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Ezetimibe undergoes glucuronidation that results in the active metabolite ezetimibe phenoxy-glucuronide (ezetimibe-glucuronide). This phase-II metabolite was shown to interact with the clinically relevant hepatic transporter organic anion transporting polypeptide (OATP) 1B1. In recent years, coproporphyrin I (CPI) was established as a Tier 1 biomarker for OATP1B-mediated interactions among other endogenous substrates like CPIII. To evaluate whether levels of the biomarker are affected by ezetimibe treatment, we assessed the impact of ezetimibe and ezetimibe-glucuronide on OATP1B1-mediated transport of CPs in vitro. Then, we quantified CP levels in serum samples of healthy volunteers treated with a single oral dose of ezetimibe (20 mg) alone or in combination with rifampin (600 mg). Results from our in vitro experiments showed a significant reduction in cellular CPI accumulation in the presence of ezetimibe-glucuronide with an IC 50 of 1.97 μM [95% CI: 1.04 to 3.96], while CPIII accumulation was impacted by 10 μM and above. In the in vivo study, we observed peak CP concentrations 1.33 h after dosing, which is closest to the t max of the ezetimibe metabolite. Co-administration of ezetimibe with rifampin resulted in even higher serum CP levels. The AUC 0-24h of CPI and CPIII increased two- and threefold, respectively, after concomitant dosing compared to ezetimibe alone. Moreover, we quantified CP levels in cumulative urine from both study phases where the renally excreted amount (A e ) of CPI and CPIII increased after ezetimibe and rifampin co-administration compared to ezetimibe alone. In conclusion, our findings indicate that rifampin co-administration results in additional inhibition of OATP1B1 in vivo., (© 2024 The Author(s). Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

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 (AUC 0-8h ) 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 IC 50 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., (© 2024 The Author(s). Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 OST α / β -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., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

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 (IC 50 )). 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 IC 50 values and unbound liver inlet concentrations (I in,max,u ), suggested in vivo OATP1B inhibition risk for drugs with R-value (1+ I in,max,u /IC 50 ) 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 IC 50 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., (© 2021 The Authors. Clinical Pharmacology & Therapeutics © 2021 American Society for Clinical Pharmacology and Therapeutics.)

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., (© 2021 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

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., (© 2021 The Authors. Clinical Pharmacology & Therapeutics © 2021 American Society for Clinical Pharmacology and Therapeutics.)

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 (CL renal ) 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 CL renal 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 CL renal 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 CL renal of CP-III, it would be better to investigate the impact of the CL renal on plasma exposure of CP-III during clinical DDI assessments., (© 2021, The American College of Clinical Pharmacology.)

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; r s  = 0.35, p < 0.0001) and with OATP1B1*15 allele (n = 126; r s  =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., (© 2021 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

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., (© 2020 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

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 (C max ) ratio and 0-16-hour area under the concentration-time curve (AUC 0-16 ) ratio relative to baseline increased with increasing GLE exposures, whereas there was a modest correlation between GLE exposure and CP-III C max ratio but no correlation with CP-III AUC 0-16 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 C max (R 2  = 0.65; P < 0.001) across individual subjects. Correlation analysis between GLE OATP1B1 R values and CP-I exposures (C max ratio and AUC 0-16 ratio) suggests that an R value of > 3 can predict a biologically meaningful inhibition of OATP1B1 when the inhibitor clinical pharmacokinetic parameters are available., (© 2020 Abbvie Inc. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

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., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

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., (© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

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, 4 β -hydroxycholesterol (4 β HC), 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 4 β HC. 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., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

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 IC 50 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., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 (C max ) 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 C max for HDA and TDA was significantly abolished in the subjects who were c.521-CC, whereas the respective fold change in AUC and C max 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., (© 2019 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

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.

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 (f T ), 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 f T (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., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Introduction: Coproporphyrin (CP) I and III have recently been proposed as endogenous clinical biomarkers to predict organic anion-transporting polypeptide 1B (OATP1B)-mediated drug-drug interactions (DDIs). In the present study, we first investigated the in vitro selectivity of CPI and CPIII towards drug uptake and efflux transporters. We then assessed the in vivo biomarker sensitivity towards OATP1B inhibition., Methods: To assess transporter selectivity, incubations with CPI and CPIII were performed in vitro, using single transporter-expressing and control systems. Furthermore, CPI and CPIII plasma concentrations were determined from participants of three independent clinical trials who were administered with either a strong, moderate, or mild clinical OATP1B inhibitor., Results: Our results show that CPI and CPIII are substrates of OATP1B1, OATP1B3, the multidrug resistance-associated protein (MRP) 2, and MRP3. No substrate interaction was shown for other prominent drug transporters that have been associated with clinical DDIs. Results from clinical studies demonstrated that changes in CPI and CPIII plasma levels were predictive for moderate (two to threefold area under the concentration-time curve [AUC] increase) and strong (≥ fivefold increases) clinical OATP1B inhibition. Furthermore, CPI, but not CPIII, concentration changes were predictive for a mild clinically observed DDI where CPI AUC increases of 1.4-fold were comparable with those observed for pitavastatin as victim drug (AUC increases of 1.5-fold)., Conclusion: Our results demonstrate the selectivity of CPI and CPIII towards the OATP1B/MRP pathway, and the herein reported data further underline the potential of CPI and CPIII as selective and sensitive clinical biomarkers to quantify OATP1B-mediated DDIs.

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 (K i,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 K i,u,OATP1Bs and biosynthesis rate of CP-I affected the magnitude of the interaction. K i,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 K i,u,OATP1B values (ratio of in vitro K i,u(statin) /in vitro K i,u(CP-I) ). In conclusion, PBPK modeling of CP-I supports dynamic prediction of OATP1B-mediated DDIs., (© 2018 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

This study evaluated coproporphyrin I (CPI) as a selective endogenous biomarker of OATP1B-mediated drug-drug interactions (DDIs) relative to clinical probe rosuvastatin using nonlinear mixed-effect modeling. Plasma and urine CPI data in the presence/absence of rifampicin were modeled to describe CPI synthesis, elimination clearances, and obtain rifampicin in vivo OATP Ki. The biomarker showed stable interoccasion baseline concentrations and low interindividual variability (<25%) in subjects with wildtype SLCO1B1. Biliary excretion was the dominant CPI elimination route (maximal >85%). Estimated rifampicin in vivo unbound OATP Ki (0.13 μM) using CPI data was 2-fold lower relative to rosuvastatin. Model-based simulations and power calculations confirmed sensitivity of CPI to identify moderate and weak OATP1B inhibitors in an adequately powered clinical study. Current analysis provides the most detailed evaluation of CPI as an endogenous OATP1B biomarker to support optimal DDI study design; further pharmacogenomic and DDI data with a panel of inhibitors are required., (© 2017 The Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Background: An increase in naturally-occurring porphyrins has been described in the blood of subjects bearing different kinds of tumors, including colorectal, and this is probably related to a systemic alteration of heme metabolism induced by tumor cells. The aim of our study was to develop an artificial neural network (ANN) classifier for early detection of colorectal adenocarcinoma based on plasma porphyrin accumulation and risk factors., Methods: We measured the endogenous fluorescence of blood plasma in 100 colorectal adenocarcinoma patients and 112 controls using a conventional spectrofluorometer. Height, weight, personal and family medical history, use of alcohol, red meat, vegetables and tobacco were all recorded. An ANN model was built up from demographic data and from the integral of the fluorescence emission peak in the range 610-650 nm. We used the Receiver Operating Characteristic (ROC) curve to assess performance in distinguishing colorectal adenocarcinoma patients and controls. A liquid chromatography-high resolution mass spectrometry (LC-HRMS) analytical method was employed to identify the agents responsible for native fluorescence., Results: The fluorescence analysis indicated that the integral of the fluorescence emission peak in the range 610-650 nm was significantly higher in colorectal adenocarcinoma patients than controls (p < 0.0001) and was weakly correlated with the TNM staging (Spearman's rho = 0.224, p = 0.011). LC-HRMS measurements showed that the agents responsible for the fluorescence emission were mainly protoporphyrin-IX (PpIX) and coproporphyrin-I (CpI). The overall accuracy of our ANN model was 88% (87% sensitivity and 90% specificity) with an area under the ROC curve of 0.83., Conclusions: These results confirm that tumor cells accumulate a diagnostic level of endogenous porphyrin compounds and suggest that plasma porphyrin concentrations, indirectly measured through fluorescence analysis, may be useful, together with risk factors, as a clinical decision support tool for the early detection of colorectal adenocarcinoma. Our future efforts will be aimed at examining how plasma porphyrin accumulation correlates with survival and response to therapy.

Aim: Coproporphyrins (CP-I and CP-III) have been identified as possible biomarkers to predict human hepatic organic anion-transporting polypeptides-mediated-drug-interactions for a new drug entering clinical development., Results: The method is applicable to quantify plasma CP-I and CP-III within 0.078-15.0 nM. The results identify and address a number of challenges encountered with porphyrin assays such as photodegradation and interferences. To overcome interferences from ubiquitous porphyrins, a surrogate matrix was used to prepare calibration standards. Quality controls were prepared in plasma and surrogate matrix to ensure parallelism between surrogate matrix and plasma., Conclusion: A robust UHPLC-MS/MS assay was developed and validated for CP-I and CP-III in plasma, and is currently applied to clinical studies to confirm suitability of Coproporphyrins as a potential substitute for drug-drug interaction study.

Aim: A validated LC-MS/MS assay for the quantitation of coproporphyrin-I and -III (CP-I, CP-III) in human plasma has been developed to understand the utility of both as possible endogenous biomarkers for organic anion-transporting polypeptides (OATP)-mediated drug-drug interactions (DDIs)., Materials and Methods:  Human plasma extracts were analyzed for CP-I and CP-III using a Sciex API 6500+ mass spectrometer. Results: The assay was utilized for plasma samples from a clinical DDI study involving a new chemical entity that presented as an OATP inhibitor in vitro. A formal DDI study, with a probe drug (atorvastatin), was also included as part of the clinical study., Conclusion: Changes in CP-I area under the plasma concentration versus time curve (AUC 0-48 h ) were observed, which were similar to the AUC ratio obtained with atorvastatin. These results support the idea that plasma CP-I may have utility in Phase I by supporting the rapid assessment of OATP inhibition risk.

Coproporphyrins are proposed as endogenous biomarkers of hepatic Organic Anion Transporting Polypeptide (OATP)1B functional activity. In this study, a new sample extraction method based on a mixed-mode anion exchange sorbent (SPE clean-up using Oasis 30mg Max 96 well plates) was developed for absolute quantification of coproporphyrin I and III (CP-I and CP-III) in human plasma. Chromatographic separation was performed with an Ace Excel 2 C18 PFP, 3μm, 2.1×150mm, maintained at 60°C. A 10mM ammonium formate containing 0.1% HCOOH and acetonitrile (100%) was used as mobile phase A and B, respectively. Mass transition, m/z 655.3→596.3 was selected to monitor CP-I and CP-III, while m/z 659.3→600.3 transition was used for the stable isotope labelled internal standard. Optimization of the liquid chromatography tandem mass spectrometry method ensured a lower limit of quantification (LLOQ) of 20pg/mL. Both CP-I and CP-III had extraction recoveries of 70%. The calibration range was 0.02-100ng/mL for both CP-I and CP-III, yielding calibration curves with correlation coefficients greater than 0.988. Inter day precision (CV<9%) and accuracy (84.3-103.9%) complied with the recommendation of the European Bioanalytical Forum. The optimized method was used to analyse plasma samples originating from three independent clinical studies. Obtained CP-I and CP-III plasma baseline levels in healthy volunteers were in good agreement with previously published data. Moreover, CP-I and CP-III plasma levels in human subjects dosed with a clinically confirmed OATP inhibitor were significantly increased compared to their baseline levels. These data demonstrate the potential of CP-I and CP-III as endogenous biomarkers to predict the drug-drug interaction (DDI) related to hepatic OATP1B inhibition. Stability of CP-I and CP-III in plasma and solvents under different processing and storage conditions was also evaluated., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aim: Coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) in plasma and urine have been proposed as biomarkers for assessing drug-drug interactions involving hepatic drug transporters such as organic anion-transporting peptides (OATP), 1B1 and 1B3. Materials & methods: Plasma and urine extracts were analyzed for CP-I/CP-III using a TripleTOF API6600 mass spectrometer. Results: Previously unreported, CP-I/CP-III doubly charged ions (m/z 328.14) were used as precursor ions to improve the assay sensitivity and selectivity over the singly charged precursor ions (m/z 655.28). Levels of CP-I and CP-III measured ranged 0.45-1.1 and 0.050-0.50 ng/ml in plasma and 5-35 and 1-35 ng/ml in urine, respectively., Conclusion: The described highly selective and sensitive CP-I/CP-III LC-HRMS assay offers options for earlier characterization and clinical safety projections for OATP1B1/3-mediated drug-drug interactions along with pharmacokinetic analyses of a new chemical entity as part of first-in-human clinical studies.

Multiple endogenous compounds have been proposed as candidate biomarkers to monitor organic anion transporting polypeptide (OATP) function in preclinical species or humans. Previously, we demonstrated that coproporphyrins (CPs) I and III are appropriate clinical markers to evaluate OATP inhibition and recapitulate clinical drug-drug interactions (DDIs). In the present study, we investigated bile acids (BAs) dehydroepiandrosterone sulfate (DHEAS), hexadecanedioate (HDA), and tetradecanedioate (TDA) in plasma as endogenous probes for OATP inhibition and compared these candidate probes to CPs. All probes were determined in samples from a single study that examined their behavior and their association with rosuvastatin (RSV) pharmacokinetics after administration of an OATP inhibitor rifampin (RIF) in healthy subjects. Among endogenous probes examined, RIF significantly increased maximum plasma concentration ( C max ) and area under the concentration-time curve (AUC) (0-24h) of fatty acids HDA and TDA by 2.2- to 3.2-fold. For the 13 bile acids in plasma examined, no statistically significant changes were detected between treatments. Changes in plasma DHEAS did not correlate with OATP1B inhibition by RIF. On the basis of the magnitude of effects for the endogenous compounds that demonstrated significant changes from baseline over interindividual variations, the overall rank order for the AUC change was found to be CP I > CP III > HDA ≈ TDA ≈ RSV > > BAs. Collectively, these results reconfirmed that CPs are novel biomarkers suitable for clinical use. In addition, HDA and TDA are useful for OATP functional assessment. Since these endogenous markers can be monitored in conjunction with pharmacokinetics analysis, the CPs and fatty acid dicarboxylates, either alone or in combination, offer promise of earlier diagnosis and risk stratification for OATP-mediated DDIs., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

In the present study, an open-label, three-treatment, three-period clinical study of rosuvastatin (RSV) and rifampicin (RIF) when administered alone and in combination was conducted in 12 male healthy subjects to determine if coproporphyrin I (CP-I) and coproporphyrin III (CP-III) could serve as clinical biomarkers for organic anion transporting polypeptide 1B1 (OATP1B1) and 1B3 that belong to the solute carrier organic anion gene subfamily. Genotyping of the human OATP1B1 gene was performed in all 12 subjects and confirmed absence of OATP1B1*5 and OATP1B1*15 mutations. Average plasma concentrations of CP-I and CP-III prior to drug administration were 0.91 ± 0.21 and 0.15 ± 0.04 nM, respectively, with minimum fluctuation over the three periods. CP-I was passively eliminated, whereas CP-III was actively secreted from urine. Administration of RSV caused no significant changes in the plasma and urinary profiles of CP-I and CP-III. RIF markedly increased the maximum plasma concentration (Cmax) of CP-I and CP-III by 5.7- and 5.4-fold (RIF) or 5.7- and 6.5-fold (RIF+RSV), respectively, as compared with the predose values. The area under the plasma concentration curves from time 0 to 24 h (AUC0-24h) of CP-I and CP-III with RIF and RSV increased by 4.0- and 3.3-fold, respectively, when compared with RSV alone. In agreement with this finding, Cmax and AUC0-24h of RSV increased by 13.2- and 5.0-fold, respectively, when RIF was coadministered. Collectively, we conclude that CP-I and CP-III in plasma and urine can be appropriate endogenous biomarkers specifically and reliably reflecting OATP inhibition, and thus the measurement of these molecules can serve as a useful tool to assess OATP drug-drug interaction liabilities in early clinical studies., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Inhibition of organic anion-transporting polypeptide (OATP)1B function can lead to serious clinical drug-drug interactions, thus a thorough evaluation of the potential for this type of interaction must be completed during drug development. Therefore, sensitive and specific biomarkers for OATP function that could be used in conjunction with clinical studies are currently in demand. In the present study, preclinical evaluations were conducted to characterize the suitability of coproporphyrins (CPs) I and III as markers of hepatic OATP functional activity. Active uptake of CPs I and III was observed in human embryonic kidney (HEK) 293 cells singly expressing human OATP1B1 (hOATP1B1), hOATP1B3, cynomolgus monkey OATP1B1 (cOATP1B1), or cOATP1B3, as well as human and monkey hepatocytes. Cyclosporin A (100 mg/kg, oral) markedly increased the area under the curve (AUC) plasma concentrations of CPs I and III by 2.6- and 5.2-fold, while rifampicin (15 mg/kg, oral) increased the AUCs by 2.7- and 3.6-fold, respectively. As the systemic exposure increased, the excretion of both isomers in urine rose from 1.6- to 4.3-fold in monkeys. In agreement with this finding, the AUC of rosuvastatin (RSV) in cynomolgus monkeys increased when OATP1B inhibitors were coadministered. In Oatp1a/1b gene cluster knockout mice (Oatp1a/1b(-/-)), CPs in plasma and urine were significantly increased compared with wild-type animals (7.1- to 18.4-fold; P < 0.001), which were also in agreement with the changes in plasma RSV exposure (14.6-fold increase). We conclude that CPs I and III in plasma and urine are novel endogenous biomarkers reflecting hepatic OATP function, and the measurements have the potential to be incorporated into the design of early clinical evaluation., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

A 31-year-old woman presented with recurrent swelling, burning, prickling and itching of the arms and the hands which appeared after UV exposure in the summer. Simultaneously she often had chills, malaise, nausea and circulatory problems lasting for hours. She did not have erythema or wheals. She had been seen by a number of specialties but to no avail. Her deceased mother suffered from the same symptoms. On presentation she had no skin lesions. Laboratory testing showed a dramatic increase in total protoporphyrin, enabling us to diagnose erythropoietic protoporphyria. This is a rare, genetic metabolic disturbance in hematopoiesis, whose true prevalence is probably underestimated. Because of abnormal or absent function of ferrochelatase, protoporphyrin accumulates in blood, erythrocytes and tissue usually causing photosensitivity as the first clinical symptom. The prognosis depends on the severity of liver involvement. Because of the marked restrictions on activity, especially in avoiding UV exposition, the quality of life of the patients and their families are strongly influenced. The odyssey of our patient and her mother in finding a diagnosis demonstrates impressively that the EPP is an important photosensitizing disease which must not be forgotten.

The species and concentrations of porphyrins in erythrocytes are of great importance in the clinical screening and diagnosis of porphyrias. However, it is difficult to analyze them simultaneously by conventional spectrofluorimetry. In this paper, we proposed a novel, simple and rapid method for the simultaneous determination of three diagnostically important porphyrins in human erythrocytes, protoporphyrin IX (PP), coproporphyrin III (CP) and zinc protoporphyrin IX (ZnPP), using hyphenated techniques based on derivative matrix isopotential synchronous fluorescence spectrometry and nonlinear variable-angle synchronous fluorescence spectrometry (DMI-NLVASFS). The spectral overlapping problems were well resolved and these three components were determined in one scanning without spectral compensation factors and chromatographic separation. The detection limits were 0.58, 0.21, 0.05 nmol L(-1) for PP, CP and ZnPP, respectively. Only 30s was needed for a single scanning and the recoveries were from 73% to 105% in erythrocytes. The Bland-Altman analysis indicates no significant difference between the proposed DMI-NLVASFS method and conventional fluorimetry method. The PP level of the erythropoietic protoporphyria (EPP) patients was significantly higher than that of healthy volunteers. This method can determine PP, CP and ZnPP simultaneously in a single scanning, thus providing a potential tool for the clinical analysis of porphyrins in human erythrocytes and the differential diagnosis of porphyrias., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Fluorescence spectroscopy provides high sensitivity in quantitative analysis. However, due to spectral interference, it is difficult to determine the individual components of fluorescent multi-component mixtures in such complicated and important body matrices as blood, urine and feces without any pre-separation. In this study, a simple and rapid approach based on non-linear variable-angle synchronous fluorescence spectrometry coupled with partial least squares analysis (NLVASF/PLS) was developed for the simultaneous determination of protoporphyrin IX (PP), uroporphyrin III (UP) and coproporphyrin III (CP). The detection limits were 0.18, 0.29 and 0.24 nmol L(-1) for protoporphyrin IX (PP), uroporphyrin III (UP) and coproporphyrin III (CP), respectively. The individual components of blood porphyrins were quantified, by this method, simultaneously in one scan with only about 30s. The recoveries of this method were above 80% in human whole blood samples. This method provided a potential tool for the determination of porphyrins in whole blood and the differential diagnosis of porphyria, especially for rapid routine screening of large number of samples., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Two Thai women who are siblings presented with a history of recurrent pruritic vesicles on dorsum of both hands and extensor surface of forearms where the sun-exposed areas are. The excoriated vesicles were healed with depressed scars. They had no previous history of intense abdominal pain, seizure, or psychiatric disorder Urinary porphyrins were analyzed by High Performance Liquid Chromatography (HPLC). The level of coproporphyrin III was detected to be higher than the uroporphyrin level. Fluorescence emission scanning of both patients' plasma was performed and demonstrated typical emission peak at 626 nm, that confirmed the diagnosis of variegate porphyria.

50 Patients with chronic renal failure undergoing hemodialysis with or without porphyria cutanea tarda (PCT)-like skin changes were investigated. The total porphyrin amount in erythrocytes, plasma and dialysate and the distribution of porphyrin metabolites in plasma and dialysate were measured. In plasma, the group of patients with skin changes (referred as PCU = porphyria cutanea uremica) showed significantly increased uroporphyrin levels as compared to the non-symptomatic group. In addition, significant differences concerning the ratio uro-/coproporphyrin in plasma were shown: non-symptomatic patients with 0.87, as opposed to the PCU group with 3.7. Considerable differences between the level of vitamin ingestion were identified between the groups. Patients with PCU took distinctly less vitamins C, E and B than patients without symptoms.

Background: Identification of porphyrias relies on the measurement of different porphyrins in urine, feces and plasma. Separation of porphyrin isomers is essential for the differential diagnosis of some porphyrias., Method: Separation of naturally occurring porphyrins was achieved on a Chromolith RP-18 column with fluorimetric detection using a methanol/ammonium acetate gradient mobile phase. Fecal and plasma porphyrins were extracted with acetonitrile and water at different pH values., Results: Eight porphyrins including protoporphyrin eluted within 20 min with good resolution of each of the I and III positional isomer pairs for standards, urine and plasma, and within 50 min for feces. Improvement of the extraction method for fecal and plasmatic porphyrins resulted in high recovery (up to 89%) and reliable quantification of protoporphyrin., Conclusions: The present RP-HPLC method is specific and efficient for routine analysis of porphyrins in human urine, feces and plasma.

The porphyrin compound in human body mainly includes hemoglobin, protoheme, protoporphyrin, coproporphyrin, uroporphyrin and so on. As we know, the metabolism of the porphyrin in human body depends on the physical conditions. It was found that the relative content of protoporphyrin (PPIX) and carotenoid in blood varied with the developing of the malignant tumor. A system, including Ti: sapphire laser, optical fiber delivery system, fluorescence probe, spectrometer CCD and computer, has been developed to examine the porphyrin in human superficial skin tissue without trauma. It was shown that the fluorescence spectrum of the porphyrin obtained from the superficial skin tissue of human earlobe has the same feature as that from the aqueous solution with PP IX.

We studied the effect of arsenic exposure on the haem biosynthetic pathway in the rat and humans. Significant increases in protoporphyrin IX, coproporphyrin III, coproporphyrin I were observed in the blood, liver and kidney, and in the urine of rats after a single dose of arsenic. The level of increase was dependent on the arsenic species present. Most of porphyrin concentrations in the tissues increased within 24 hr and urinary excretion elevated within 48 hr. In the human study, we collected urine samples from 113 people who live in Xing Ren of Guizhou Province, a coal-borne arsenicosis endemic area in southwest of PR China and from 30 people who live in Xing Yi (about 80 km southwest of Xing Ren) where arsenicosis is not prevalent. We analyzed the urinary porphyrins using HPLC. Results indicate that all urinary porphyrins were higher in the arsenic exposed group than those in the control group. Women, children and older age people spend much of their time indoors, they had greater increases of urinary arsenic and porphyrins. They were the higher risk groups among the study subjects. A positive correlation between the urinary arsenic levels and porphyrin concentrations demonstrated the effect of arsenic on haem biosynthesis. Significant alteration in the porphyrin excretion profiles of the younger age (<20 y) arsenic exposed group suggested that porphyrins could be used as early warning biomarkers for chronic exposure to arsenic.

We studied the antitumor effects of photodynamic therapy (PDT) with Zincphyrin, coproporphyrin III with zinc, derived from Streptomyces sp. AC8007, in vitro and in vivo. The photokilling effect of Zincphyrin in the presence of 0.78-100 microg/ml with visible light of 27.2 mW x min/cm2 for 10 min was lower than the hematoporphyrin (Hp) used as a control with L5178Y or sarcoma-180 cells. On the other hand, Zincphyrin apparently reduced tumor growth after intraperitoneal injection at doses of 12.5-50 mg/kg with light irradiation of 75.48 mW x min/cm2 for 10 min in sarcoma-180-bearing mice. Although no mice treated with Zincphyrin died, Hp did cause the death of mice. In B-16 melanoma-bearing mice, both Zincphyrin and Hp had a similar phototherapic effect. Further improvement of the phototherapic effect was observed with the continuous administration of Zincphyrin at 12.5 mg/kg per day for 3 days. The concentration of Zincphyrin in the serum reached a maximum level of 16 microg/ml within 20 min, and the concentration remained at 4.2 microg/ml at 1 hour after the onset of treatment, indicating its rapid action in the body. No animals died after the intraperitoneal administration of Zincphyrin at 100 mg/kg plus exposure to light of 10 mW x min/cm2 for 2 hours, and the body weight of the mice did not decrease. In contrast, all animals receiving 100 mg/kg of Hp under the same conditions died. These results indicate that Zincphyrin would be a useful photosensitizer with low phototoxicity.

Objective: To evaluate the usefulness of maternal plasma zinc-coproporphyrin (ZCP) level as a marker for intrauterine passage of meconium., Methods: A pilot study consisting of 10 pregnancies with meconium-stained amniotic fluid and 10 pregnancies with clear amniotic fluid was used. The corresponding plasma and amniotic fluid levels of ZCP were measured using spectrofluorometry. ZCP levels in plasma and amniotic fluid were compared between the two groups and the relation between plasma and amniotic fluid ZCP levels in the clear and meconium-stained groups was assessed using Spearman rank-order correlation., Results: Mean amniotic fluid ZCP was significantly higher in the meconium-stained amniotic fluid as compared to the clear amniotic fluid group. Although mean plasma ZCP levels were higher in the meconium-stained amniotic fluid group, this difference was not statistically significant. There was no significant correlation between plasma ZCP levels and amniotic fluid ZCP, but we could categorize patients according to plasma ZCP levels into four categories with different risks for having meconium-stained amniotic fluid., Conclusions: Plasma ZCP might be a promising test for prediction of intrauterine passage of meconium in high-risk patients if confirmed by larger studies. The implications of this prediction on management remain unknown.

We studied the accuracy and precision of a HPLC method for determination of porphyrins in urine. A commercial standard solution appeared to contain less porphyrins than indicated by the manufacturer, since calibration resulted in lower concentrations of uroporphyrin and coproporphyrin: 16% and 8%, respectively. Coefficients of variation for the measurement of uro-, hepta-, copro I and copro III porphyrins in samples of patients with and without porphyria were often much less than 15%. Comparison of measurements with and without calibrated standards revealed differences for uroporphyrin and coproporphyrin of 27% and 5%, respectively. Recovery of added uroporphyrin and coproporphyrin was 99%. The main cause of the variability in test results was apparently the improperly calibrated standard solutions. The precision of porphyrin measurements was not influenced by the type of porphyria.

A patient with porphyria cutanea tarda developed distinctive sclerodermoid changes with extensive dystrophic calcification and ulcerations in the sclerotic areas. In addition to these skin complications, over the course of 20 years other internal problems also appeared, such as diabetes mellitus, fatty cirrhosis and ocular inflammation. These severe clinical complications were related to omission of treatment and the alcohol abuse.

Objective: Blue light phototherapy is commonly administered to neonates as treatment of indirect hyperbilirubinemia, often in conjunction with blood transfusions to treat hemolytic anemia. We observed a distinctive cutaneous complication of phototherapy in six neonates with hyperbilirubinemia., Methodology: We studied the clinical and histologic characteristics of the eruption, as well as the porphyrin levels in affected neonates. Five of the patients had erythroblastosis fetalis; the other had profound anemia from twin-twin transfusion. All of the neonates developed purpuric patches at sites of maximal exposure to the phototherapy lights, with dramatic sparing at shielded sites within 24 hours after initiation of the phototherapy. On discontinuation of phototherapy, all eruptions cleared within 1 week. Examination of skin biopsy sections showed purpura without significant inflammation or keratinocyte necrosis. Plasma porphyrins (copro- and proto-) were elevated in the two patients in which they were assessed., Conclusions: The distribution of the eruption in areas exposed to light and presence of circulating porphyrins suggest that porphyrinemia may underlie the light-induced purpuric eruption. Additional studies will be required to determine definitively the mechanisms of both the purpuric phototherapy-induced eruption and the development of increased blood porphyrin levels in these transfused neonates.

Protoporphyrin accumulates in tissues after administration of delta-aminolevulinic acid, and can be used as a photosensitizer for photodynamic therapy. To determine the distribution of porphyrins in a large animal model after administration of this porphyrin precursor, delta-aminolevulinic acid was administered to anesthetized dogs (100 mg/kg body weight intravenously) and porphyrin concentrations were measured in tissues (liver, pancreas, prostate, bladder, muscle and skin), plasma and urine for 6-10 h. Porphyrins increased markedly (up to 50-fold) in plasma within 1 h, were still markedly increased at 8 h, and consisted mostly of coproporphyrin III and protoporphyrin. Tissue porphyrin concentrations increased more slowly, were highest in liver, pancreas and prostate 7-10 h after delta-aminolevulinic acid administration, and were predominantly protoporphyrin. Maximum porphyrin concentrations in liver were 3- and 4-fold higher than in pancreas and prostate, respectively. Urinary delta-aminolevulinic acid excretion increased and was greatest 2-4 h after dosing; urinary porphobilinogen and porphyrins increased more gradually and remained increased up to at least 8 h. Coproporphyrin III was the predominant porphyrin in urine at all times, but hepta-, hexa- and pentacarboxyl porphyrins increased proportionally after administration of delta-aminolevulinic acid. These results indicate that porphyrins accumulate in plasma as well as tissues and urine after administration of delta-aminolevulinic acid, and may contribute to tumor necrosis during photodynamic therapy.

To elucidate the sex difference in porphyrin metabolic disorders induced by lead exposure, we determined plasma delta-aminolevulinic acid (ALA), urinary ALA, and urinary coproporphyrin (CP) in 298 lead-exposed workers (160 males and 138 females), and compared the data thus obtained. The use of fluorometric high-performance liquid chromatography (HPLC) method which is highly sensitive and specific made possible the measurement of ALA in a small volume (50 microliters) of plasma. The concentrations (mean +/- SD) of lead in blood (males: 55.1 +/- 12.9 micrograms/dl; females: 54.7 +/- 13.5 micrograms/dl) indicated that the intensity of occupational exposure to lead was almost equal in the two groups. However, the elevation of plasma ALA concentration and the increased urine ALA and CP excretion among these lead workers were much higher in females than in males, confirming the finding of a sex difference in the biological effect of human exposure. The difference in urine CP excretion was especially pronounced, the mean concentration of urinary CP in the female workers being 3.5-5 times higher than that in the male workers.

Raised plasma uroporphyrin levels were found in all the 14 patients with end-stage renal disease studied, 7 of whom were on continuous ambulatory peritoneal dialysis (CAPD) and 7 on hemodialysis (HD). The elevation observed in the HD group was higher than that noted in the CAPD group; 18-fold in the former and 13-fold in the latter. The difference in uroporphyrin plasma levels between the two dialysis populations might be explained, at least partially, by the reduced activity of uroporphyrinogen decarboxylase (UROD), the enzyme which converts uroporphyrinogen to coproporphyrinogen. A decrease of 48% was noted in the HD group, whereas no change was observed in the CAPD group. A significant negative correlation (r = -0.37, p < 0.01) was found between the concentration of uroporphyrin in plasma and the activity of UROD. In view of the data shown, it is suggested that erythrocyte UROD activity should be interpreted with caution in HD patients suspected of having porphyria cutanea tarda.