Dihydro-stilbene gigantol relieves CCl4-induced hepatic oxidative stress and inflammation in mice via inhibiting C5b-9 formation in the liver.

In general, anti-inflammatory treatment is considered for multiple liver diseases despite the etiology. But current drugs for alleviating liver inflammation have defects, making it necessary to develop more potent and safer drugs for liver injury. In this study, we screened a series of (dihydro-)stilbene or (dihydro-)phenanthrene derivatives extracted from Pholidota chinensis for their potential biological activities. Among 31 compounds, the dihydro-stilbene gigantol exerted most potent protective effects on human hepatocytes against lithocholic acid toxicity, and exhibited solid antioxidative and anti-inflammatory effect in vitro. In mice with CCl4-induced acute liver injury, pre-administration of gigantol (10, 20, 40 mg· kg-1· d-1, po, for 7 days) dose-dependently decreased serum transaminase levels and improved pathological changes in liver tissues. The elevated lipid peroxidation and inflammatory responses in the livers were also significantly alleviated by gigantol. The pharmacokinetic studies showed that gigantol was highly concentrated in the mouse livers, which consisted with its efficacy in preventing liver injury. Using a label-free quantitative proteomic analysis we revealed that gigantol mainly regulated the immune system process in liver tissues of CCl4-treated mice, and the complement and coagulation cascades was the predominant pathway; gigantol markedly inhibited the expression of complement component C9, which was a key component for the formation of terminal complement complex (TCC) C5b-9. These results were validated by immunohistochemistry (IHC) or real time-PCR. Confocal microscopy analysis showed that gigantol significantly inhibited the vascular deposition of TCC in the liver. In conclusion, we demonstrate for the first time that oral administration of gigantol potently relieves liver oxidative stress and inflammation, possibly via a novel mechanism of inhibiting the C5b-9 formation in the liver.

Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2.

Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF's active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5-100 µmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC50 value of 4.91 µmol/L. TER (5 µmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells' uptake of acyclovir and increased the plasma concentration.

Different structures of berberine and five other protoberberine alkaloids that affect P-glycoprotein-mediated efflux capacity.

Berberine, berberrubine, thalifendine, demethyleneberberine, jatrorrhizine, and columbamine are six natural protoberberine alkaloid (PA) compounds that display extensive pharmacological properties and share the same protoberberine molecular skeleton with only slight substitution differences. The oral delivery of most PAs is hindered by their poor bioavailability, which is largely caused by P-glycoprotein (P-gp)-mediated drug efflux. Meanwhile, P-gp undergoes large-scale conformational changes (from an inward-facing to an outward-facing state) when transporting substrates, and these changes might strongly affect the P-gp-binding specificity. To confirm whether these six compounds are substrates of P-gp, to investigate the differences in efflux capacity caused by their trivial structural differences and to reveal the key to increasing their binding affinity to P-gp, we conducted a series of in vivo, in vitro, and in silico assays. Here, we first confirmed that all six compounds were substrates of P-gp by comparing the drug concentrations in wild-type and P-gp-knockout mice in vivo. The efflux capacity (net efflux) ranked as berberrubine > berberine > columbamine ~ jatrorrhizine > thalifendine > demethyleneberberine based on in vitro transport studies in Caco-2 monolayers. Using molecular dynamics simulation and molecular docking techniques, we determined the transport pathways of the six compounds and their binding affinities to P-gp. The results suggested that at the early binding stage, different hydrophobic and electrostatic interactions collectively differentiate the binding affinities of the compounds to P-gp, whereas electrostatic interactions are the main determinant at the late release stage. In addition to hydrophobic interactions, hydrogen bonds play an important role in discriminating the binding affinities.

Optimized Hepatocyte-Like Cells with Functional Drug Transporters Directly-Reprogrammed from Mouse Fibroblasts and their Potential in Drug Disposition and Toxicology.

BACKGROUND/AIMS: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep) cells directly reprogrammed from mice fibroblast cells were fully characterized. METHODS: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α) and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed. RESULTS: The expression and function of p-glycoprotein (P-gp), bile salt efflux pump (Bsep), multidrug resistance-associated protein 2 (Mrp2), Na+-dependent taurocholate cotransporting polypeptide (Ntcp), and organic anion transporter polypedtides (Oatps) in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α) and treatment with selected inducers. In vitro intrinsic biliary clearances (CLb,int) of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid) and DPDPE ([D-Pen2,5] enkephalin hydrate) correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs) (r2 = 0.984). Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes. CONCLUSION: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening.

Inhibition of hepatic cytochrome P450 enzymes and sodium/bile acid cotransporter exacerbates leflunomide-induced hepatotoxicity.

AIM: Leflunomide is an immunosuppressive agent marketed as a disease-modifying antirheumatic drug. But it causes severe side effects, including fatal hepatitis and liver failure. In this study we investigated the contributions of hepatic metabolism and transport of leflunomide and its major metabolite teriflunomide to leflunomide induced hepatotoxicity in vitro and in vivo. METHODS: The metabolism and toxicity of leflunomide and teriflunomide were evaluated in primary rat hepatocytes in vitro. Hepatic cytochrome P450 reductase null (HRN) mice were used to examine the PK profiling and hepatotoxicity of leflunomide in vivo. The expression and function of sodium/bile acid cotransporter (NTCP) were assessed in rat and human hepatocytes and NTCP-transfected HEK293 cells. After Male Sprague-Dawley (SD) rats were administered teriflunomide (1,6, 12 mg · kg(-1) · d(-1), ig) for 4 weeks, their blood samples were analyzed. RESULTS: A nonspecific CYPs inhibitor aminobenzotriazole (ABT, 1 mmol/L) decreased the IC50 value of leflunomide in rat hepatocytes from 409 to 216 µmol/L, whereas another nonspecific CYPs inhibitor proadifen (SKF, 30 µmol/L) increased the cellular accumulation of leflunomide to 3.68-fold at 4 h. After oral dosing (15 mg/kg), the plasma exposure (AUC0-t) of leflunomide increased to 3-fold in HRN mice compared with wild type mice. Administration of leflunomide (25 mg·kg(-1) · d(-1)) for 7 d significantly increased serum ALT and AST levels in HRN mice; when the dose was increased to 50 mg·kg(-1) · d(-1), all HRN mice died on d 6. Teriflunomide significantly decreased the expression of NTCP in human hepatocytes, as well as the function of NTCP in rat hepatocytes and NTCP-transfected HEK293 cells. Four-week administration of teriflunomide significantly increased serum total bilirubin and direct bilirubin levels in female rats, but not in male rats. CONCLUSION: Hepatic CYPs play a critical role in detoxification process of leflunomide, whereas the major metabolite teriflunomide suppresses the expression and function of NTCP, leading to potential cholestasis.

[Absorption, distribution, metabolism, excretion and toxicity of biologics and its application in pharmacokinetic modeling].

Recently, more and more attentions of drug development are placed to macromolecules, such as monoclonal antibodies, proteins, etc. It has become one of the most promising areas in drug research and development in 21st Century. In terms of the structure and the ADMET (absorption, distribution, metabolism, excretion and toxicity), macromolecules is different from small molecule drugs, which lead to a distinct modeling strategy. The characterization of biologics ADMET processes and its application in the PK model selection of macromolecules are reviewed in this paper.

Timosaponin A3 induces hepatotoxicity in rats through inducing oxidative stress and down-regulating bile acid transporters.

AIM: To investigate the mechanisms underlying the hepatotoxicity of timosaponin A3 (TA3), a steroidal saponin from Anemarrhena asphodeloides, in rats. METHODS: Male SD rats were administered TA3 (100 mg·kg(-1)·d(-1), po) for 14 d, and the blood and bile samples were collected after the final administration. The viability of a sandwich configuration of cultured rat hepatocytes (SCRHs) was assessed using WST-1. Accumulation and biliary excretion index (BEI) of d8-TCA in SCRHs were determined with LC-MS/MS. RT-PCR and Western blot were used to analyze the expression of relevant genes and proteins. ROS and ATP levels, and mitochondrial membrane potential (MMP) were measured. F-actin cytoskeletal integrity was assessed under confocal microscopy. RESULTS: TA3 administration in rats significantly elevated the total bile acid in serum, and decreased bile acid (BA) component concentrations in bile. TA3 inhibited the viability of the SCRHs with an IC50 value of 15.21±1.73 µmol/L. Treatment of the SCRHs with TA3 (1-10 µmol/L) for 2 and 24 h dose-dependently decreased the accumulation and BEI of d8-TCA. The TA3 treatment dose-dependently decreased the expression of BA transporters Ntcp, Bsep and Mrp2, and BA biosynthesis related Cyp7a1 in hepatocytes. Furthermore, the TA3 treatment dose-dependently increased ROS generation and HO-1 expression, decreased the ATP level and MMP, and disrupted F-actin in the SCRHs. NAC (5 mmol/L) significantly ameliorated TA3-induced effects in the SCRHs, whereas mangiferin (10-200 µg/mL) almost blocked TA3-induced ROS generation. CONCLUSION: TA3 triggers liver injury through inducing ROS generation and suppressing the expression of BA transporters. Mangiferin, an active component in Anemarrhena, may protect hepatocytes from TA3-induced hepatotoxicity.

Opposite regulation of hepatic breast cancer resistance protein in type 1 and 2 diabetes mellitus.

Previous studies on diabetes have reported controversial results with regard to transporters in liver. The present study aimed to explore changes in hepatic breast cancer resistance protein (BCRP) expression and functions, as well as the possible underlying mechanisms, in type 2 diabetic patients, type 1 (streptozotocin-induced), and type 2 (Goto Kakizaki) diabetic rats. Protein and mRNA levels of human (h) and rat (r) BCRP were investigated using Western blot and quantitative polymerase chain reaction analyses. Functions of liver rBCRP were evaluated using rosuvastatin. Sandwich cultured rat hepatocytes (SCRH) were cultured with d-glucose, insulin, or oleic acid for 72 h, and rBCRP mRNA was detected. The effect of oleic acid on rBCRP function in SCRH was also investigated using rosuvastatin. Results showed that liver rBCRP mRNA levels decreased to 20% in type 1 diabetic rats, whereas that in diabetic patients and GK rats significantly increased threefold and twentyfold, respectively. No changes were observed in h/rBCRP protein levels of type 2 diabetic patients and GK rats. The functions of rBCRP significantly declined in type 1 diabetic rats but showed no significant changes in GK rats. The data from SCRH indicated that d-glucose decreased rBCRP mRNA level to 60%. Oleic acid increased rBCRP mRNA in SCRH by approximately eightfold but decreased rBCRP function to 50%. Therefore, h/rBCRP expression and functions were oppositely regulated in type 1 and type 2 diabetes mellitus subjects. Alternations in d-glucose, insulin, and free fatty acid levels in plasma might contribute to the changes in h/rBCRP expression and functions.

Development and validation of a sensitive quantification method for hematoporphyrin monomethyl ether in plasma using high-performance liquid chromatography with fluorescence detection.

A rapid, sensitive, precise and specific method for determination of hematoporphyrin monomethyl ether (HMME), a novel photodynamic therapy (PDT) drug, was developed and validated using high-performance liquid chromatography (HPLC) with fluorescence detection. HMME was isolated from the plasma by a single-step liquid-liquid extraction with ethyl acetate. The analyte and internal standard fluorescein were baseline separated on a Diamonsil C(18) analytical column (4.6 x 150 mm, 5 microm) and analyzed using a fluorescence detector with the excitation and emission wavelengths set at 395 and 613 nm, respectively. The method was linear in the concentration range 0.025-5 microg/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL. The inter- and intra-day accuracies and precisions were all within 10% and the mean recoveries of HMME and fluorescein were 95 +/- 3.7 and 90 +/- 2.3%, respectively. The analyte was stable during all sample storage, preparation and analysis periods. This method was successfully applied to a pharmacokinetic study after a single-dose intravenous administration of HMME (5 mg/kg) to beagle dogs. This method was reproducible and sensitive enough for the pharmacokinetic study of HMME. Based on the results of the pharmacokinetic study, we suggest that a rather long light-avoiding time is essential for patients under HMME therapy.

The antihyperglycaemic activity of berberine arises from a decrease of glucose absorption.

The mechanism of action of berberine as an antihyperglycaemic agent was investigated in the Caco-2 cell line. Berberine was found to effectively inhibit the activity of disaccharidases in Caco-2 cells. It also decreased sucrase activity after preincubation with Caco-2 cells for 72 hours. However gluconeogenesis and glucose consumption of Caco-2 cells were not influenced. 2-Deoxyglucose transporting through Caco-2 cell monolayers was decreased by berberine but the effect was not statistically significant. These results suggest that the antihyperglycaemic activity of berberine is at least partly due to its ability to inhibit alpha-glucosidase and decrease glucose transport through the intestinal epithelium.

[Inhibitory action of berberine on glucose absorption].

AIM: To study the absorption characteristics of berberine and its influence on glucose absorption. METHODS: Rat recirculating perfusion model was used to study berberine absorption characteristics and Caco-2 cell model was used to explore the influence of berberine on disaccharidase, using HPLC to assay the appearance of glucose to indicate enzyme activities. RESULTS: Berberine was found to be hardly absorbed in the intestine (less than 5% in 2.5 h). However, sucrase and maltase activities were found to be inhibited by berberine, its ID50 to sucrase is 1.830 mg.L-1, and showed no dose dependent influence on maltase activity. Berberine also showed influence on glucose absorption. However, this effect is not significant. CONCLUSION: Berberine may act as an alpha-glucosidase inhibitor, which is its main mechanism in diabetes treatment.

Cyclosporin A enhanced protection of nimodipine against brain damage induced by hypoxia-ischemia in mice and rats.

AIM: To study whether P-glycoprotein (P-gp) inhibitor cyclosporin A (CsA) enhanced the protection of nimodipine (NMD) against brain damage. METHOD: (1) After mice were given ip NMD alone or co-administration of CsA, survival time of mice were recorded following decapitation and ip injection of NaNO2, respectively. (2) After rats were given ip NMD alone or co-administration of CsA, 20 min forebrain ischemia induced by the technique of two-carotid occlusion plus hypovolemic hypotension. Following reperfusion of 1 h, the content of malondialdehyde (MDA), lactic acid (LA) and activity of lactic dehydrogenase (LDH) in cortex tissue were measured. (3) NMD level in brain was also determined after ip injection of NMD 2 mg/kg alone and co-administration of CsA, respectively. RESULTS: NMD showed potent pharmacological activity in the three models. The survival time of mice by decapitation and ip NaNO2 were significantly (P <0.05) prolonged after co-administration of CsA. In rat forebrain ischemia/reperfusion, levels of MDA, LA, and LDH by co-administration of CsA were greatly modified, compared with those of NMD alone group. The level of NMD in rat brain was increased markedly after co-administration of CsA. CONCLUSION: P-gp inhibitor CsA may enhance the protection of NMD against brain damage.

The involvement of P-glycoprotein in berberine absorption.

Berberine is an important ingredient in a number of traditional Chinese medicines but has been shown to have poor bioavailability in the dog. The aim of this study was to use the P-glycoprotein (P-glycoprotein) inhibitors cyclosporin A, verapamil and the monoclonal antibody C219 in in vivo and in vitro models of intestinal absorption to determine the role of P-glycoprotein in berberine absorption. In the rat recirculating perfusion model, berberine absorption was improved 6-times by P-glycoprotein inhibitors. In the rat everted intestinal sac model, berberine serosal-to-mucosal transport was significantly decreased by cyclosporin A. In Ussing-type chambers, the rate of serosal-to-mucosal transport across rat ileum was 3-times greater than in the reverse direction and was significantly decreased by cyclosporin A. In Caco-2 cells, berberine uptake was significantly increased by P-glycoprotein inhibitors and by monoclonal antibody C219. P-glycoprotein appears to contribute to the poor intestinal absorption of berberine which suggests P-glycoprotein inhibitors could be of therapeutic value by improving its bioavailability.