Prediction of Hepatobiliary Clearances and Hepatic Concentrations of Transported Drugs in Humans Using Rosuvastatin as a Model Drug.

To assess efficacy and toxicity of a drug in humans, it is important to measure the tissue concentration of a drug at the target site. For a drug that is transported into or out of the tissue, the tissue unbound steady-state concentration can be dramatically different from its corresponding unbound steady-state plasma concentration. Because routine measurement of drug tissue concentrations is not possible, using rosuvastatin as a model transporter substrate drug, we compared the ability of the proteomics-informed relative expression factor (REF) approach and sandwich-cultured human hepatocytes (SCH) to accurately predict rosuvastatin human hepatobiliary clearances and hepatic concentrations. REF-predicted rosuvastatin biliary clearance (CLbile ), estimated using BCRP-overexpressing, MDR1-overexpressing, and MRP2-overexpressing vesicles, together with our previously published REF-predicted rosuvastatin hepatic sinusoidal uptake clearance (CLuptake ) and physiologically scaled sinusoidal passive uptake and efflux clearance (CLs,efflux ), were used to predict rosuvastatin hepatic concentrations. For SCH, the estimated rosuvastatin CLbile , CLuptake , and CLs,efflux were scaled using physiological scaling. The REF-predicted CLbile (6.39 ± 1.56 mL/minute) and hepatic rosuvastatin area under the concentration-time curve (AUC) fell within our a priori defined success criterion, i.e., within twofold of the observed positron emission tomography-imaged values. In contrast, as expected, SCH dramatically overpredicted (predicted/observed ratio P/O = 8.38-10.41) rosuvastatin CLbile , and underpredicted hepatic AUC (P/O = 0.08-0.14). For both approaches, predictions were improved by using the parallel tube model vs. well-stirred model. Overall, using rosuvastatin as a model drug, this study demonstrates the success of the REF approach in predicting in vivo CLbile and hepatic concentration of drugs, and highlights the shortcomings of the SCH approach in making such predictions.

Effect of food intake on the pharmacokinetics of rivoceranib in healthy subjects.

Rivoceranib is a selective inhibitor of VEGFR-2 being developed for the treatment of solid tumor. The objective of the study was to evaluate the effect of food on bioavailability as well as single- and multiple-dose pharmacokinetics (PKs) of 81 and 201 mg doses of rivoceranib. The study was conducted as a two-part study. In Part 1 (single ascending dose (SAD), open-label, crossover study design), 2 oral doses of rivoceranib (81 mg or 201 mg) were given to all healthy subjects with a minimum 3-day washout period between dosing. Part 2 was a multiple ascending dose (MAD), open-label, crossover design where subjects were divided based on 81 and 201 mg doses. Both doses were administered with and without food in a crossover manner for the SAD and MAD parts. 24 healthy subjects completed Part 1 and 20 subjects completed Part 2. For the 81 mg dose in the SAD and MAD parts of the study, their food effect was not observed. For the 201 mg dose in both parts, food appeared to increase bioavailability by 20%-30% in Part 1, and 30%-40% in Part 2. Median tmax value was delayed when rivoceranib was administered with food at each dose level in both parts of the study. Dose proportionality was confirmed only for the AUC0-∞  value from Part 1-fasted cohort but inconclusive for Cmax  and AUC parameters under other dosing regimens. In conclusion, rivoceranib when taken with food delays tmax appears to increase bioavailability at 201 mg dose.

Absence of ethnic difference on single-dose pharmacokinetics of rivoceranib between healthy male Caucasian, Japanese, and Chinese subjects.

Rivoceranib (known in China as apatinib) is a selective vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitor which inhibits angiogenesis in solid tumors. The aim of study was to evaluate potential pharmacokinetic (PK) differences between the Caucasian, Japanese, and Chinese populations. An open-label, single-dose, parallel-design PK study of rivoceranib was conducted in Caucasian, Japanese, and Chinese subjects. A total of 18 healthy males were recruited to each group (54 total), and 201 mg rivoceranib tablets (as 250 mg rivoceranib mesylate) were administered orally to subjects. Plasma samples were collected, and rivoceranib plasma concentration was determined using LC-MS/MS. For PK analysis, non-compartmental and compartmental analyses were performed. Intrinsic factors (CYP2C19 and CYP3A4 genotype) were also examined. Non-compartmental analysis showed no significant difference in AUC0-t , AUC0-∞ , Cmax , tmax , and t1/2 . Apparent clearance and volume of distribution were different across the three populations; however, the extent of this difference does not require dose modification. For compartmental modeling, a two-compartment model was used to fit the plasma concentrations. No significant difference was observed in absorption, elimination, and intercompartmental transfer rate constants among the three groups. The present study shows no major ethnic PK differences between Caucasian, Japanese, and Chinese populations.

Successful Prediction of Positron Emission Tomography-Imaged Metformin Hepatic Uptake Clearance in Humans Using the Quantitative Proteomics-Informed Relative Expression Factor Approach.

Predicting transporter-mediated in vivo hepatic drug clearance (CL) from in vitro data (IVIVE) is important in drug development to estimate first-in-human dose and the impact of drug interactions and pharmacogenetics on hepatic drug CL. For IVIVE, one can use human hepatocytes and the traditional milligrams of protein content per gram of liver tissue (MGPGL) approach. However, this approach has been found to consistently underpredict the observed in vivo hepatic drug CL. Therefore, we hypothesized that using transporter-expressing cells and the relative expression factor (REF), determined using targeted quantitative proteomics, will accurately predict in vivo hepatic CL of drugs. We have successfully tested this hypothesis in rats with rosuvastatin, which is transported by hepatic Organic anion transporting polypeptides (OATPs). Here, we tested this hypothesis for another drug and another transporter; namely, organic cation transporter (OCT)1-mediated hepatic distributional CL of metformin. First, we estimated the in vivo metformin hepatic sinusoidal uptake CL (CLh,s,in) of metformin by reanalysis of previously published human positron emission tomography imaging data. Next, using the REF approach, we predicted the in vivo metformin CLh,s,in using OCT1-transporter-expressing HEK293 cells or plated human hepatocytes. Finally, we compared this REF-based prediction with that using the MGPGL approach. The REF approach accurately predicted the in vivo metformin hepatic CLh,s,in, whereas the MGPGL approach considerably underpredicted the in vivo metformin CLh,s,in Based on these and previously published data, the REF approach appears to be superior to the MGPGL approach for a diverse set of drugs transported by different transporters. SIGNIFICANCE STATEMENT: This study is the first to use organic cation transporter 1-expressing cells and plated hepatocytes to compare proteomics-informed REF approach with the traditional MGPGL approach to predict hepatic uptake CL of metformin in humans. The proteomics-informed REF approach, which corrected for plasma membrane abundance, accurately predicted the positron emission tomography-imaged metformin hepatic uptake CL, whereas the MGPGL approach consistently underpredicted this CL.

The essential role of the transporter ABCG2 in the pathophysiology of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is an inherited disease caused by loss-of-function mutations of ferrochelatase, an enzyme in the heme biosynthesis pathway that converts protoporphyrin IX (PPIX) into heme. PPIX accumulation in patients with EPP leads to phototoxicity and hepatotoxicity, and there is no cure. Here, we demonstrated that the PPIX efflux transporter ABCG2 (also called BCRP) determines EPP-associated phototoxicity and hepatotoxicity. We found that ABCG2 deficiency decreases PPIX distribution to the skin and therefore prevents EPP-associated phototoxicity. We also found that ABCG2 deficiency protects against EPP-associated hepatotoxicity by modulating PPIX distribution, metabolism, and excretion. In summary, our work has uncovered an essential role of ABCG2 in the pathophysiology of EPP, which suggests the potential for novel strategies in the development of therapy for EPP.

Mechanisms of CYP3A Induction During Pregnancy: Studies in HepaRG Cells.

Activity of CYP3A, an enzyme responsible for metabolism of many marketed drugs, is induced by ~ 2-fold in pregnant women. Through studies in sandwich-cultured human hepatocytes (SCHH) and HepaRG cells, our laboratory has shown that this induction is likely mediated by the increase in cortisol plasma concentrations during pregnancy. Cortisol, at plasma concentrations observed during the third trimester (~ 800 nM), either alone or in combination with other pregnancy-related hormones, induces CYP3A activity in SCHH and HepaRG cells when cultured in dexamethasone-free media. To determine the mechanism(s) by which cortisol induces CYP3A activity, HepaRG cells were pre-incubated in dexamethasone-free medium and then incubated for 72 h with cortisol (798 nM). Glucocorticoid receptor (GR), pregnane X receptor (PXR), and CYP3A4 or CYP3A5 were knocked down using siRNA, and mRNA expression of these genes was measured. CYP3A4, and not CYP3A5, was found to be the dominant contributor to total CYP3A activity in control- and cortisol-treated HepaRG cells. Constitutive mRNA expression of CYP3A4 in HepaRG cells was regulated by both PXR and GR whereas constitutive expression of CYP3A5 in HepaRG cells was regulated by GR alone. Cortisol-mediated CYP3A4 induction in HepaRG cells was primarily mediated by GR-dependent PXR induction pathway and to a smaller extent via a PXR-independent pathway. Cortisol-mediated CYP3A5 induction was regulated by GR-dependent PXR-independent pathway. These data indicate that PXR plays a central role in cortisol-mediated induction of CYP3A activity during pregnancy and suggests that other enzymes and transporters, such as CYP2B6 and P-glycoprotein, may also be induced during pregnancy via the same mechanism(s).

Liver metabolomics in a mouse model of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a genetic disease that results from the defective mutation in the gene encoding ferrochelatase (FECH), the enzyme that converts protoporphyrin IX (PPIX) to heme. Liver injury and even liver failure can occur in EPP patients because of PPIX accumulation in the liver. The current study profiled the liver metabolome in an EPP mouse model caused by a Fech mutation (Fech-mut). As expected, we observed the accumulation of PPIX in the liver of Fech-mut mice. In addition, our metabolomic analysis revealed the accumulation of bile acids and ceramide (Cer) in the liver of Fech-mut mice. High levels of bile acids and Cer are toxic to the liver. Furthermore, we found that the major phosphatidylcholines (PC) in the liver and the ratio of total PC to PPIX in the bile were decreased in Fech-mut mice compared to wild type mice. A decrease of the ratio of PC to PPIX in the bile can potentiate the accumulation of PPIX in the liver because PC increases PPIX solubility and excretion. These metabolomic findings suggest that the accumulation of PPIX, together with the disruption of the homeostasis of bile acids, Cer, and PC, contributes to EPP-associated liver injury.

Metabolism of KO143, an ABCG2 inhibitor.

The ATP-binding cassette sub-family G member 2 (ABCG2) plays an important role in modulating drug disposition and endobiotic homeostasis. KO143 is a potent and relatively selective ABCG2 inhibitor. We found that the metabolic stability of KO143 was very poor in human liver microsomes (HLM). Our further studies illustrated that the tert-butyl ester group in KO143 can be rapidly hydrolyzed and removed by carboxylesterase 1. This metabolic pathway was confirmed as a major pathway of KO143 metabolism in both HLM and mice. K1 is an analog of KO143 without the ester group. We found that the metabolic stability of K1 was significantly improved in HLM when compared to KO143. These data suggest that the ester group in KO143 is the major cause of the poor metabolic stability of KO143. The data from this study can be used to guide the development of KO143 analogs with better metabolic properties.

Clinical Drug-Drug Interaction Evaluations to Inform Drug Use and Enable Drug Access.

Clinical drug-drug interactions (DDIs) can occur when multiple drugs are taken by the same patient. Significant DDIs can result in clinical toxicity or treatment failure. Therefore, DDI assessment is an integral part of drug development and the benefit-risk assessment of new therapies. Regulatory agencies including the Food and Drug Administration, the European Medicines Agency, and the Pharmaceuticals and Medical Devices Agency of Japan have made recommendations in their DDI guidance documents on various methodologies (in vitro, in silico, and clinical) to assess DDI potential and inform patient management strategies. This commentary focuses on clinical DDI evaluation for the purpose of drug development and regulatory evaluation.

Chronic Treatment with Isoniazid Causes Protoporphyrin IX Accumulation in Mouse Liver.

Isoniazid (INH) can cause hepatotoxicity. In addition, INH is contraindicated in patients suffering from porphyrias. Our metabolomic analysis revealed that chronic treatment with INH in mice causes a hepatic accumulation of protoporphyrin IX (PPIX). PPIX is an intermediate in the heme biosynthesis pathway, and it is also known as a hepatotoxin. We further found that INH induces delta-aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. We also found that INH downregulates ferrochelatase (FECH), the enzyme that converts PPIX to heme. In summary, this study illustrated that chronic treatment with INH causes PPIX accumulation in mouse liver in part through ALAS1 induction and FECH downregulation. This study also highlights that drugs can disrupt the metabolic pathways of endobiotics and increase the risk of liver damage.

Protoporphyrin IX: the Good, the Bad, and the Ugly.

Protoporphyrin IX (PPIX) is ubiquitously present in all living cells in small amounts as a precursor of heme. PPIX has some biologic functions of its own, and PPIX-based strategies have been used for cancer diagnosis and treatment (the good). PPIX serves as the substrate for ferrochelatase, the final enzyme in heme biosynthesis, and its homeostasis is tightly regulated during heme synthesis. Accumulation of PPIX in human porphyrias can cause skin photosensitivity, biliary stones, hepatobiliary damage, and even liver failure (the bad and the ugly). In this work, we review the mechanisms that are associated with the broad aspects of PPIX. Because PPIX is a hydrophobic molecule, its disposition is by hepatic rather than renal excretion. Large amounts of PPIX are toxic to the liver and can cause cholestatic liver injury. Application of PPIX in cancer diagnosis and treatment is based on its photodynamic effects.

A metabolomic perspective of griseofulvin-induced liver injury in mice.

Griseofulvin (GSF) causes hepatic porphyria in mice, which mimics the liver injury associated with erythropoietic protoporphyria (EPP) in humans. The current study investigated the biochemical basis of GSF-induced liver injury in mice using a metabolimic approach. GSF treatment in mice resulted in significant accumulations of protoporphyrin IX (PPIX), N-methyl PPIX, bile acids, and glutathione (GSH) in the liver. Metabolomic analysis also revealed bioactivation pathways of GSF that contributed to the formation of GSF-PPIX, GSF-GSH and GSF-proline adducts. GSF-PPIX is the precursor of N-methyl PPIX. A six-fold increase of N-methyl PPIX was observed in the liver of mice after GSF treatment. N-methyl PPIX strongly inhibits ferrochelatase, the enzyme that converts PPIX to heme, and leads to PPIX accumulation. Excessive PPIX in the liver results in bile duct blockage and disturbs bile acid homeostasis. The accumulation of GSH in the liver was likely due to Nrf2-mediated upregulation of GSH synthesis. In summary, this study provides the biochemical basis of GSF-induced liver injury that can be used to understand the pathophysiology of EPP-associated liver injury in humans.

Nuclear receptors in herb-drug interactions.

Widespread usage of herbs as supplements or medicines raises the potential of herb-drug interactions (HDIs). Basically, HDIs occur by pharmacokinetic and/or pharmacodynamic pathways. Nuclear receptors (NRs) are a class of transcription factors whose role in drug interactions has been defined. A large number of herbs activate NRs, resulting in HDIs. NR-mediated HDIs are similar to drug-drug interactions, but are more complicated because of the presence of multiple compounds in herbs. Dosage and therapeutic sequence as well as various other factors, including the patient's gender, age, and genetic makeup, may affect outcomes of NR-mediated HDIs.