Clinical significance of CYP2C19 polymorphisms on the metabolism and pharmacokinetics of 11ß-hydroxysteroid dehydrogenase type-1 inhibitor BMS-823778.

AIMS: BMS-823778 is an inhibitor of 11ß-hydroxysteroid dehydrogenase type-1, and thus a potential candidate for Type 2 diabetes treatment. Here, we investigated the metabolism and pharmacokinetics of BMS-823778 to understand its pharmacokinetic variations in early clinical trials. METHODS: The metabolism of BMS-823778 was characterized in multiple in vitro assays. Pharmacokinetics were evaluated in healthy volunteers, prescreened as CYP2C19 extensive metabolizers (EM) or poor metabolizers (PM), with a single oral dose of [14 C]BMS-823778 (10 mg, 80 µCi). RESULTS: Three metabolites (<5%) were identified in human hepatocytes and liver microsomes (HLM) incubations, including two hydroxylated metabolites (M1 and M2) and one glucuronide conjugate (M3). As the most abundant metabolite, M1 was formed mainly through CYP2C19. M1 formation was also correlated with CYP2C19 activities in genotyped HLM. In humans, urinary excretion of dosed radioactivity was significantly higher in EM (68.8%; 95% confidence interval 61.3%, 76.3%) than in PM (47.0%; 43.5%, 50.6%); only small portions (<2%) were present in faeces or bile from both genotypes. In plasma, BMS-823778 exposure in PM was significantly (5.3-fold, P = 0.0097) higher than in EM. Furthermore, total radioactivity exposure was significantly higher (P < 0.01) than BMS-823778 exposure in all groups, indicating the presence of metabolites. M1 was the only metabolite observed in plasma, and much lower in PM. In urine, the amount of M1 and its oxidative metabolite in EM was 7-fold of that in PM, while more glucuronide conjugates of BMS-823778 and M1 were excreted in PM. CONCLUSIONS: CYP2C19 polymorphisms significantly impacted systemic exposure and metabolism pathways of BMS-823778 in humans.

Bile Salt Homeostasis in Normal and Bsep Gene Knockout Rats with Single and Repeated Doses of Troglitazone.

The interference of bile acid secretion through bile salt export pump (BSEP) inhibition is one of the mechanisms for troglitazone (TGZ)-induced hepatotoxicity. Here, we investigated the impact of single or repeated oral doses of TGZ (200 mg/kg/day, 7 days) on bile acid homoeostasis in wild-type (WT) and Bsep knockout (KO) rats. Following oral doses, plasma exposures of TGZ were not different between WT and KO rats, and were similar on day 1 and day 7. However, plasma exposures of the major metabolite, troglitazone sulfate (TS), in KO rats were 7.6- and 9.3-fold lower than in WT on day 1 and day 7, respectively, due to increased TS biliary excretion. With Bsep KO, the mRNA levels of multidrug resistance-associated protein 2 (Mrp2), Mrp3, Mrp4, Mdr1, breast cancer resistance protein (Bcrp), sodium taurocholate cotransporting polypeptide, small heterodimer partner, and Sult2A1 were significantly altered in KO rats. Following seven daily TGZ treatments, Cyp7A1 was significantly increased in both WT and KO rats. In the vehicle groups, plasma exposures of individual bile acids demonstrated variable changes in KO rats as compared with WT. WT rats dosed with TGZ showed an increase of many bile acid species in plasma on day 1, suggesting the inhibition of Bsep. Conversely, these changes returned to base levels on day 7. In KO rats, alterations of most bile acids were observed after seven doses of TGZ. Collectively, bile acid homeostasis in rats was regulated through bile acid synthesis and transport in response to Bsep deficiency and TGZ inhibition. Additionally, our study is the first to demonstrate that repeated TGZ doses can upregulate Cyp7A1 in rats.

Coproporphyrins I and III as Functional Markers of OATP1B Activity: In Vitro and In Vivo Evaluation in Preclinical Species.

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.

Coproporphyrins in Plasma and Urine Can Be Appropriate Clinical Biomarkers to Recapitulate Drug-Drug Interactions Mediated by Organic Anion Transporting Polypeptide Inhibition.

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.

Application of Static Models to Predict Midazolam Clinical Interactions in the Presence of Single or Multiple Hepatitis C Virus Drugs.

Asunaprevir (ASV), daclatasvir (DCV), and beclabuvir (BCV) are three drugs developed for the treatment of chronic hepatitis C virus infection. Here, we evaluated the CYP3A4 induction potential of each drug, as well as BCV-M1 (the major metabolite of BCV), in human hepatocytes by measuring CYP3A4 mRNA alteration. The induction responses were quantified as induction fold (mRNA fold change) and induction increase (mRNA fold increase), and then fitted with four nonlinear regression algorithms. Reversible inhibition and time-dependent inhibition (TDI) on CYP3A4 activity were determined to predict net drug-drug interactions (DDIs). All four compounds were CYP3A4 inducers and inhibitors, with ASV demonstrating TDI. The curve-fitting results demonstrated that fold increase is a better assessment to determine kinetic parameters for compounds inducing weak responses. By summing the contribution of each inducer, the basic static model was able to correctly predict the potential for a clinically meaningful induction signal for single or multiple perpetrators, but with over prediction of the magnitude. With the same approach, the mechanistic static model improved the prediction accuracy of DCV and BCV when including both induction and inhibition effects, but incorrectly predicted the net DDI effects for ASV alone or triple combinations. The predictions of ASV or the triple combination could be improved by only including the induction and reversible inhibition but not the ASV CYP3A4 TDI component. Those results demonstrated that static models can be applied as a tool to help project the DDI risk of multiple perpetrators using in vitro data.

Involvement of Drug Transporters in Organ Toxicity: The Fundamental Basis of Drug Discovery and Development.

Membrane transporters play a pivotal role in many organs to maintain their normal physiological functions and contribute significantly to drug absorption, distribution, and elimination. Knowledge gained from gene modified animal models or human genetic disorders has demonstrated that interruption of the transporter activity can lead to debilitating diseases or organ toxicities. Herein we describe transporter associated diseases and organ toxicities resulting from transporter gene deficiency or functional inhibition in the liver, kidney, gastrointestinal tract (GIT), and central nervous system (CNS). While proposing additional transporters as targets for drug-induced organ toxicity, strategies and future perspectives are discussed for transporter risk assessment in drug discovery and development.

Biliary excretion of pravastatin and taurocholate in rats with bile salt export pump (Bsep) impairment.

The bile salt export pump (BSEP) is expressed on the canalicular membrane of hepatocytes regulating liver bile salt excretion, and impairment of BSEP function may lead to cholestasis in humans. This study explored drug biliary excretion, as well as serum chemistry, individual bile acid concentrations and liver transporter expressions, in the SAGE Bsep knockout (KO) rat model. It was observed that the Bsep protein in KO rats was decreased to 15% of that in the wild type (WT), as quantified using LC-MS/MS. While the levels of Ntcp and Mrp2 were not significantly altered, Mrp3 expression increased and Oatp1a1 decreased in KO animals. Compared with the WT rats, the KO rats had similar serum chemistry and showed normal liver transaminases. Although the total plasma bile salts and bile flow were not significantly changed in Bsep KO rats, individual bile acids in plasma and liver demonstrated variable changes, indicating the impact of Bsep KO. Following an intravenous dose of deuterium labeled taurocholic acid (D4-TCA, 2 mg/kg), the D4-TCA plasma exposure was higher and bile excretion was delayed by approximately 0.5 h in the KO rats. No differences were observed for the pravastatin plasma concentration-time profile or the biliary excretion after intravenous administration (1 mg/kg). Collectively, the results revealed that these rats have significantly lower Bsep expression, therefore affecting the biliary excretion of endogenous bile acids and Bsep substrates. However, these rats are able to maintain a relatively normal liver function through the remaining Bsep protein and via the regulation of other transporters. Copyright © 2016 John Wiley & Sons, Ltd.

Functional significance of conserved cysteines in the human organic cation transporter 2.

The significance of conserved cysteines in the human organic cation transporter 2 (hOCT2), namely the six cysteines in the long extracellular loop (loop cysteines) and C474 in transmembrane helix 11, was examined. Uptake of tetraethylammonium (TEA) and 1-methyl-4-phenypyridinium (MPP) into Chinese hamster ovary cells was stimulated >20-fold by hOCT2 expression. Both cell surface expression and transport activity were reduced considerably following mutation of individual loop cysteines (C51, C63, C89, C103, and C143), and the C89 and C103 mutants had reduced Michaelis constants (K(t)) for MPP. The loop cysteines were refractory to interaction with thiol-reactive biotinylation reagents, except after pretreatment of intact cells with dithiothreitol or following cell membrane solubilization. Reduction of disulfide bridge(s) did not affect transport, but labeling the resulting free thiols with maleimide-PEO(2)-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the K(t) value for MPP. In contrast, the K(t) value associated with TEA transport was reduced sevenfold in the C474A mutant, and the C474F mutant failed to transport TEA. This study shows that some but not all of the six extracellular loop cysteines exist within disulfide bridge(s). Each loop cysteine is important for plasma membrane targeting, and their mutation can influence substrate binding. The effect of C474 mutation on TEA transport suggests that it contributes to a TEA binding surface. Given that TEA and MPP are competitive inhibitors, the differential effects of C474 modification on TEA and MPP binding suggest that the binding surfaces for each are distinct, but overlapping in area.

Expression of organic anion transporter 2 in the human kidney and its potential role in the tubular secretion of guanine-containing antiviral drugs.

The organic anion transporters 1 and 3 (OAT1 and OAT3) and organic cation transporter 2 (OCT2) are important for renal tubular drug secretion. In contrast, evidence for OAT2 expression in the human kidney is limited, and its role in renal drug transport is unknown. Both mRNA (real-time polymerase chain reaction) and protein (Western blotting) for OAT2 were detected in renal cortex from eight donors, and interindividual variability in protein levels was 3-fold. OAT2 protein in the renal cortex was localized (by immunohistochemistry) to the basolateral domain of tubules, as were OAT1 and OAT3. The absolute abundance of OAT2 mRNA was similar to that of OAT1 mRNA and 3-fold higher than that of OCT2 mRNA but 10-fold lower than that of OAT3 mRNA. A previous observation that OAT2 transports cGMP led us to examine whether acyclovir, ganciclovir, and penciclovir are OAT2 substrates; they are guanine-containing antivirals that undergo active tubular secretion. Transport of the antivirals into human embryonic kidney cells was stimulated 10- to 20-fold by expression of OAT2, but there was little to no transport of the antivirals by OAT1, OAT3, or OCT2. The K(m) values for acyclovir, ganciclovir, and penciclovir transport were 94, 264, and 277 µM, respectively, and transport efficiencies were relatively high (6-24 µl · min(-1) · mg protein(-1)). This study provides definitive evidence for the expression of OAT2 in the human kidney and is the first to demonstrate that OAT2, compared with OAT1, OAT3, or OCT2, has a preference for antiviral drugs mainly eliminated in the urine via active secretion.

Characterization of the inhibitory effects of N-butylpyridinium chloride and structurally related ionic liquids on organic cation transporters 1/2 and human toxic extrusion transporters 1/2-k in vitro and in vivo.

Ionic liquids (ILs) are a class of salts that are expected to be used as a new source of solvents and for many other applications. Our previous studies revealed that selected ILs, structurally related organic cations, are eliminated exclusively in urine as the parent compound, partially mediated by renal transporters. This study investigated the inhibitory effects of N-butylpyridinium chloride (NBuPy-Cl) and structurally related ILs on organic cation transporters (OCTs) and multidrug and toxic extrusion transporters (MATEs) in vitro and in vivo. After Chinese hamster ovary cells expressing rat (r) OCT1, rOCT2, human (h) OCT2, hMATE1, or hMATE2-K were constructed, the ability of NBuPy-Cl, 1-methyl-3-butylimidazolium chloride (Bmim-Cl), N-butyl-N-methylpyrrolidinium chloride (BmPy-Cl), and alkyl substituted pyridinium ILs to inhibit these transporters was determined in vitro. NBuPy-Cl (0, 0.5, or 2 mg/kg per hour) was also infused into rats to assess its effect on the pharmacokinetics of metformin, a substrate of OCTs and MATEs. NBuPy-Cl, Bmim-Cl, and BmPy-Cl displayed strong inhibitory effects on these transporters (IC(50) = 0.2-8.5 µM). In addition, the inhibitory effects of alkyl-substituted pyridinium ILs on OCTs increased dramatically as the length of the alkyl chain increased. The IC(50) values were 0.1, 3.8, 14, and 671 µM (hexyl-, butyl-, and ethyl-pyridinium and pyridinium chloride) for rOCT2-mediated metformin transport. Similar structurally related inhibitory kinetics were also observed for rOCT1 and hOCT2. The in vivo coadministration study revealed that NBuPy-Cl reduced the renal clearance of metformin in rats. These results demonstrate that ILs compete with other substrates of OCTs and MATEs and could alter the in vivo pharmacokinetics of such substrates.

Application of a PBPK model to elucidate the changes of systemic and liver exposures for rosuvastatin, carotegrast, and bromfenac followed by OATP inhibition in monkeys.

The impact of organic anion-transporting polypeptide (OATP) inhibition on systemic and liver exposures of three OATP substrates was investigated in cynomolgus monkeys. A monkey physiologically-based pharmacokinetic (PBPK) model was constructed to describe the exposure changes followed by OATP functional attenuation. Rosuvastatin, bromfenac, and carotegrast were administered as a single intravenous cassette dose (0.5 mg/kg each) in monkeys with and without predosing with rifampin (RIF; 20 mg/kg) orally. The plasma exposure of rosuvastatin, bromfenac, carotegrast, and OATP biomarkers, coproporphyrin I (CP-I) and CP-III were increased 2.3, 2.1, 9.1, 5.4, and 8.8-fold, respectively, when compared to the vehicle group. The liver to plasma ratios of rosuvastatin and bromfenac were reduced but the liver concentration of the drugs remained unchanged by RIF treatment. The liver concentrations of carotegrast, CP-I, and CP-III were unchanged at 1 h but increased at 6 h in the RIF-treated group. The passive permeability, active uptake, and biliary excretion were characterized in suspended and sandwich-cultured monkey hepatocytes and then incorporated into the monkey PBPK model. As demonstrated by the PBPK model, the plasma exposure is increased through OATP inhibition while liver exposure is maintained by passive permeability driven from an elevated plasma level. Liver exposure is sensitive to the changes of metabolism and biliary clearances. The model further suggested the involvement of additional mechanisms for hepatic uptakes of rosuvastatin and bromfenac, and of the inhibition of biliary excretion for carotegrast, CP-I, and CP-III by RIF. Collectively, impaired OATP function would not reduce the liver exposure of its substrates in monkeys.

Reprogrammed mesenchymal stem cells derived from iPSCs promote bone repair in steroid-associated osteonecrosis of the femoral head.

BACKGROUND: Cellular therapy based on mesenchymal stem cells (MSCs) is a promising novel therapeutic strategy for the osteonecrosis of the femoral head (ONFH), which is gradually becoming popular, particularly for early-stage ONFH. Nonetheless, the MSC-based therapy is challenging due to certain limitations, such as limited self-renewal capability of cells, availability of donor MSCs, and the costs involved in donor screening. As an alternative approach, MSCs derived from induced pluripotent stem cells (iPSCs), which may lead to further standardized-cell preparations. METHODS: In the present study, the bone marrow samples of patients with ONFH (n = 16) and patients with the fracture of the femoral neck (n = 12) were obtained during operation. The bone marrow-derived MSCs (BMSCs) were isolated by density gradient centrifugation. BMSCs of ONFH patients (ONFH-BMSCs) were reprogrammed to iPSCs, following which the iPSCs were differentiated into MSCs (iPSC-MSCs). Forty adult male rats were randomly divided into following groups (n = 10 per group): (a) normal control group, (b) methylprednisolone (MPS) group, (c) MPS + BMSCs treated group, and (d) MPS + iPSC-MSC-treated group. Eight weeks after the establishment of the ONFH model, rats in BMSC-treated group and iPSC-MSC-treated group were implanted with BMSCs and iPSC-MSCs through intrabone marrow injection. Bone repair of the femoral head necrosis area was analyzed after MSC transplantation. RESULTS: The morphology, immunophenotype, in vitro differentiation potential, and DNA methylation patterns of iPSC-MSCs were similar to those of normal BMSCs, while the proliferation of iPSC-MSCs was higher and no tumorigenic ability was exhibited. Furthermore, comparing the effectiveness of iPSC-MSCs and the normal BMSCs in an ONFH rat model revealed that the iPSC-MSCs was equivalent to normal BMSCs in preventing bone loss and promoting bone repair in the necrosis region of the femoral head. CONCLUSION: Reprogramming can reverse the abnormal proliferation, differentiation, and DNA methylation patterns of ONFH-BMSCs. Transplantation of iPSC-MSCs could effectively promote bone repair and angiogenesis in the necrosis area of the femoral head.

Small molecule splicing modifiers with systemic HTT-lowering activity.

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the huntingtin (HTT) gene. Consequently, the mutant protein is ubiquitously expressed and drives pathogenesis of HD through a toxic gain-of-function mechanism. Animal models of HD have demonstrated that reducing huntingtin (HTT) protein levels alleviates motor and neuropathological abnormalities. Investigational drugs aim to reduce HTT levels by repressing HTT transcription, stability or translation. These drugs require invasive procedures to reach the central nervous system (CNS) and do not achieve broad CNS distribution. Here, we describe the identification of orally bioavailable small molecules with broad distribution throughout the CNS, which lower HTT expression consistently throughout the CNS and periphery through selective modulation of pre-messenger RNA splicing. These compounds act by promoting the inclusion of a pseudoexon containing a premature termination codon (stop-codon psiExon), leading to HTT mRNA degradation and reduction of HTT levels.

In vitro model systems to investigate bile salt export pump (BSEP) activity and drug interactions: A review.

The bile salt export pump protein (BSEP), expressed on the canalicular membranes of hepatocytes, is primarily responsible for the biliary excretion of bile salts. The inhibition of BSEP transport activity can lead to an increase in intracellular bile salt levels and liver injury. This review discusses the various in vitro assays currently available for assessing the effect of drugs or other chemical entities to modulate BSEP transport activity. BSEP transporter assays use one of the following platforms: Xenopus laevis oocytes; canalicular membrane vesicles (CMV); BSEP-expressed membrane vesicles; cell lines expressing BSEP; sandwich cultured hepatocytes (SCH); and hepatocytes in suspension. Two of these, BSEP-expressed insect membrane vesicles and sandwich cultured hepatocytes, are the most commonly used assays. BSEP membrane vesicles prepared from transfected insect cells are useful for assessing BSEP inhibition or substrate specificity and exploring mechanisms of BSEP-associated genetic diseases. This model can be applied in a high-throughput format for discovery-drug screening. However, experimental results from use of membrane vesicles may lack physiological relevance and the model does not allow for investigation of in situ metabolism in modulation of BSEP activity. Hepatocyte-based assays that use the SCH format provide results that are generally more physiologically relevant than membrane assays. The SCH model is useful in detailed studies of the biliary excretion of drugs and BSEP inhibition, but due to the complexity of SCH preparation, this model is used primarily for determining biliary clearance and BSEP inhibition in a limited number of compounds. The newly developed hepatocyte in suspension assay avoids many of the complexities of the SCH method. The use of pooled cryopreserved hepatocytes in suspension minimizes genetic variance and individual differences in BSEP activity and also provides the opportunity for higher throughput screening and cross-species comparisons.

[Effect of Spatholobus suberectus on the bone marrow cells and related cytokines of mice].

OBJECTIVE: To study the effect of Spatholobus suberectus on proliferation and the hematonic mechanism. METHOD: The techniques of culture of hematopoietic cell and hematopoietic growth factor (HGF) assay were used. RESULT: Spatholobus suberectus could obviously promote the proliferation of bone marrow cells in healthy and anaemic mice. The culture media of spleen cell, macrophage, lung and skeletal muscle treated with S. suberectus had much stronger stimulating effects on hematopoietic cells. CONCLUSION: S. suberectus may enhance hematopoiesis by directly or indirectly stimulating stroma cell in hematopoietic inductive microenvironment and muscle tissue to secrete some HGF (Epo, GM-CSF, IL, and MK-CSF). This is one of the biological mechanisms for hematonic effect of S. suberectus.

[The influence of Bazhen decoction on hematopoietic modulator in anaemic mice].

This study was designed to evaluate the effect of Bazhen decoction on bone marrow depression induced by cyclophosphamide (CY) in mice. An experimental model of mouse bone marrow injury was established through cyclophosphamide induced and the following phenomena were observed. The techniques of culture of hematopoietic progenitor cell and hematopoietic growth factor assay were used. Bazhen decoction could obviously promote the proliferation of bone marrow cells of anaemic mice. The culture media of spleen cell, macrophage, lung and skeletal muscle treated with Bazhen decoction had much stronger stimulating effects on hematopoietic cells. The bone marrow cells of the anaemic mice could yield TNF through Bazhen decoction treatment. It was suggested that Bazhen decoction is clinically a hopeful drug used to cure bone marrow depression and attenuate the side effects of CY.