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.

Significant increases in detection capability for assessment of organic anion transporting polypeptide-mediated drug-drug interaction in cynomolgus monkeys by modifying pretreatment of Coproporphyrin I and III, and glycochenodeoxycholate-3-sulfate in plasma.

BACKGROUND: Coproporphyrin I (CP-I), Coproporphyrin III (CP-III), and glycochenodeoxycholate-3-sulfate (GCDCA-S) act as endogenous substrates of Organic Anion Transporting Polypeptide (OATP) 1B and have been considered for application in OATP1B-mediated drug‒drug interaction (DDI) risk assessments. Prior assays of the endogenous OATP substrates might exhibit reduced DDI detection capability and possibly overlook low DDI risk. We pioneered a simultaneous assay of the three substrates in monkey plasma using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) and applied it to monkey studies to identify lower DDI risk. RESULTS: The methodology development indicated that precursors of CP-I/III were oxidized to form CP-I/III, diminishing the detection capability in DDI risk assessments. A precursor eliminated analytical (PEA) method was developed to eliminate the precursors through solid-phase extraction. This method aimed to prevent the oxidation of CP-I/III precursors by incorporating edaravone. For comparison, a precursor oxidized analytical (POA) method was also developed, wherein the precursors of CP-I/III were fully oxidized to CP-I/III. The PEA method achieved high sensitivity for CP-I/III and GCDCA-S, with lower quantification limits of 0.01 ng mL-1 and 0.5 ng mL-1, respectively. Both methods ensured that the validation parameters met the acceptance criteria. The two methods were applied to a monkey study, with CP-I/III showcasing notably enhanced DDI detection capabilities through the novel PEA method in comparison to the POA method. SIGNIFICANCE: This study's methodology has future implications for OATP-mediated DDI risk assessment using endogenous substrates. The novel PEA method can identify lower OATP-mediated DDI risks for drugs that the current methods cannot detect. Our method is likely applicable in clinical settings, and its utility should be assessed in clinical trials.

Repression of Organic Anion Transporting Polypeptide (OATP) 1B Expression and Increase of Plasma Coproporphyrin Level as Evidence for OATP1B Downregulation in Cynomolgus Monkeys Treated with Chenodeoxycholic Acid.

Farnesoid X receptor (FXR) is a nuclear receptor known to markedly alter expression of major transporters and enzymes in the liver. However, its effects toward organic anion transporting polypeptides (OATP) 1B1 and 1B3 remain poorly characterized. Therefore, the present study was aimed at determining the effects of chenodeoxycholic acid (CDCA), a naturally occurring FXR agonist, on OATP1B expression in cynomolgus monkeys. Multiple administrations of 50 and 100 mg/kg of CDCA were first shown to significantly repress mRNA expression of SLCO1B1/3 approximately 60% to 80% in monkey livers. It also suppressed cytochrome P450 (CYP)7A1-mRNA and induced OSTα/ß-mRNA, which are well known targets of FXR and determinants of bile acid homeostasis. CDCA concomitantly decreased OATP1B protein abundance by approximately 60% in monkey liver. In contrast, multiple doses of 15 mg/kg rifampin (RIF), a pregnane X receptor agonist, had no effect on hepatic OATP1B protein, although it induced the intestinal P-glycoprotein and MR2 proteins by ∼2-fold. Moreover, multiple doses of CDCA resulted in a steady ∼2- to 10-fold increase of the OATP1B biomarkers coproporphyrins (CPs) in the plasma samples collected prior to each CDCA dose. Additionally, 3.4- to 11.2-fold increases of CPI and CPIII areas under the curve were observed after multiple administrations compared with the single dose and vehicle administration dosing groups. Taken together, these data suggest that CDCA represses the expression of OATP1B1 and OATP1B3 in monkeys. Further investigation of OATP1B downregulation by FXR in humans is warranted, as such downregulation effects may be involved in bile acid homeostasis and potential drug interactions in man. SIGNIFICANCE STATEMENT: Using gene expression and proteomics tools, as well as endogenous biomarker data, for the first time, we have demonstrated that OATP1B expression was suppressed and its activity was reduced in the cynomolgus monkeys following oral administration of 50 and 100 mg/kg/day of chenodeoxycholic acid (CDCA), a Farnesoid X receptor agonist, for 8 days. These results lead to a better understanding of OATP1B downregulation by CDCA and its role on bile acid and drug disposition.

Biomarker-Informed Model-Based Risk Assessment of Organic Anion Transporting Polypeptide 1B Mediated Drug-Drug Interactions.

Quantitative prediction of drug-drug interactions (DDIs) involving organic anion transporting polypeptide (OATP)1B1/1B3 inhibition is limited by uncertainty in the translatability of experimentally determined in vitro inhibition potency (half-maximal inhibitory concentration (IC50 )). This study used an OATP1B endogenous biomarker-informed physiologically-based pharmacokinetic (PBPK) modeling approach to predict the effect of inhibitor drugs on the pharmacokinetics (PKs) of OATP1B substrates. Initial static analysis with about 42 inhibitor drugs, using in vitro IC50 values and unbound liver inlet concentrations (Iin,max,u ), suggested in vivo OATP1B inhibition risk for drugs with R-value (1+ Iin,max,u /IC50 ) above 1.5. A full-PBPK model accounting for transporter-mediated hepatic disposition was developed for coproporphyrin I (CP-I), an endogenous OATP1B biomarker. For several inhibitors (cyclosporine, diltiazem, fenebrutinib, GDC-0810, itraconazole, probenecid, and rifampicin at 3 different doses), PBPK models were developed and verified against available CP-I plasma exposure data to obtain in vivo OATP1B inhibition potency-which tend to be lower than the experimentally measured in vitro IC50 by about 2-fold (probenecid and rifampicin) to 37-fold (GDC-0810). Models verified with CP-I data are subsequently used to predict DDIs with OATP1B probe drugs, rosuvastatin and pitavastatin. The predicted and observed area under the plasma concentration-time curve ratios are within 20% error in 55% cases, and within 30% error in 89% cases. Collectively, this comprehensive study illustrates the adequacy and utility of endogenous biomarker-informed PBPK modeling in mechanistic understanding and quantitative predictions of OATP1B-mediated DDIs in drug development.

Performance of Plasma Coproporphyrin I and III as OATP1B1 Biomarkers in Humans.

A previous study in 356 healthy Finnish volunteers showed that glycochenodeoxycholate 3-O-glucuronide (GCDCA-3G) and glycodeoxycholate 3-O-glucuronide (GDCA-3G) are promising biomarkers of organic anion transporting polypeptide 1B1 (OATP1B1). In the same cohort, we now evaluated the performances of two other OATP1B1 biomarkers, coproporphyrin I (CPI) and III (CPIII), and compared them with GCDCA-3G and GDCA-3G. Based on decreased (*5 and *15) and increased (*14 and *20) function SLCO1B1 haplotypes, we stratified the participants to poor, decreased, normal, increased, and highly increased OATP1B1 function groups. Fasting plasma CPI concentration was 68% higher in the poor (95% confidence interval, 44%, 97%; P = 1.74 × 10-10 ), 7% higher in the decreased (0%, 15%; P = 0.0385), 10% lower in the increased (3%, 18%; P = 0.0087), and 23% lower in the highly increased (1%, 40%; P = 0.0387) function group than in the normal function group. CPIII concentration was 27% higher (7%, 51%; P = 0.0071) in the poor function group than in the normal function group. CPI and CPIII detected poor OATP1B1 function with areas under the precision-recall curve (AUPRC) of 0.388 (95% confidence interval, 0.197, 0.689) and 0.0798 (0.0485, 0.203), and receiver operating characteristic curve (AUROC) of 0.888 (0.851, 0.919) and 0.731 (0.682, 0.776). The AUPRC and AUROC of GCDCA-3G were, however, 0.389 (0.258, 0.563) and 0.100 (-0.0046, 0.204; P = 0.0610) larger than those of CPI, and 0.697 (0.555, 0.831) and 0.257 (0.141, 0.373; P < 0.0001) larger than those of CPIII. In conclusion, these data indicate that plasma CPI outperforms CPIII in detecting altered OATP1B1 function, but GCDCA-3G is an even more sensitive OATP1B1 biomarker than CPI.

Factors Influencing Plasma Coproporphyrin-I Concentration as Biomarker of OATP1B Activity in Patients With Rheumatoid Arthritis.

Organic anion transporting polypeptides (OATPs) 1B are drug transporters mainly expressed in the sinusoidal membrane. In previous reports, genetic factor, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), which is one of the uremic toxins, inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) decreased OATP1B1 activity in vitro, but in vivo effects of these factors have not been elucidated. Plasma coproporphyrin-I (CP-I) is spotlighted as a highly accurate endogenous substrate of OATP1B. This study focused on patients with rheumatoid arthritis (RA) and evaluated the influence of several factors comprising gene polymorphisms, uremic toxins, and inflammatory cytokines on OATP1B activity using plasma CP-I concentration. Thirty-seven outpatients with RA who satisfied the selection criteria were analyzed at the time of recruitment (baseline) and at the next visit. OATP1B1*15 carriers tended to have higher CP-I concentration compared with noncarriers. Plasma CP-I correlated positively with CMPF concentration, but did not correlate with IL-6 or TNF-α concentration. Multiple logistic regression analysis by stepwise selection identified plasma CMPF concentration and OATP1B1*15 allele as significant factors independently affecting plasma CP-I concentration at baseline and at the next visit, respectively. In conclusion, the present results suggest that inflammatory cytokines do not have clinically significant effects on OATP1B activity, whereas the effects of genetic polymorphisms and uremic toxins should be considered.

Relationship of hemoglobin level and plasma coproporphyrin-I concentrations as an endogenous probe for phenotyping OATP1B.

Plasma coproporphyrin-I (CP-I) concentration is used as a sensitive and selective endogenous probe for phenotyping organic anion transporting polypeptides 1B (OATP1B) activity in many studies. CP-I is produced in the process of heme synthesis, but the relationship between plasma CP-I concentrations and heme synthesis activity is unknown. In this study, we evaluated the relationship between plasma CP-I concentration and hemoglobin level as a biomarker of heme synthesis activity. The data of 391 subjects selected from the Japanese general population were analyzed. One hundred twenty-six participants had OATP1B1*15 allele, 11 of whom were homozygous (OATP1B1*15/*15). Multiple regression analysis identified hemoglobin level as an independent variable associated with plasma CP-I concentration (p < 0.0001). A significant positive correlation was observed between hemoglobin level and plasma CP-I concentration in participants without OATP1B1*15 allele (n = 265; rs  = 0.35, p < 0.0001) and with OATP1B1*15 allele (n = 126; rs  =0.27, p = 0.0022). However, Kruskal-Wallis test showed no large difference in Kruskal-Wallis statistics between the distribution of plasma CP-I concentrations and that of ratio of plasma CP-I to hemoglobin among six OATP1B1 polymorphism groups. These findings suggest that the hemoglobin level seems to reflect biosynthesis of CP-I. However, correction by hemoglobin level is not required when using basal plasma CP-I concentration for phenotyping OATP1B activity.

Further Evaluation of Coproporphyrins as Clinical Endogenous Markers for OATP1B.

Coproporphyrins (CP-I and CP-III) in plasma are considered potential markers for assessing liver organic anion-transporting polypeptide transporter OATP1B activity and monitoring OATP1B-mediated drug-drug interactions (DDIs) in clinical settings. However, the effect of altered renal clearance (CLrenal ) on CP-I and CP-III plasma exposure has rarely been examined. Therefore, the purpose of this study is to further evaluate CP-I and CP-III as clinical endogenous markers for OATP1B activity and to investigate the impact of CLrenal on DDI assessments for the first time. In this study, 18 healthy participants were recruited to receive RO7049389 (a potential inhibitor of OATP1B) 800 mg twice daily for 6 days and a single dose of pitavastatin (a probe drug of OATP1B) before and after RO7049389 treatment. Plasma concentrations of pitavastatin, CP I, CP III, and the amounts of CP-I and CP-III excreted in urine were measured. Seventeen healthy participants completed the study. After multiple doses of RO7049389, the area under the plasma concentration-time curve from time 0 to 12 hours of pitavastatin increased 1.95-fold (90% confidence interval [CI], 1.58-2.41), while for CP-I and CP-III it increased 3.00-fold (90%CI, 2.35-3.82) and 2.84-fold (90%CI, 2.22-3.65), respectively. Concurrently, the CLrenal of CP-I decreased by 31% (90%CI, 23%-39%), and that of CP-III decreased by 70% (90%CI, 61%-77%). In conclusion, CP-I and CP-III in plasma display the potential to be applied as endogenous markers for the evaluation of OATP1B inhibition in clinical trials. While renal transporters contribute significantly to the CLrenal of CP-III, it would be better to investigate the impact of the CLrenal on plasma exposure of CP-III during clinical DDI assessments.

Substantially Increased Plasma Coproporphyrin-I Concentrations Associated With OATP1B1*15 Allele in Japanese General Population.

Coproporphyrin-I (CP-I) in plasma is a sensitive and specific endogenous probe for phenotyping organic anion transporting polypeptides 1B (OATP1B, encoded by SLCO1B). A few small-scale studies suggested that plasma CP-I concentration is affected by OATP1B1 polymorphism, but detailed studies are lacking. In this large-scale study, we measured plasma CP-I concentrations in 391 subjects from the Japanese general population, and evaluated the relationship between plasma CP-I concentrations and OATP1B1 polymorphisms to further assess the utility of plasma CP-I concentrations as an endogenous OATP1B probe. Plasma CP-I concentrations were 0.45 ± 0.12, 0.47 ± 0.16, 0.47 ± 0.20, 0.50 ± 0.15, 0.54 ± 0.14, and 0.74 ± 0.31 ng/mL in participants with OATP1B1*1b/*1b (n = 103), *1a/*1b (n = 122), *1a/*1a (n = 40), *1b/*15 (n = 74), *1a/*15 (n = 41), and *15/*15 (n = 11), respectively, showing an ascending rank order with significant difference (P < 0.0001). Post hoc analysis revealed significant increases in plasma CP-I concentration in OATP1B1*1b/*15 (P = 0.036), *1a/*15 (P = 0.0005), and *15/*15 (P = 0.0003) groups compared with the OATP1B1*1b/*1b group. There was no significant difference among OATP1B genotypes in plasma concentration of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, a uremic toxin reported to decrease OATP1B activity in vivo. These findings confirm the utility of plasma CP-I concentrations as an endogenous biomarker for phenotyping of OATP1B activity. Plasma CP-I concentration is potentially useful for the study of drug-drug interactions via OATP1B or individual dose adjustment of OATP1B substrates.

Coproporphyrin I Can Serve as an Endogenous Biomarker for OATP1B1 Inhibition: Assessment Using a Glecaprevir/Pibrentasvir Clinical Study.

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are involved in the disposition of a variety of commonly prescribed drugs. The evaluation of OATP1B1/1B3 inhibition potential by investigational drugs is of interest during clinical drug development due to various adverse events associated with increased exposures of their substrates. Regulatory guidance documents on the in vitro assessment of OATP1B1/1B3 inhibition potential are conservative with up to a third of predictions resulting in false positives. This work investigated the utility of OATP1B1/1B3 endogenous biomarkers, coproporphyrin (CP)-I and CP-III, to assess clinical inhibition of OATP1B1/1B3 and potentially eliminate the need for prospective clinical drug-drug interaction (DDI) studies. Correlations between CP-I exposures and various OATP1B1 static DDI predictions were also evaluated. Glecaprevir/pibrentasvir (GLE/PIB) 300/120 mg fixed-dose combination is known to cause clinical inhibition of OATP1B1/1B3. In a clinical study evaluating the relative bioavailability of various formulations of GLE/PIB regimen, CP-I peak plasma concentration (Cmax ) ratio and 0-16-hour area under the concentration-time curve (AUC0-16 ) ratio relative to baseline increased with increasing GLE exposures, whereas there was a modest correlation between GLE exposure and CP-III Cmax ratio but no correlation with CP-III AUC0-16 ratio. This suggests that CP-I is superior to CP-III as an endogenous biomarker for evaluation of OATP1B1 inhibition. There was a significant correlation between CP-I and GLE Cmax (R2  = 0.65; P < 0.001) across individual subjects. Correlation analysis between GLE OATP1B1 R values and CP-I exposures (Cmax ratio and AUC0-16 ratio) suggests that an R value of > 3 can predict a biologically meaningful inhibition of OATP1B1 when the inhibitor clinical pharmacokinetic parameters are available.

Transporter Activity Changes in Nonalcoholic Steatohepatitis: Assessment with Plasma Coproporphyrin I and III.

Expression and functional changes in the organic anion transporting polypeptide (OATP)-multidrug resistance-associated protein (MRP) axis of transporters are well reported in patients with nonalcoholic steatohepatitis (NASH). These changes can impact plasma and tissue disposition of endo- and exogenous compounds. The transporter alterations are often assessed by administration of a xenobiotic or by transporter proteomic analysis from liver biopsies. Using gene expression, proteomics, and endogenous biomarkers, we show that the gene expression and activity of OATP and MRP transporters are associated with disease progression and recovery in humans and in preclinical animal models of NASH. Decreased OATP and increased MRP3/4 gene expression in two cohorts of patients with steatosis and NASH, as well as gene and protein expression in multiple NASH rodent models, have been established. Coproporphyrin I and III (CP I and III) were established as substrates of MRP4. CP I plasma concentration increased significantly in four animal models of NASH, indicating the transporter changes. Up to a 60-fold increase in CP I plasma concentration was observed in the mouse bile duct-ligated model compared with sham controls. In the choline-deficient amino acid-defined high-fat diet (CDAHFD) model, CP I plasma concentrations increased by >3-fold compared with chow diet-fed mice. In contrast, CP III plasma concentrations remain unaltered in the CDAHFD model, although they increased in the other three NASH models. These results suggest that tracking CP I plasma concentrations can provide transporter modulation information at a functional level in NASH animal models and in patients. SIGNIFICANCE STATEMENT: Our analysis demonstrates that multidrug resistance-associated protein 4 (MRP4) transporter gene expression tracks with nonalcoholic steatohepatitis (NASH) progression and intervention in patients. Additionally, we show that coproporphyrin I and III (CP I and III) are substrates of MRP4. CP I plasma and liver concentrations increase in different diet- and surgery-induced rodent NASH models, likely explained by both gene- and protein-level changes in transporters. CP I and III are therefore potential plasma-based biomarkers that can track NASH progression in preclinical models and in humans.

PBPK Model of Coproporphyrin I: Evaluation of the Impact of SLCO1B1 Genotype, Ethnicity, and Sex on its Inter-Individual Variability.

Coproporphyrin I (CPI) is an endogenous biomarker of OATP1B activity and associated drug-drug interactions. In this study, a minimal physiologically-based pharmacokinetic model was developed to investigate the impact of OATP1B1 genotype (c.521T>C), ethnicity, and sex on CPI pharmacokinetics and interindividual variability in its baseline. The model implemented mechanistic descriptions of CPI hepatic transport between liver blood and liver tissue and renal excretion. Key model parameters (e.g., endogenous CPI synthesis rate, and CPI hepatic uptake clearance) were estimated by fitting the model simultaneously to three independent CPI clinical datasets (plasma and urine data) obtained from white (n = 16, men and women) and Asian-Indian (n = 26, all men) subjects, with c.521 variants (TT, TC, and CC). The optimized CPI model successfully described the observed data using c.521T>C genotype, ethnicity, and sex as covariates. CPI hepatic active was 79% lower in 521CC relative to the wild type and 42% lower in Asian-Indians relative to white subjects, whereas CPI synthesis was 23% higher in male relative to female subjects. Parameter sensitivity analysis showed marginal impact of the assumption of CPI synthesis site (blood or liver), resulting in comparable recovery of plasma and urine CPI data. Lower magnitude of CPI-drug interaction was simulated in 521CC subjects, suggesting the risk of underestimation of CPI-drug interaction without prior OATP1B1 genotyping. The CPI model incorporates key covariates contributing to interindividual variability in its baseline and highlights the utility of the CPI modeling to facilitate the design of prospective clinical studies to maximize the sensitivity of this biomarker.

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.

Detection of Weak Organic Anion-Transporting Polypeptide 1B Inhibition by Probenecid with Plasma-Based Coproporphyrin in Humans.

Probenecid (PROB) is a clinical probe inhibitor of renal organic anion transporter (OAT) 1 and OAT3 that inhibits in vitro activity of hepatic drug transporters OATP1B1 and OATP1B3. It was hypothesized that PROB could potentially affect the disposition of OATP1B drug substrates. The plasma levels of the OATP1B endogenous biomarker candidates, including coproporphyrin I (CPI), CPIII, hexadecanedioate (HDA), and tetradecanedioate (TDA), were examined in 14 healthy subjects treated with PROB. After oral administration with 1000 mg PROB alone and in combination with furosemide (FSM), AUC (0-24 h) values were 1.39 ± 0.21-fold and 1.57 ± 0.41-fold higher than predose levels for CPI and 1.34 ± 0.16-fold and 1.45 ± 0.57-fold higher for CPIII. Despite increased systemic exposures, no decreases in CPI and CPIII renal clearance were observed (0.97 ± 0.38-fold and 1.16 ± 0.51-fold for CPI, and 1.34 ± 0.53-fold and 1.50 ± 0.69-fold for CPIII, respectively). These results suggest that the increase of CP systemic exposure is caused by OATP1B inhibition. Consistent with this hypothesis, PROB inhibited OATP1B1- and OATP1B3-mediated transport of CPI in a concentration-dependent manner, with IC50 values of 167 ± 42.0 and 76.0 ± 17.2 µM, respectively, in transporter-overexpressing human embryonic kidney cell assay. The inhibition potential was further confirmed by CPI and CPIII hepatocyte uptake experiments. In contrast, administration of PROB alone did not change AUC (0-24 h) of HDA and TDA relative to prestudy levels, although the administration of PROB in combination with FSM increased HDA and TDA levels compared with FSM alone (1.02 ± 0.18-fold and 0.90 ± 0.20-fold vs. 1.71 ± 0.43-fold and 1.62 ± 0.40-fold). Taken together, these findings indicate that PROB displays weak OATP1B inhibitory effects in vivo and that coproporphyrin is a sensitive endogenous probe of OATP1B inhibition. This study provides an explanation for the heretofore unknown mechanism responsible for PROB's interaction with other xenobiotics. SIGNIFICANCE STATEMENT: This study suggested that PROB is a weak clinical inhibitor of OATP1B based on the totality of evidence from the clinical interaction between PROB and CP and the in vitro inhibitory effect of PROB on OATP1B-mediated CP uptake. It demonstrates a new methodology of utilizing endogenous biomarkers to evaluate complex drug-drug interaction, providing explanation for the heretofore unknown mechanism responsible for PROB's inhibition. It provides evidence to strengthen the claim that CP is a sensitive circulating endogenous biomarker of OATP1B inhibition.

Simultaneous quantification of coproporphyrin-I and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry.

In chronic kidney disease (CKD), organic anion transporting polypeptide (OATP)1B activity is reduced by mechanisms involving 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), a uremic toxin. Coproporphyrin-I (CP-I) is a sensitive and specific endogenous probe for phenotyping OATP1B activity and a potentially useful tool to individualize the dosage of OATP1B substrates. In this study, we developed and validated an assay for simultaneous quantification of CP-I and CMPF in human plasma using ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). The samples were prepared by solid phase extraction, and then subjected to UHPLC-MS/MS quantification. The assay fulfilled the requirements of the US Food and Drug Administration (FDA) guideline for assay validation, with a lower limit of quantification of 0.1 for CP-I and 50 ng/mL for CMPF. Recovery rates from human plasma ranged from 97.3%-109.8% for CP-I, and 94.1%-113.3% for CMPF. Matrix effects corrected by internal standards varied between 107.2 % and 119.3 % for CP-I, and between 90.4 % and 107.4 % for CMPF. The validated assay was applied to measurement of plasma CP-I and CMPF concentrations in 10 healthy volunteers, 14 stage 3-5 CKD patients, and 14 stage 5D CKD patients. The concentrations measured in all samples were within the calibration ranges. Our novel method may be clinically useful for simultaneous measurement of plasma CP-I and CMPF concentrations in human samples, and contribute to reveal the in vivo relationship of OATB1B activity with accumulation of CMPF in CKD patients.

Organic Anion Transporter Polypeptide 1B1 Polymorphism Modulates the Extent of Drug-Drug Interaction and Associated Biomarker Levels in Healthy Volunteers.

Understanding transporter-mediated drug-drug interactions is an integral part of risk assessment in drug development. Recent studies support the use of hexadecanedioate (HDA), tetradecanedioate (TDA), coproporphyrin (CP)-I, and CP-III as clinical biomarkers for evaluating organic anion-transporting polypeptide (OATP)1B1 (SLCO1B1) inhibition. The current study investigated the effect of OATP1B1 genotype c.521T>C (OATP1B1-Val174Ala) on the extent of interaction between cyclosporin A (CsA) and pravastatin, and associated endogenous biomarkers of the transporter (HDA, TDA, CP-I, and CP-III), in 20 healthy volunteers. The results show that the levels of each clinical biomarker and pravastatin were significantly increased in plasma samples of the volunteers following administration of pravastatin plus CsA compared with pravastatin plus placebo. The overall fold change in the area under the concentration-time curve (AUC) and maximum plasma concentration (Cmax ) was similar among the four biomarkers (1.8-2.5-fold, paired t-test P value < 0.05) in individuals who were homozygotes or heterozygotes of the major allele, c.521T. However, the fold change in AUC and Cmax for HDA and TDA was significantly abolished in the subjects who were c.521-CC, whereas the respective fold change in AUC and Cmax for pravastatin and CP-I and CP-III were slightly weaker in individuals who were c.521-CC compared with c.521-TT/TC genotypes. In addition, this study provides the first evidence that SLCO1B1 c.521T>C genotype is significantly associated with CP-I but not CP-III levels. Overall, these results suggest that OATP1B1 genotype can modulate the effects of CsA on biomarker levels; the extent of modulation differs among the biomarkers.

Recovery of OATP1B Activity after Living Kidney Transplantation in Patients with End-Stage Renal Disease.

PURPOSE: Recently, several studies have shown that renal failure decreases the metabolic clearance of drugs and the transportation capability of some drug transporters. However, whether organic anion transporting polypeptide (OATP)1B activities decrease in renal failure remains unknown. In this study, we measured plasma concentrations of coproporphyrin-I (CP-I), a specific endogenous OATP1B probe, in patients with end stage renal disease before and after living kidney transplantation and evaluated the effect of renal function on OATP1B activity. METHODS: This prospective study recruited 13 patients with end-stage renal disease. Plasma CP-I concentrations were measured before and 7, 14, 30 and 90 days after living kidney transplantation. RESULTS: Plasma CP-I concentrations decreased over time after living kidney transplantation and showed significant difference on day 90 compared with before living kidney transplantation [1.12 ± 0.59 vs 0.65 ± 0.27 ng/mL, p < 0.05 (95% CI of difference - 0.927, -0.013)]. A significant negative correlation was observed between estimated glomerular filtration rate and plasma CP-I concentration (r = -0.30, p < 0.05), suggesting recovery of OATP1B activity with improvement in renal function. CONCLUSIONS: OATP1B activity may decrease in renal failure and dose adjustment of OATP1B substrates may be needed in patients with renal failure.

Comprehensive Evaluation of the Utility of 20 Endogenous Molecules as Biomarkers of OATP1B Inhibition Compared with Rosuvastatin and Coproporphyrin I.

Endogenous biomarkers can be clinically relevant tools for the assessment of transporter function in vivo and corresponding drug-drug interactions (DDIs). The aim of this study was to perform systematic evaluation of plasma data obtained for 20 endogenous molecules in the same healthy subjects (n = 8-12) in the absence and presence of organic anion transporting polypeptide (OATP) inhibitor rifampicin (600 mg, single dose). The extent of rifampicin DDI magnitude [the ratio of the plasma concentration-time area under the curve (AUCR)], estimated fraction transported (fT), and baseline variability was compared across the biomarkers and relative to rosuvastatin and coproporphyrin I (CPI). Out of the 20 biomarkers investigated tetradecanedioate (TDA), hexadecanedioate (HDA), glycocholic acid, glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA), and coproporphyrin III (CPIII) showed the high AUCR (2.1-8.5) and fT (0.5-0.76) values, indicative of substantial OATP1B-mediated transport. A significant positive correlation was observed between the individual GDCA and TDCA AUCRs and the magnitude of rosuvastatin-rifampicin interaction. The CPI and CPIII AUCRs were significantly correlated, but no clear trend was established with the rosuvastatin AUCR. Moderate interindividual variability (15%-62%) in baseline exposure and AUCR was observed for TDA, HDA, and CPIII. In contrast, bile acids demonstrated high interindividual variability (69%-113%) and significant decreases in baseline plasma concentrations during the first 4 hours. This comprehensive analysis in the same individuals confirms that none of the biomarkers supersede CPI in the evaluation of OATP1B-mediated DDI risk. Monitoring of CPI and GDCA/TDCA may be beneficial for dual OATP1B/sodium-taurocholate cotransporting polypeptide inhibitors with consideration of challenges associated with large inter- and intraindividual variability observed for bile acids. Benefit of monitoring combined biomarkers (CPI, one bile acid and one fatty acid) needs to be confirmed with larger data sets and against multiple OATP1B clinical probes and perpetrators.

PBPK Modeling of Coproporphyrin I as an Endogenous Biomarker for Drug Interactions Involving Inhibition of Hepatic OATP1B1 and OATP1B3.

The aim of the present study was to establish a physiologically based pharmacokinetic (PBPK) model for coproporphyrin I (CP-I), a biomarker supporting the prediction of drug-drug interactions (DDIs) involving hepatic organic anion transporting polypeptide 1B (OATP1B), using clinical DDI data with an OATP1B inhibitor rifampicin (300 and 600 mg, orally). The in vivo inhibition constants of rifampicin used as initial input parameters for OATP1Bs (Ki,u,OATP1Bs ) and multidrug resistance-associated protein two-mediated biliary excretion were estimated as 0.23 and 0.87 µM, respectively, from previous reports. Sensitivity analysis demonstrated that the Ki,u,OATP1Bs and biosynthesis rate of CP-I affected the magnitude of the interaction. Ki,u,OATP1Bs values optimized by nonlinear least-squares fitting were ~0.5-fold of the initial value. It was determined that the blood concentration-time profiles of four statins were well-predicted using corrected individual Ki,u,OATP1B values (ratio of in vitro Ki,u(statin) /in vitro Ki,u(CP-I) ). In conclusion, PBPK modeling of CP-I supports dynamic prediction of OATP1B-mediated DDIs.