暂无摘要。

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

OBJECTIVE: Encorafenib is a kinase inhibitor indicated for the treatment of patients with unresectable or metastatic melanoma or metastatic colorectal cancer, respectively, with selected BRAF V600 mutations. A clinical drug-drug interaction (DDI) study was designed to evaluate the effect of encorafenib on rosuvastatin, a sensitive substrate of OATP1B1/3 and breast cancer resistance protein (BCRP), and bupropion, a sensitive CYP2B6 substrate. Coproporphyrin I (CP-I), an endogenous substrate for OATP1B1, was measured in a separate study to deconvolute the mechanism of transporter DDI.
METHODS: DDI study participants received a single oral dose of rosuvastatin (10 mg) and bupropion (75 mg) on days - 7, 1, and 14 and continuous doses of encorafenib (450 mg QD) and binimetinib (45 mg BID) starting on day 1. The CP-I data were collected from participants in a phase 3 study who received encorafenib (300 mg QD) and cetuximab (400 mg/m2 initial dose, then 250 mg/m2 QW). Pharmacokinetic and pharmacodynamic analysis was performed using noncompartmental and compartmental methods.
RESULTS: Bupropion exposure was not increased, whereas rosuvastatin Cmax and area under the receiver operating characteristic curve (AUC) increased approximately 2.7 and 1.6-fold, respectively, following repeated doses of encorafenib and binimetinib. Increase in CP-I was minimal, suggesting that the primary effect of encorafenib on rosuvastatin is through BCRP. Categorization of statins on the basis of their metabolic and transporter profile suggests pravastatin would have the least potential for interaction when coadministered with encorafenib.
CONCLUSIONS: The results from these clinical studies suggest that encorafenib does not cause clinically relevant CYP2B6 induction or inhibition but is an inhibitor of BCRP and may also inhibit OATP1B1/3 to a lesser extent. Based on these results, it may be necessary to consider switching statins or reducing statin dosage accordingly for coadministration with encorafenib.
BACKGROUND: ClinicalTrials.gov NCT03864042, registered 6 March 2019.

Understanding the reaction mechanism of enzymes at the molecular level is generally a difficult task, since many parameters affect the turnover. Often, due to high reactivity and formation of transient species or intermediates, detailed information on enzymatic catalysis is obtained by means of model substrates. Whenever possible, it is essential to confirm a reaction mechanism based on substrate analogues or model systems by using the physiological substrates. Here we disclose the ferrous iron incorporation mechanism, in solution, and in crystallo, by the coproporphyrin III-coproporphyrin ferrochelatase complex from the firmicute, pathogen, and antibiotic resistant, Listeria monocytogenes. Coproporphyrin ferrochelatase plays an important physiological role as the metalation represents the penultimate reaction step in the prokaryotic coproporphyrin-dependent heme biosynthetic pathway, yielding coproheme (ferric coproporphyrin III). By following the metal titration with resonance Raman spectroscopy and x-ray crystallography, we prove that upon metalation the saddling distortion becomes predominant both in the crystal and in solution. This is a consequence of the readjustment of hydrogen bond interactions of the propionates with the protein scaffold during the enzymatic catalysis. Once the propionates have established the interactions typical of the coproheme complex, the distortion slowly decreases, to reach the almost planar final product.

In recent years, the identification of endogenous substrates as biomarkers became an uprising topic. Particularly coproporphyrins (CPs), byproducts of heme biosynthesis, are intensely investigated as biomarkers for predicting interactions with the organic anion transporting polypeptide (OATP) 1B transporters. In the context of drug-drug interactions, several preclinical and clinical studies assessed the effect of the OATP1B-index inhibitor rifampin on CPI levels. However, rifampin is not only a \"perpetrator\" drug of transporters but is also known for its interaction with the nuclear receptor pregnane X receptor (PXR) leading to the efficient induction of PXR-target genes. These include hemoproteins like cytochrome P450 enzymes but also the δ-aminolevulinate synthase 1, which is the rate-limiting enzyme in heme biosynthesis. In this study, we showed that quantification of CPs in clinical serum samples was possible after long-term storage at -20°C. We quantified CPI, CPIII, and heme levels in clinical serum samples (at selected timepoints) that originated from a trial investigating the interaction potential of repeated rifampin administration in 12 healthy participants. In samples collected at the assumed time to maximum concentration of rifampin, higher CP levels were observed compared to baseline. Increased levels persisted even 14 h after discontinuation of rifampin. No impact on heme serum levels was observed. We found a correlation between CP isomers at baseline and at 14 h after rifampin intake. In summary, we show that multiple doses of rifampin affect CP levels. However, besides inhibition of hepatic OATP function there is evidence for an interaction with CP levels beyond the transporter level.

Dubin-Johnson syndrome (DJS) presents during the neonatal period with a phenotype that overlaps with a broad list of causes of neonatal cholestasis (NC), which makes the identification of DJS challenging for clinicians. We conducted a case-controlled study to investigate the utility of urinary coproporphyrins (UCP) I% as a potential diagnostic biomarker.
We reviewed our database of 533 cases of NC and identified 28 neonates with disease-causing variants in ATP-binding cassette-subfamily C member 2 (ABCC2) gene \"Cases\" (Study period 2008-2019). Another 20 neonates with cholestasis due to non-DJS diagnoses were included as \"controls.\" Both groups underwent UCP analysis to measure CP isomer I percentage (%).
Serum alanine aminotransferase (ALT) levels were within the normal range in 26 patients (92%) and mildly elevated in 2 patients. ALT levels were significantly lower in neonates with DJS than in NC from other causes (P < 0.001). The use of normal serum ALT levels to predict DJS among neonates with cholestasis had a sensitivity of 93%, specificity 90%, positive predictive value (PPV) 34%, and negative predictive value (NPV) 99.5%. The median UCPI% was significantly higher in DJS patients [88%, interquartile range (IQR) 1-IQR3, 84.2%-92.7%] than in NC from other causes [67%, (IQR1-IQR3, 61%-71.5%; Confidence interval 0.18-0.28; P< 0.001)]. The use of UCPI% >80% to predict DJS had a sensitivity, specificity, PPV, and NPV of 100%.
Based on the results from our study, we propose sequencing of the ABCC2 gene in neonates with normal ALT, presence of cholestasis and UCP1% >80%.

Isotretinoin [13-cis-retinoic acid (13cisRA)] is widely used for the treatment of neuroblastoma and acne. It acts via regulating gene transcription through binding to retinoic acid receptors. Yet, the potential for isotretinoin to cause transcriptionally mediated drug-drug interactions (DDIs) has not been fully explored. We hypothesized that isotretinoin and its active metabolites all-trans-retinoic acid (atRA) and 4-oxo-13cisRA would alter the transcription of enzymes and transporters in the human liver via binding to nuclear receptors. The goal of this study was to define the DDI potential of isotretinoin and its metabolites resulting from transcriptional regulation of cytochrome P450 and transporter mRNAs. In human hepatocytes (n = 3), 13cisRA, atRA, and 4-oxo-13cisRA decreased OATP1B1, CYP1A2, CYP2C9, and CYP2D6 mRNA and increased CYP2B6 and CYP3A4 mRNA in a concentration-dependent manner. The EC50 values for OATP1B1 mRNA downregulation ranged from 2 to 110 nM, with maximum effect (Emax ) ranging from 0.17- to 0.54-fold. Based on the EC50 and Emax values and the known circulating concentrations of 13cisRA and its metabolites after isotretinoin dosing, a 55% decrease in OATP1B1 activity was predicted in vivo. In vivo DDI potential was evaluated clinically in participants dosed with isotretinoin for up to 32 weeks using coproporphyrin-I (CP-I) as an OATP1B1 biomarker. CP-I steady-state serum concentrations were unaltered following 2, 8, or 16 weeks of isotretinoin treatment. These data show that isotretinoin and its metabolites alter transcription of multiple enzymes and transporters in vitro, but translation of these changes to in vivo drug-drug interactions requires clinical evaluation for each enzyme. SIGNIFICANCE STATEMENT: Isotretinoin and its metabolites alter the mRNA expression of multiple cytochrome P450s (CYPs) and transporters in human hepatocytes, suggesting that isotretinoin may cause clinically significant drug-drug interactions (DDIs). Despite the observed changes in organic anion transporting polypeptide 1B1 (OATP1B1) mRNA in human hepatocytes, no clinical DDI was observed when measuring a biomarker, coproporphyrin-I. Further work is needed to determine whether these findings can be extrapolated to a lack of a DDI with CYP1A2, CYP2B6, and CYP2C9 substrates.

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.

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.

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.