor syndrome and the methods proposed in this study would enable this in a simple way.

Polymorphisms in solute carrier genes (SLC19A1, SLCO1B1, and SLCO1B3) predicts survival and toxicity in North Indian lung cancer patients undergoing platinum-based doublet chemotherapy.

WHAT IS KNOWN AND OBJECTIVE: Solute Carrier (SLC) transporters are known mediators of drug disposition that facilitate the influx of substrates and various chemotherapeutic agents into cells. Polymorphisms in the SLC19A1, SLCO1B1, and SLCO1B3 gene influence the prognosis in the cancer patients, but little is known about their role in lung cancer in Asians. So, the current study aims to investigate the polymorphisms in SLC19A1, SLCO1B1, and SLCO1B3 genes in Northern Indian lung cancer patients. METHODS: Patients with lung cancer who had a confirmed histology and cytology diagnosis were enrolled in the study. SLC polymorphisms were assessed by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) for variations in SLC19A1 (G80 A), SLCO1B1 (A388 G, T521 C), and SLCO1B3 (A1683-5676 G). RESULTS AND DISCUSSION: Our results showed that mutant genotype for SLC19A1 G80 A polymorphism had higher median survival time (MST) compared to wild genotype. ADCC patients with mutant genotype showed better survival compared to wild genotype for SLC19A1 G80 A. SCLC patients G80 A polymorphism showed increased survival in patients with mutant genotype (p = 0.04). In SLCO1B3, A1683-5676 G patients carrying heterozygous alleles and administered with platinum and docetaxel showed inferior survival (p = 0.006). In T521 C variant, patients with carrier genotype had reduced chances of developing anaemia (p = 0.04). Patients with SLC19A1 and SLCO1B3 variants showed lower incidence of thrombocytopenia and nephrotoxicity. WHAT IS NEW AND CONCLUSION: Our findings imply that Solute Carrier gene polymorphisms modulate the overall survival in lung cancer patients undergoing platin-based doublet chemotherapy, also these polymorphisms have a modifying impact on the associated adverse events/toxicity.

Effect of rare coding variants of charged amino acid residues on the function of human organic anion transporting polypeptide 1B3 (SLCO1B3).

Human organic anion transporting polypeptide 1B3 (OATP1B3, gene symbol SLCO1B3) is a liver-specific uptake transporter. Its function was reported to be largely affected by some positively charged amino acid residues. However, so far the effect of naturally occurring genetic variants of charged residues on OATP1B3's function has not been explored yet. Therefore, in the present study nonsynonymous single nucleotide variants that led to the replacement of charged residues of OATP1B3 were investigated. Our results demonstrated that rare coding variants c.542G > A (p.R181H) and c.592G > A (p.D198N) had a great effect on the function of OATP1B3 mainly due to their influence on protein's surface expression. Further mutation studies showed that a negatively charged residue at position 198 was indispensable to the proper expression of OATP1B3 on the plasma membrane, while a positively charged reside at position 181 was not a must. Structural modeling indicated that R181 is located at the center of putative transmembrane domain 4 (TM4) and its side chain faces towards TM2 instead of towards the substrate translocation pathway, whereas D198 is located at the border of TM4 and intracellular loop 2 and may electrostatically repulse negatively charged phospholipid head groups. In conclusion, our results indicated that rare coding variants that cause changes of charged amino acid residues might have large influence on the function and expression of OATP1B3.

Diltiazem on tacrolimus exposure and dose sparing in Chinese pediatric primary nephrotic syndrome: impact of CYP3A4, CYP3A5, ABCB1, and SLCO1B3 polymorphisms.

PURPOSE: To evaluate the role of diltiazem on tacrolimus sparing in pediatric primary nephrotic syndrome (PNS) and its relation to CYP3A4, CYP3A5, ABCB1, and SLCO1B3 polymorphisms. METHODS: The PNS children treated with tacrolimus and with steady-state trough concentration (C0) were retrospectively collected. The impacts of diltiazem on tacrolimus dose-adjusted C0 (C0/D), target concentration achievement, and required dose were evaluated. Meanwhile, the relationship between the polymorphisms (including CYP3A4*1G, CYP3A5*3, ABCB1-C3435T, and SCLO1B3) and dose-sparing effect were investigated. RESULTS: A total of 71 children with 535 concentrations, including 16 children with concomitant diltiazem, were involved. Significantly increased C0/D (94.0 vs 83.8 ng/mL per mg/kg, p = 0.038) and lower required daily dose of tacrolimus (0.056 vs 0.064 mg/kg, p = 0.003) were observed in patients co-administered with diltiazem. Subpopulation carrying CYP3A4*1G, CYP3A5*1, ABCB1-3435TT, or SLCO1B3-699AA was presented with enhanced increment in tacrolimus C0/D by 38.8-102.9%. CONCLUSION: Moderate effect of diltiazem on tacrolimus sparing, which might relate to the polymorphisms of CYP3A4, CYP3A5, ABCB1, and SLCO1B3, was documented.

Development of a Rat Sandwich-Cultured Hepatocytes Model Expressing Functional Human Organic Anion Transporting Polypeptide (OATP) 1B3: A Potential Screening Tool for Liver-Targeting Compounds.

PURPOSE: Organic anion transporting polypeptide (OATP) 1B3 transports many clinically important drugs, including statins, from blood into the liver. It exclusively expresses in human liver under normal physiological conditions. There is no rodent ortholog of human OATP1B3. Tissue targeting of therapeutic molecules mediated by transporters, including liver-targeting via liver-specific OATPs, is an emerging area in drug development. Sandwich-cultured primary hepatocytes (SCH) are a well characterized in vitro model for assessment of hepatic drug uptake and biliary excretion. The current study was designed to develop a novel rat SCH model expressing human OATP1B3 to study the hepatic disposition of OATP1B3 substrates. METHODS: Primary rat hepatocytes transduced with adenoviral vectors expressing FLAG-tagged OATP1B3 (Ad-OATP1B3), a control vector Ad-LacZ, or that were non-transduced were cultured in a sandwich configuration. FLAG immunoblot and immunofluorescence-staining determined expression and localization of OATP1B3. Uptake of [3H]-cholecystokinin octapeptide (CCK-8), a specific OATP1B3 substrate, was determined. Taurocholate (TC) is a substrate routinely used in SCH to assess biliary excretion via bile canaliculi (BC) and is also a substrate of OATP1B3. [3H]-TC accumulation in cells+BC, cells, biliary excretion index (BEI) and in vitro Clbiliary were determined using B-CLEAR® technology. RESULTS: OATP1B3 protein was extensively expressed and primarily localized on the plasma membrane in day 4 Ad-OATP1B3-transduced rat SCH. [3H]-CCK-8 accumulation in cells+BC was significantly greater (~5-13 folds, p<0.001) in day 4 SCH with vs. without Ad-OATP1B3-transduction. Expressing OATP1B3 in rat SCH significantly increased [3H]-TC accumulation in cells+BC and cells, without affecting BEI and in vitro Clbiliary. CONCLUSIONS: Rat SCH expressing human OATP1B3-is a novel in vitro model allowing simultaneous assessment of hepatic uptake, hepatocellular accumulation and biliary excretion process of a human OATP1B3 substrate. This model could be a potential tool for screening for liver-targeting compounds mediated by OATP1B3.

Organic Anion Transporting Polypeptide 1B1 Is a Potential Reporter for Dual MR and Optical Imaging.

Membrane proteins responsible for transporting magnetic resonance (MR) and fluorescent contrast agents are of particular importance because they are potential reporter proteins in noninvasive molecular imaging. Gadobenate dimeglumine (Gd-BOPTA), a liver-specific MR contrast agent, has been used globally for more than 10 years. However, the corresponding molecular transportation mechanism has not been validated. We previously reported that the organic anion transporting polypeptide (OATP) 1B3 has an uptake capability for both MR agents (Gd-EOB-DTPA) and indocyanine green (ICG), a clinically available near-infrared (NIR) fluorescent dye. This study further evaluated OATP1B1, another polypeptide of the OATP family, to determine its reporter capability. In the OATP1B1 transfected 293T transient expression model, both Gd-BOPTA and Gd-EOB-DTPA uptake were confirmed through 1.5 T MR imaging. In the constant OAPT1B1 and OATP1B3 expression model in the HT-1080 cell line, both HT-1080-OAPT1B1 and HT-1080-OATP1B3 were observed to ingest Gd-BOPTA and Gd-EOB-DTPA. Lastly, we validated the ICG uptake capability of both OATP1B1 and OATP1B3. OAPT1B3 exhibited a superior ICG uptake capability to that of OAPT1B1. We conclude that OATP1B1 is a potential reporter for dual MR and NIR fluorescent molecular imaging, especially in conjunction with Gd-BOPTA.

Altered Profile of E1-S Transporters in Endometrial Cancer: Lower Protein Levels of ABCG2 and OSTß and Up-Regulation of SLCO1B3 Expression.

Endometrial cancer (EC) is associated with increased estrogen actions. Locally, estrogens can be formed from estrone-sulphate (E1-S) after cellular uptake by organic anion-transporting polypeptides (OATP) or organic anion transporters (OAT). Efflux of E1-S is enabled by ATP Binding Cassette transporters (ABC) and organic solute transporter (OST)αß. Currently, 19 E1-S transporters are known but their roles in EC are not yet understood. Here, we analysed levels of E1-S transporters in Ishikawa (premenopausal EC), HEC-1-A (postmenopausal EC), HIEEC (control) cell lines, in EC tissue, examined metabolism of steroid precursor E1-S, studied effects of OATPs' inhibition and gene-silencing on E1-S uptake, and assessed associations between transporters and histopathological data. Results revealed enhanced E1-S metabolism in HEC-1-A versus Ishikawa which could be explained by higher levels of OATPs in HEC-1-A versus Ishikawa, especially 6.3-fold up-regulation of OATP1B3 (SLCO1B3), as also confirmed by immunocytochemical staining and gene silencing studies, lower ABCG2 expression and higher levels of sulfatase (STS). In EC versus adjacent control tissue the highest differences were seen for ABCG2 and SLC51B (OSTß) which were 3.0-fold and 2.1-fold down-regulated, respectively. Immunohistochemistry confirmed lower levels of these two transporters in EC versus adjacent control tissue. Further analysis of histopathological data indicated that SLCO1B3 might be important for uptake of E1-S in tumours without lymphovascular invasion where it was 15.6-fold up-regulated as compared to adjacent control tissue. Our results clearly indicate the importance of E1-S transporters in EC pathophysiology and provide a base for further studies towards development of targeted treatment.

Organic Anion Transporting Polypeptide-Mediated Hepatic Uptake of Glucuronide Metabolites of Androgens.

We previously established that androgen glucuronides are effluxed by multidrug resistance-associated proteins 2 and 3. However, no data exist on the mechanism of hepatic uptake of these metabolites. The first goal of this study was to explore the role of hepatic uptake transporters and characterize transport kinetics of glucuronides of testosterone (TG), dihydrotestosterone (DHTG), androsterone (AG), and etiocholanolone (EtioG) using cell lines overexpressing organic anion transporting polypeptides (OATP1B1, OATP1B3, and OATP2B1). Using a quantitative proteomics-guided approach, we then estimated the fractional contribution of individual OATPs in hepatic uptake of these glucuronides. The transport screening assays revealed that the glucuronides were primarily taken up by OATP1B1 and OATP1B3. The K m values for OATP1B1-mediated uptake were low for EtioG (6.2 µM) as compared with AG, TG, and DHTG (46.2, 56.7, and 71.3 µM, respectively), whereas the K m value for OATP1B3-mediated uptake for EtioG, AG, DHTG, and TG were 19.8, 29.3, 69.6, and 110.4 µM, respectively. Both OATP1B1 and OATP1B3 exhibited the highest transport rate toward AG as compared with other glucuronides. When adjusted for the transporter abundance in human livers, EtioG and DHTG were predicted to be transported by both OATP1B1 and OATP1B3, whereas TG and AG were preferentially (>68%) transported by OATP1B3. Collectively, this report elucidates the mechanisms of hepatic uptake of androgen glucuronides. Perturbation of these processes by genetic polymorphisms, disease conditions, or drug interactions can lead to changes in enterohepatic recycling of androgens. TG and AG can be further investigated as potential biomarkers of OATP1B3 inhibition. SIGNIFICANCE STATEMENT: This is the first study to elucidate the mechanism of hepatic uptake of androgen glucuronides and estimate the fractional contribution of individual OATPs using quantitative proteomics. Our results show that both OATP1B1 and OATP1B3 are responsible for the hepatic uptake of major circulating testosterone glucuronides. The apparent higher selectivity of OATP1B3 toward testosterone glucuronide and androsterone glucuronide can be leveraged for establishing these metabolites as clinical biomarkers of OATP1B3 activity.

Absence of OATP1B (Organic Anion-Transporting Polypeptide) Induction by Rifampin in Cynomolgus Monkeys: Determination Using the Endogenous OATP1B Marker Coproporphyrin and Tissue Gene Expression.

Organic anion-transporting polypeptide (OATP) 1B induction is an evolving mechanism of drug disposition and interaction. However, there are contradictory reports describing OATP1B expression in hepatocytes and liver biopsies after administration of an inducer. This study investigated the in vivo effects of the common inducer rifampin (RIF) on the activity and expression of cynomolgus monkey OATP1B1 and OATP1B3 transporters, which are structurally and functionally similar their human OATP1B counterparts. Multiple doses of oral RIF (15 mg/kg) resulted in a steady 3.9-fold increase of CYP3A biomarker, 4ß-hydroxycholesterol (4ßHC), in the plasma samples collected before each RIF dose during the treatment period (i.e., predose). In contrast, the predose plasma levels of OATP1B biomarkers coproporphyrin (CP) I and CPIII did not change when compared with RIF treatment. The trough concentration, area under plasma concentration-time curve (AUC), and half-life of RIF decreased markedly during RIF treatment, suggesting that RIF induced its own clearance. Consequently, RIF treatment increased CPI and CPIII AUCs substantially after a single administration and, to a lesser extent, after multiple administrations compared with preadministration AUCs. In addition, OATP1B1 and OATP1B3 mRNA expressions were not modulated by RIF treatment (0.85-1.3-fold), whereas CYP3A8 expression was increased 3.7-5.0-fold, which correlated well with the predose levels of CP and 4ßHC. Rifampin treatment showed 2.0-3.3-fold increases in P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) expression in the small intestine. Collectively, these findings indicate that monkey OATP1B and OATP1B3 are not induced by RIF, and further investigation of OATP1B induction by RIF and other nuclear receptor activators in humans is warranted. SIGNIFICANCE STATEMENT: In this study, combined endogenous biomarker and gene expression data suggested that RIF did not induce OATP1B in cynomolgus monkeys. For the first time, the study determines transporter gene expression in the nonhuman primate liver, gut, and kidney tissues after administration of RIF for 7 days, leading to a better understanding of the induction of OATP1B and other major drug transporters. Finally, it provides evidence to strengthen the claim that coproporphyrin is a suitable endogenous probe of OATP1B activity.

Impact of SLCO1B3 polymorphisms on clinical outcomes in lung allograft recipients receiving mycophenolic acid.

Single-nucleotide polymorphisms (SNPs) in genes involved in mycophenolic acid (MPA) metabolism have been shown to contribute to variable MPA exposure, but their clinical effects are unclear. We aimed to determine if SNPs in key genes in MPA metabolism affect outcomes after lung transplantation. We performed a retrospective cohort study of 275 lung transplant recipients, 228 receiving mycophenolic acid and a control group of 47 receiving azathioprine. Six SNPs known to regulate MPA exposure from the SLCO, UGT and MRP2 families were genotyped. Primary outcome was 1-year survival. Secondary outcomes were 3-year survival, nonminimal (≥A2 or B2) acute rejection, and chronic lung allograft dysfunction (CLAD). Statistical analyses included time-to-event Kaplan-Meier with log-rank test and Cox regression modeling. We found that SLCO1B3 SNPs rs4149117 and rs7311358 were associated with decreased 1-year survival [rs7311358 HR 7.76 (1.37-44.04), p = 0.021; rs4149117 HR 7.28 (1.27-41.78), p = 0.026], increased risk for nonminimal acute rejection [rs4149117 TT334/T334G: OR 2.01 (1.06-3.81), p = 0.031; rs7311358 GG699/G699A: OR 2.18 (1.13-4.21) p = 0.019] and lower survival through 3 years for MPA patients but not for azathioprine patients. MPA carriers of either SLCO1B3 SNP had shorter survival after CLAD diagnosis (rs4149117 p = 0.048, rs7311358 p = 0.023). For the MPA patients, Cox regression modeling demonstrated that both SNPs remained independent risk factors for death. We conclude that hypofunctional SNPs in the SLCO1B3 gene are associated with an increased risk for acute rejection and allograft failure in lung transplant recipients treated with MPA.

Changes in Organic Anion Transporting Polypeptide Uptake in HEK293 Overexpressing Cells in the Presence and Absence of Human Plasma.

Generating accurate in vitro data is crucial for in vitro to in vivo extrapolation and pharmacokinetic predictions. The use of human embryonic kidney (HEK) 293 cells overexpressing organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 in protein-free buffer and 100% human plasma incubations was explored for the uptake of four OATP substrates: pravastatin, rosuvastatin, repaglinide, and pitavastatin. Differences were observed for each parameter [unbound Michaelis constant (K m,u), V max, intrinsic clearance (CLint), and unbound passive diffusion Pdif,u] obtained from the buffer and plasma incubations in both cells, and the fold differences were greatest for the highly protein bound compounds. The fold change in K m,u values ranged from 1.91 to 619, and the fold change in V max values ranged from 1.22 to 97.4. As a result, in both cells, the CLint values generated in the plasma incubations were higher by 0.762- to 31.7-fold than the values generated in the protein-free buffer. The passive diffusion was also higher in the plasma incubations for all four compounds, with a fold difference range of 1.73-23.4. These shifts in the presence and absence of human plasma suggest that plasma proteins may play a role in both the active uptake and passive diffusion processes. The results also support the idea of a transporter-induced protein-binding shift, where high protein binding may not limit the uptake of compounds that have high affinity for transporters. The addition of plasma to incubations leading to higher CLint values for transporter substrates helps mitigate the underprediction commonly noted with in vitro to in vivo extrapolation. SIGNIFICANCE STATEMENT: The current investigation brings a new perspective on how to mitigate the underprediction commonly noted with in vitro to in vivo extrapolation for OATP substrates by using HEK293 cells overexpressing OATP1B1 and OATP1B3. It also supports the idea of a transporter-induced protein-binding shift, where high protein binding may not limit the uptake of compounds that have high affinity for transporters.

Elucidation of OATP1B1 and 1B3 transporter function using transgenic rodent models and commonly known single nucleotide polymorphisms.

The clearance of many drugs from the blood into the liver, such as the statins, is dependent on the organic anion transporting polypeptides (OATPs). Patients with *5 and *15 polymorphisms of OATP1B1 remove less of the statin as it traverses the liver and thus more reaches the rest of the body, including the skeletal muscle where it can cause myalgia, myopathy, and rhabdomyolysis. OATP1B1 polymorphisms also affect the pharmacokinetics of anticancer drugs (methotrexate, taxanes, and doxorubicin) and numerous anti-hypertensive drugs. In contrast to OATP1B1, OATP1B3 does not appear to have polymorphisms of known physiological and pharmacological significance, except for Rotor patients, who have both defective OATP1B1 and OATP1B3 transport function. OATP1B1 and OATP1B3 also play important roles in the hepatic uptake of many endogenous molecules, such as bile acids, bilirubin, and coproporphyrins. However, the transport of individual bile acids is not well understood. Complete deficiency of OATP1B1 and 1B3 function in Rotor syndrome disrupts the hepatic reuptake of conjugated bilirubin with a corresponding clinical presentation as mild hyperbilirubinemia. Interestingly, cholecystokinin is only transported into the liver by OATP1B3. Hepatotoxicants such as the mushroom toxin phalloidin and the cyanobacterias toxin microcystin-LR are transported by the OATP1Bs as they are not hepatotoxic in Oatp1b2 "knock-out" mice. In conclusion, the OATP1Bs are important in the hepatic uptake of endogenous chemicals, drugs, and toxicants. Because there are polymorphisms of OATP1B1, knowledge of the genotype/phenotype is of importance in diagnosing and treatment of patients.

Genetic variants of SLCO1B7 are of relevance for the transport function of OATP1B3-1B7.

The family of Organic Anion Transporting Polypeptides are known to facilitate the transmembrane transport. OATP1B3-1B7 is a novel member of the OATP1B-subfamily, and is encoded by SLCO1B3-SLCO1B7 readthrough deriving from the genes SLCO1B3 and SLCO1B7 on chromosome 12. The resulting protein is expressed in the smooth endoplasmatic reticulum of hepatocytes, is functional, and transports dehydroepiandrosterone-sulfate (DHEAS). In the gene area encoding for the 1B7-part of the protein, there are coding polymorphisms. It was the aim of this study to test the frequency and the impact of these genetic variants on transport activity. The minor allele frequency (MAF) of the coding polymorphisms was determined in a cohort of 192 individuals. DHEAS transport function was determined by applying the vTF-7 based heterologous expression system using plasmids encoding for OATP1B3-1B7 or the respective variants. The genetic variants 641 T (MAF 0.021), 1073 G (MAF 0.169) and 1775 A (MAF 0.013) significantly reduced DHEAS accumulation in cells transfected with OATP1B3-1B7, albeit without significantly influencing expression of the transporter as determined by Western blot analysis and immunofluorescence after heterologous expression. Genotyping revealed complete linkage of the variants 884A, 1073 G and 1501C. Presence of the haplotype abolished the DHEAS-transport function of OATP1B3-1B7. Naturally and frequently occurring genetic variants located within the gene region of SLCO1B7 encoding for the 1B7-part of OATP1B3-1B7 influence the in vitro function of this member of the OATP1B-family. With their functional characterisation, we provide the basis for pharmacogenetic studies, which may help to understand the in vivo relevance of this transporter.

Epistane, an anabolic steroid used for recreational purposes, causes cholestasis with elevated levels of cholic acid conjugates, by upregulating bile acid synthesis (CYP8B1) and cross-talking with nuclear receptors in human hepatocytes.

Anabolic-androgenic steroids are testosterone derivatives, used by body-builders to increase muscle mass. Epistane (EPI) is an orally administered 17α-alkylated testosterone derivative with 2a-3a epithio ring. We identified four individuals who, after EPI consumption, developed long-lasting cholestasis. The bile acid (BA) profile of three patients was characterized, as well the molecular mechanisms involved in this pathology. The serum BA pool was increased from 14 to 61-fold, basically on account of primary conjugated BA (cholic acid (CA) conjugates), whereas secondary BA were very low. In in vitro experiments with cultured human hepatocytes, EPI caused the accumulation of glycoCA in the medium. Moreover, as low as 0.01 µM EPI upregulated the expression of key BA synthesis genes (CYP7A1, by 65% and CYP8B1, by 67%) and BA transporters (NTCP, OSTA and BSEP), and downregulated FGF19. EPI increased the uptake/accumulation of a fluorescent BA analogue in hepatocytes by 50-70%. Results also evidenced, that 40 µM EPI trans-activated the nuclear receptors LXR and PXR. More importantly, 0.01 µM EPI activated AR in hepatocytes, leading to an increase in the expression of CYP8B1. In samples from a human liver bank, we proved that the expression of AR was positively correlated with that of CYP8B1 in men. Taken together, we conclude that EPI could cause cholestasis by inducing BA synthesis and favouring BA accumulation in hepatocytes, at least in part by AR activation. We anticipate that the large phenotypic variability of BA synthesis enzymes and transport genes in man provide a putative explanation for the idiosyncratic nature of EPI-induced cholestasis.