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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., Here the authors describe the discovery of a class of small molecule splicing modifiers which are orally bioavailable, cross the blood-brain barrier, and lower levels of huntingtin in a mouse model of Huntington’s disease (HD).

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.

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.

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.

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.

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.

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.

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.

Studies were conducted to characterize the effect of dose and route of administration on the disposition of N-butylpyridinium chloride (NBuPy-Cl), an ionic liquid with solvent properties. Urine was the major route of NBuPy-Cl excretion after intravenous (5 mg/kg), single oral (0.5, 5, or 50 mg/kg), or repeated oral (50 mg/kg/day, 5 days) administration to male F-344 rats and single oral (50 mg/kg) administration to female B6C3F1 mice. Depending on the vehicle, absorption after dermal application (5 mg/kg, 125 μg/cm2) was 10 to 35% at 96 h. After the single intravenous dose, the blood concentration of NBuPy-Cl decreased in a biphasic manner with an elimination half-life of 2.2 h and a clearance of 7 ml/min. After single oral administration of NBuPy-Cl (50 mg/kg), maximum blood concentration was reached at 1.3 h, and the bioavailability was determined to be 47% at 6 h based on the blood toxicokinetics and 67% at 72 h based on urinary excretion. In all the urine and blood samples, only the parent compound was detected. Coadministration of NBuPy-Cl and inulin (by intravenous injection) revealed that the clearance of NBuPy-Cl exceeded the rat glomerular filtration rate. After incubation with Chinese hamster ovary cells expressing human organic cation transporter 2 (hOCT2), NBuPy-Cl was transported effectively (Kt = 18 μM), and also a potent inhibitor of hOCT2 mediated tetraethylammonium transport (IC50 = 2.3 μM). In summary, NBuPy-Cl is partially absorbed from the gastrointestinal tract and eliminated rapidly in the urine as parent compound most likely by renal glomerular filtration and OCT2-mediated secretion.

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 ( Kt) 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-PEO2-biotin did. Mutation of C474 to an alanine or phenylalanine did not affect the Ktvalue for MPP. In contrast, the Ktvalue 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.

Studies were conducted to characterize the effects of dose and route of administration on the disposition of 1-butyl-1-methylpyrrolidinium (BmPy-Cl) in male Fischer-344 rats. After a single oral administration of [(14)C]BmPy-Cl (50 mg/kg), BmPy-Cl in the blood decreased rapidly after C(max) of 89.1 min with a distribution half-life (t(1/2)(alpha)) of 21 min, an elimination half-life (t(1/2)(beta)) of 5.6 h, and a total body clearance of 7.6 ml/min. After oral administration (50, 5, and 0.5 mg/kg), 50 to 70% of the administered radioactivity was recovered in the feces, with the remainder recovered in the urine. Serial daily oral administrations of [(14)C]BmPy-Cl (50 mg/kg/day for 5 days) did not result in a notable alteration in disposition or elimination. After each administration, 88 to 94% of the dose was eliminated in a 24-h period, with 63 to 76% of dose recovered in the feces. Intravenous administration of [(14)C]BmPy-Cl (5 mg/kg) resulted in biphasic elimination. Oral systemic bioavailability was 43.4%, approximately equal to the dose recovered in urine after oral administration (29-38%). Total dermal absorption of [(14)C]BmPy-Cl (5 mg/kg) was moderate when it was applied in dimethylformamide-water (34 + or - 13%), variable in water (22 + or - 8%), or minimal in ethanol-water (13 + or - 1%) vehicles. Urine was the predominant route of elimination regardless of vehicle. Only parent [(14)C]BmPy-Cl was detected in the urine after all doses and routes of administration. BmPy-Cl was found to be a substrate for (K(t) = 37 microM) and inhibitor of (IC(50/tetraethylammonium) = 0.5 microM) human organic cation transporter 2. In summary, BmPy-Cl is moderately absorbed, extracted by the kidney, and eliminated in the urine as parent compound, independent of dose, number, or route of administration.

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.