Simultaneous assessment of absorption and pharmacokinetic characteristics of four active flavonoids from Chimonanthus nitens Leaf Granules using LC-MS determination: in vivo and in vitro.

A sensitive UPLC-HRMS method was developed and validated for simultaneous quantification of four active flavonoids from Chimonanthus nitens Leaf Granules (CNLG) in biological matrix. The method was utilized in pharmacokinetic study of the four flavonoids in rats as well as other evaluation assays in vitro. It was revealed that rutin, nicotiflorin, and astragalin had poor oral bioavailability in rats possibly due to low intestinal permeability and metabolism in intestinal flora. Kaempferol underwent rapid glucuronidation and sulphation in rat plasma with medium permeability coefficient. The results provided valuable data for future research and development of CNLG flavonoids.

[Intestinal absorption components and absorption characteristics of Wuwei Qingzhuo San by everted intestinal sac models].

This study investigated the absorption profile of Wuwei Qingzhuo San in different intestinal segments and the absorption characteristics of its alkaloids(piperine, piperanine, piperlonguminine, and dihydropiperlonguminine). The everted gut sac model was established, and the chemical components of Wuwei Qingzhuo San in different intestinal segments were detected by UPLC-Q-TOF-MS. The content of piperine, piperanine, piperlonguminine, and dihydropiperlonguminine in intestinal absorption fluid was determined by UPLC-Q-TRAP-MS and the absorption parameters were calculated. The absorption characteristics in different intestinal segments at different time were analyzed. As a result, 27, 27, 8, and 6 absorbent components from Wuwei Qingzhuo San were detected in the intestinal cyst fluid of jejunum, ileum, duodenum, and colon by UPLC-Q-TOF-MS technology, respectively. It was also found that piperine, piperanine, piperlonguminine, and dihydropiperlonguminine from Wuwei Qingzhuo San showed linear absorption in various intestinal segments, with r values exceeding 0.9. In terms of absorption content, the components were ranked as piperine>piperanine>dihydropiperlonguminine>piperlonguminine in various intestinal segments, but the absorption rate and mechanism of each component varied. The results demonstrate that the absorption of the components of Wuwei Qingzhuo San in different intestinal segments is selective and is not a simple semi-permeable membrane permeation process.

OAT3 Participates in Drug-Drug Interaction between Bentysrepinine and Entecavir through Interactions with M8-A Metabolite of Bentysrepinine-In Rats and Humans In Vitro.

Bentysrepinine (Y101) is a novel phenylalanine dipeptide for the treatment of hepatitis B virus. Renal excretion played an important role in the elimination of Y101 and its metabolites, M8 and M9, in healthy Chinese subjects, although the molecular mechanisms of renal excretion and potential drug-drug interactions (DDIs) remain unclear. The present study aimed to determine the organic anion transporters (OATs) involved in the renal disposition of Y101 and to predict the potential DDI between Y101 and entecavir, the first-line agent against HBV and a substrate of OAT1/3. Pharmacokinetic studies and uptake assays using rat kidney slices, as well as hOAT1/3-HEK293 cells, were performed to evaluate potential DDI. The co-administration of probenecid (an inhibitor of OATs) significantly increased the plasma concentrations and area under the plasma concentration-time curves of M8 and M9 but not Y101, while reduced renal clearance and the cumulative urinary excretion of M8 were observed in rats. The time course of Y101 and M8 uptake via rat kidney slices was temperature-dependent. Moreover, the uptake of M8 was inhibited significantly by probenecid and benzylpenicillin, but not by p-aminohippurate or tetraethyl ammonium. M8 was found to be a substrate of hOAT3, but Y101 is not a substrate of either hOAT1 or hOAT3. Additionally, the entecavir inhibited the uptake of M8 in the hOAT3-transfected cells and rat kidney slices in vitro. Interestingly, no significant changes were observed in the pharmacokinetic parameters of Y101, M8 or entecavir, regardless of intravenous or oral co-administration of Y101 and entecavir in rats. In conclusion, M8 is a substrate of OAT3 in rats and humans. Furthermore, M8 also mediates the DDI between Y101 and entecavir in vitro, mediated by OAT3. We speculate that it would be safe to use Y101 with entecavir in clinical practice. Our results provide useful information with which to predict the DDIs between Y101 and other drugs that act as substrates of OAT3.

Effect of X-ray radiation on the pharmacokinetics of apatinib in vivo in rats.

Purpose: The "radiotherapy-pharmacokinetic" ("RT-PK") phenomenon refers to the fact that radiation can significantly alter the pharmacokinetic behavior of a drug. At present, it is not clear whether there is an "RT-PK" phenomenon that can affect apatinib during concurrent chemoradiotherapy. In this study, we used a rat irradiation model to study the effects of X-ray radiation on absorption, tissue distribution, and excretion of apatinib. Method: Healthy Sprague-Dawley (SD) rats were randomly divided into control and radiation groups. The radiation group was given an appropriate dose of abdominal X-ray radiation, while the control group was not given irradiation. After 24 h of recovery, both groups were given apatinib solution 45 mg/kg by gavage. A quantitative LC-MS/MS method was developed to determine the concentration of apatinib in the rats, so as to compare the differences between the control and radiation groups and thus investigate the modulating effect of radiation on the pharmacokinetics of apatinib in rats. Results: After abdominal X-ray irradiation, the area under the curve (AUC0-t) of apatinib in rat plasma decreased by 33.8% and 76.3% at 0.5 and 2 Gy, respectively. Clearance (CL) and volume of distribution (Vd) increased and were positively correlated with radiation dose. X-ray radiation significantly reduced the concentration of apatinib in the liver and small intestine, and there was no tissue accumulation. In excretion studies, we found that X-ray radiation reduced the cumulative excretion of apatinib in feces and urine by 11.24% and 86.17%, respectively. Conclusion: Abdominal X-ray radiation decreased plasma exposure, tissue distribution, and excretion of apatinib in rats, suggesting that the RT-PK phenomenon affects apatinib. We speculate that this RT-PK phenomenon is closely related to changes in metabolic enzymes in vivo. In clinical practice, when apatinib is combined with radiotherapy, attention should be paid to adjusting the dose of apatinib and optimizing the treatment plan to alleviate the adverse effects of this RT-PK phenomenon.

[Identification of chemical constituents in Zhachong Shisanwei Pills by UPLC-Q-TOF-MS/MS].

Zhachong Shisanwei Pills, composed of 13 Chinese medicinal materials, are used for treating the diseases such as hemiplegia, pain of muscles and bones, rheumatism, and joint pain. The chemical composition and pharmacodynamics of Zhachong Shisanwei Pills have not been reported. Ultra-performance liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS) was employed to quickly identify the chemical components of Zhachong Shisanwei Pills, which was performed with Shim-pack GIST C_(18) column(4.6 mm×150 mm, 5 µm). The gradient elution was conducted with methanol-0.05% acetic acid as the mobile phase. Electrospray ionization mass spectrometry(ESI-MS) was carried out in both positive and negative ion modes. The compounds were identidied based on accurate relative molecular weight, fragment ion species, and the MS data of reference substances and in literature. In conclusion, a total of 98 compounds were identified, including 19 organic acids, 36 flavonoids, 13 volatile oils, 8 tannins, 5 2-(2-phenylethyl)chromones, 5 amino acids, 3 sesquiterpenoids, 3 alkaloids, and 2 other compounds. This study characte-rized the chemical components of Zhachong Shisanwei Pills rapidly for the first time, laying a foundation for further research on the pharmacodynamic material basis and quality evaluation.

Mitochondrion-targeted supramolecular "nano-boat" simultaneously inhibiting dual energy metabolism for tumor selective and synergistic chemo-radiotherapy.

Rationale: Tumor energy metabolism has been a well-appreciated target of cancer therapy; however, the metabolism change of cancer cells between oxidative phosphorylation and glycolysis poses a challenge to the above. In this study, we constructed an innovative mitochondrion-targeted supramolecular "nano-boat" based on peptide self-assembly for tumor combined chemo-radiotherapy by simultaneously inhibiting the dual energy metabolism. Methods: A lipophilic self-assembled peptide and a positively charged cyclen were integrated to fabricate a brand new mitochondrion-targeted nano-platform for the first time. The indices of mitochondrial dysfunction including mitochondrial membrane potential, apoptosis proteins expression and ultrastructure change were evaluated using a JC-1 probe, western blotting and biological transmission electron microscopy, respectively. Energy metabolism assays were conducted on a Seahorse XF24 system by detecting the oxygen consumption rate and the glycolytic proton efflux rate. The radio-sensitization effect was investigated by colony formation, the comet assay, and γ-H2AX staining. Results: The supramolecular "nano-boat" could selectively kill cancer cells by much higher enrichment and reactive oxygen species generation than those in normal cells. In the cancer cells treated with the supramolecular "nano-boat", the dysfunctional morphological changes of the mitochondrial ultrastructure including swelling and pyknosis were evidently observed, and the endogenous mitochondrial apoptosis pathway was effectively triggered by abundant of cytochrome C leaking out. Concurrently, the dual metabolic pathways of glycolysis and oxidative phosphorylation were severely inhibited. More importantly, the supramolecular "nano-boat" displayed an excellent radio-sensitization effect with a sensitization enhancement ratio value as high as 2.58, and hence, in vivo efficiently combining radiotherapy yielded an enhanced chemo-radiotherapy effect. Conclusion: Our study demonstrated that the rationally designed peptide-based "nano-boat" could efficiently induce cancer cell apoptosis by the energy metabolism inhibition involving multiple pathways, which may provide the motivation for designing novel and universal mitochondria-targeted drug delivery systems for cancer therapy.

Phenotype-Based HPLC-Q-TOF-MS/MS Coupled With Zebrafish Behavior Trajectory Analysis System for the Identification of the Antidepressant Components in Methanol Extract of Anshen Buxin Six Pills.

Phenotype screening has become an important tool for the discovery of active components in traditional Chinese medicine. Anshen Buxin Six Pills (ASBX) are a traditional Mongolian medicine used for the treatment of neurosis in clinical settings. However, its antidepressant components have not been explicitly identified and studied. Here, the antidepressant effect of ASBX was evaluated in adult zebrafish. High performance liquid chromatography-mass spectrometry (HPLC-Q-TOF-MS/MS) was combined with zebrafish behavior trajectory analysis to screen and identify the antidepressant-active extract fraction and active components of ASBX. Finally, the antidepressant effect of the active ingredients were verified by the behavior, pathology, biochemical indices and protein level of adult fish. The novel tank driving test (NTDT) showed that ASBX can effectively improve the depressive effect of reserpine on zebrafish. Petroleum ether and dichloromethane extracts of ASBX were screened as antidepressant active extracts. Costunolide (COS) and dehydrocostus lactone (DHE) were screened as the active components of ASBX. COS had been shown to significantly improve the depressive behavior, nerve injury and neurotransmitter levels (5-hydroxytryptamine (5-HT) and norepinephrine (NE)) of zebrafish by inhibiting the high expression of serotonin transporter and norepinephrine transporter induced by reserpine suggesting the antidepressant effect of COS may be related to its effect on 5-HT and NE pathways. This study provided a phenotype based screening method for antidepressant components of traditional Chinese medicines, so as to realize the separation, identification and activity screening of components at the same time.

Tricarboxylate Citrate Transporter of an Oleaginous Fungus Mucor circinelloides WJ11: From Function to Structure and Role in Lipid Production.

The citrate transporter protein (CTP) plays an important role in citrate efflux from the mitochondrial matrix to cytosol that has great importance in oleaginous fungi. The cytoplasmic citrate produced after citrate efflux serves as the primary carbon source for the triacylglycerol and cholesterol biosynthetic pathways. Because of the CTP's importance, our laboratory has extensively studied its structure/function relationships in Mucor circinelloides to comprehend its molecular mechanism. In the present study, the tricarboxylate citrate transporter (Tct) of M. circinelloides WJ11 has been cloned, overexpressed, purified, kinetically, and structurally characterized. The Tct protein of WJ11 was expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system for kinetic studies. Our results showed that Tct has a high affinity for citrate with Km 0.018 mM. Furthermore, the tct overexpression and knockout plasmids were created and transformed into M. circinelloides WJ11. The mitochondria of the tct-overexpressing transformant of M. circinelloides WJ11 showed a 49% increase in citrate efflux, whereas the mitochondria of the tct-knockout transformant showed a 39% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. To elucidate the structure-function relationship of this biologically important transporter a 3D model of the mitochondrial Tct protein was constructed using homology modeling. The overall structure of the protein is V-shaped and its 3D structure is dimeric. The transport stability of the structure was also assessed by molecular dynamics simulation studies. The activity domain was identified to form hydrogen bond and stacking interaction with citrate and malate upon docking. Tricarboxylate citrate transporter has shown high binding energy of -4.87 kcal/mol to citric acid, while -3.80 kcal/mol to malic acid. This is the first report of unraveling the structural characteristics of WJ11 mitochondrial Tct protein and understanding the approach of the transporting toward its substrate. In conclusion, the present findings support our efforts to combine functional and structural data to better understand the Tct of M. circinelloides at the molecular level and its role in lipid accumulation.

Investigation of the metabolites of five major constituents from Berberis amurensis in normal and pseudo germ-free rats.

Berberis amurensis (Berberidaceae) is a traditional Chinese medicine, which is often used to treat hypertension, inflammation, dysentery and enteritis. It contains alkaloids, mainly including berberine, berbamine, magnoflorine, jatrorrhizine and palmatine. Berberis amurensis extracts (BAEs) is often orally taken. Oral herbs might be metabolized by intestinal bacteria in the small intestine. However, the interaction between the herb and the gut microbiota is still unknown. In the current study, UPLC/Q-TOF-MS/MS combined with Metabolitepilot and Peakview software was used to identify the metabolites of BAEs in anti-biotic cocktail induced pseudo germ-free rats and normal rats. As a result, a total of 46 metabolites in normal rats were detected and its main metabolic pathways include demethylation, dehydrogenation, methylation, hydroxylation, sulfation and glucuronidation. Only 29 metabolites existed in pseudo germ-free rats. Dehydrogenated metabolites (M29, M30, M34 and M36), methylated metabolites (M33, M41 and M46) and other metabolites were not detected in pseudo germ-free rats. The result implied that the intestinal bacteria have an influence on the metabolism of BAEs. Furthermore, this investigation might contribute to the understanding of the metabolism of BAEs, and further promote its clinical application.

Molecular Mechanism of Citrate Efflux by the Mitochondrial Citrate Transporter CT in Filamentous Fungus Mucor circinelloides WJ11.

The mitochondrial citrate transporter (MCT) plays an important role in citrate efflux from the mitochondria in eukaryotes, and hence provides a direct correlation between carbohydrate metabolism and lipid synthesis. Our previous studies on transporters confirmed the presence of two MCTs (TCT and CT) in oleaginous Mucor circinelloides WJ11 associated with high lipid accumulation. However, the molecular mechanism of citrate efflux from the mitochondria by MCT in M. circinelloides is still unclear. To study the citrate transport mechanism of CT, the citrate transporter gene was expressed in Escherichia coli, and its product was purified. The citrate transport activity of the protein was studied in CT reconstituted liposomes. Our results showed high efficiency of CT for [14C] citrate/citrate exchange with K m 0.01 mM at 25°C. Besides citrate, other molecules such as oxaloacetate, malate, fumarate, succinate aconitate, oxoadipate, isocitrate, and glutamate also promote citrate transport. In addition, the ct overexpression and knockout plasmids were constructed and transferred into M. circinelloides WJ11, and the mitochondria were isolated, and the transport activity was studied. Our findings showed that in the presence of 10 mM malate, the mitochondria of ct-overexpressing transformant showed 51% increase in the efflux rate of [14C] citrate, whereas the mitochondria of the ct-knockout transformant showed 18% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. This study provided the first mechanistic evidence of citrate efflux from the mitochondria by citrate transporter in oleaginous filamentous fungus M. circinelloides, which is associated with high lipid accumulation.

A coassembled peptide hydrogel boosts the radiosensitization of cisplatin.

We constructed a novel supramolecular hydrogel by carrying out a coassembly of cisplatin and short naproxen-capped peptides. This procedure boosted the radiosensitization effect of cisplatin by increasing the number of Pt-DNA adducts, arresting the cell cycle, and inhibiting cyclooxygenase-2.

In vitro metabolism and in vivo pharmacokinetics of bentysrepinine (Y101), an investigational new drug for anti-HBV-infected hepatitis: focus on interspecies comparison.

The objective of this study was to clarify the species differences of pharmacokinetics of Y101 (N-[N-benzoyl-O-(2-dimethylaminoethyl)-l-tyrosyl]-l-phenylalaninol hydrochloride), a derivative of herbal ingredient with anti-HBV hepatitis activity, in rats, dogs, monkeys and humans.The metabolic stability and metabolite identification studies using liver microsomes in vitro, plasma protein binding using a rapid equilibrium dialysis in vitro, pharmacokinetic studies in vivo were carried out to evaluate the interspecies differences. The toxicokinetic study in monkeys was also investigated.The metabolic profiles were similar in monkeys and humans, which were significant different from rats and dogs in vitro. In vitro plasma protein binding showed no major differences between species with medium to high protein binding rates. After single oral dose to rats, dogs, and monkeys, the absolute oral bioavailability of Y101 was 44.9%, 43.1%, and 19.2%, respectively. There was no accumulation for Y101 toxicokinetics in monkeys after oral administration for 90 d.The metabolic profiles indicated monkey was the very animal model for preclinical safety evaluation of Y101. Our results have demonstrated the favorable pharmacokinetics profile of Y101, which supports the clinical trials in humans.

Direct quantification of intact FIM in monkey plasma using a selective chromatography-tandem mass spectrometry method: Application in a pharmacokinetic study.

FIM protein, which consists of 155 amino acids, was developed as a novel GLP-1 analog to reduce blood glucose, and pharmacodynamic results showed that it had a certain effect when used in treating Alzheimer's disease. The molecular weight of FIM is 16,304 Da. In theory, the concentration of FIM in biological samples should be determined by the ligand binding assay method or indirectly quantified using LC-MS/MS instrumentation. However, the above methods are complex and time-consuming. In this study, we successfully developed a simpler LC-MS/MS method for directly quantifying the intact FIM protein in monkey plasma for the first time. The chromatographic separation of FIM was achieved using an InertSustain Bio C18 column with a mobile phase of acetonitrile containing 0.1% formic acid (A)-water containing 0.1% formic acid (B) at a flow rate of 0.3 ml/min. Good linearity was observed in the concentration range of 5-500 ng/ml (r2 > 0.99). The intra- and inter-day precisions (expressed as relative standard deviation, RSD) of FIM were 2.30-12.8 and 7.30-13.2%, respectively. The intra- and inter-day accuracies (expressed as a relative error, RE) were -12.7-6.55 and - 10.1-0.892%, respectively. This method was successfully applied for a pharmacokinetic study of the FIM protein in four monkeys after subcutaneous administration.

Carrier-Free Supramolecular Hydrogel Composed of Dual Drugs for Conquering Drug Resistance.

The resistance of tumor cells to anticancer drugs has become one of the principal causes of the failure in clinical chemotherapy. To overcome this issue, developing feasible drug delivery systems for effective cancer therapy is urgently needed. In this work, we construct an amphiphilic drug self-delivery system consisting of Taxol and tyroservatide (YSV) to overcome drug resistance. The carrier-free supramolecular hydrogel composed of nanofibers is formed by the involved ester bond self-hydrolysis process, which has high drug loading efficiency and facilitates the delivery of both the hydrophobic Taxol and hydrophilic YSV. Because of the dual inhibitory function of YSV on histone deacetylase and P-glycoprotein, an improved combinational anticancer effect of the molecule against drug-resistant tumor cells in vitro is achieved. Furthermore, the designed drug self-delivery system exhibited enhanced antitumor efficiency and favorable biocompatibility in vivo when administered by tail vein injection. Our study provides a new strategy for fabricating a carrier-free supramolecular hydrogel to overcome drug resistance, which might open up an alternative avenue for the tumor combinational therapy.

Metabolite identification of bentysrepinine (Y101), a novel anti-HBV agent in rats using a five-step strategy based on a combined workflow with two different platforms of liquid chromatography-tandem mass spectrometry.

Bentysrepinine (Y101), a derivative of repensine (a compound isolated from Dichondrarepens Forst), is a novel phenyalanine dipeptide inhibiting DNA-HBV and cccDNA activities and is currently under development for the treatment of hepatitis B virus (HBV)-infected hepatitis. Our previous study implied that there might be an existence of extensive metabolism of Y101 in rats. Therefore, it is necessary to perform metabolic profiling study to further evaluate its safety and drug-like properties. In this study, the metabolism of Y101 in rats was investigated by a convincible five-step strategy to characterize metabolites in plasma and that excreted into urine, bile and feces. The five-step strategy was realized by using an combined workflow on two different MS platforms, including various scan modes of liquid chromatography with hybrid quadruple-linear ion trap mass spectrometry (LC-QTRAP-MS/MS) and various post-acquiring data mining tools of liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS). QTOF MS/MS was employed as a powerful complementary tool to enable high confidence of metabolites identification using its functions of accurate MS and MS/MS fragmentation. As a result, a total of 30 metabolites were detected, including 25 phase I and 5 phase II metabolites. Among them, four primary metabolites (M6-M9) were further identified by comparing with the authentic standards chemically synthesized. The possible metabolic pathways of Y101 in rats were proposed to be amide hydrolysis, monohydroxylation, dihydroxylation, N-oxidation, demethylation, methylation, glucosidation and glucuronidation. This is the first study of the metabolism of Y101 in rats. The five-step strategy was successfully used to systematically characterize metabolites of Y101 in rats, and it would be generally applied for metabolite identification of new drug candidate.

Research Advances on Hepatotoxicity of Herbal Medicines in China.

In general, herbal medicines have been considered as safe by the general public, since they are naturally occurring and have been applied in treatment for over thousands of years. As the use of herbal medicine is rapidly increasing globally, the potential toxicity of herbal drugs, in particular drug-induced liver injury (DILI), has now become a serious medical issue. According to the literature, the authors analyzed and discussed the hepatotoxicity problem of Chinese herbal medicines (CHM), including global overview on herbal-induced liver injury (HILI), current research progress on toxic CHM, diagnosis and treatment of HILI, and modern approaches and technologies of study of hepatotoxicity. As to promote the recognition of HILI and tackle the issue, a guideline for the diagnosis and treatment of HILI has recently been drafted by Chinese scientists. As suggested by the guideline, the hepatotoxicity issue of CHM, as a matter of fact, is overestimated. Up to date, the investigation of hepatotoxicity of CHM is now booming with worldwide application of CHM. This review therefore provides useful information for investigating hepatotoxicity of herbal medicine and characterizing DILI caused by CHM. In addition, authors describe in which way further efforts should be made to study the rationale of CHM and liver injury.

[The in vitro transport mechanism of bentysrepinine: a novel anti-hepatitis B virus drug candidate].

Bentysrepinine (Y101), a derivative of phenylalanine dipeptide, is a novel drug candidate for the treatment of hepatitis B virus (HBV) infection. Our previous preclinical pharmacokinetic study showed that its in vivo absorption and distribution characteristics were probably related to transmembrane transport after Y101 was administered intragastically in rats. In this study, Caco-2 and MDCK-MDR1 cell models were used to investigate interactions between Y101 and P-gp through the apparent permeation coefficient (P(app)) and efflux ratio (RE); the results showed that Y101 was a substrate of P-gp. In addition, gene-transfected cell models, HEK293-h OATP1B1, HEK293-h OATP2B1 and CHO-PEPT1 were used to evaluate the affinity to OATP1B1, OATP2B1 and PEPT1. The results suggest that Y101 has a weak inhibitory effect on OATP1B1 and OATP2B1, and Y101 may not be substrates of OATP1B1, OATP2B1 or PEPT1. The above results can be used to explain the in vivo absorption and distribution characteristics, and to provide a scientific basis for the further development of Y101.

[The in vitro inhibition and induction of cytochrome P450 activities by bentysrepinine: a novel candidate of anti-hepatitis B virus drug].

Bentysrepinine (Y101), a derivative of phenyalanine dipeptide, has a novel mechanism in the treatment of hepatitis B virus (HBV) infection with a good anti-HBV effect. In the present study, a fluorometric-based high throughput method using cytochrome P450 (CYP) screening kit was adopted to evaluate in vitro inhibition potential of Y101 on CYP isoenzymes by calculating remaining enzyme activities and inhibitory potential (IC(50) values) using the determined values of fluorescence intensity. The result showed that Y101 exhibited little activity in the inhibition of CYP1A2, CYP3A4, CYP2C9, CYP2C19 and CYP2D6 (IC(50) > 100 µmol·L(-1)). Y101 was used to treat human primary hepotocytes for 72 h, and the enzyme activities of CYP1A2, CYP2B6 and CYP3A4 were determined with a cocktail of probe substrates for the three CYP isoforms. The metabolites were simultaneously determined using a LC-MS/MS method. Y101 had no activity in the induction of CYP1A2, CYP2B6 and CYP3A4 on the basis of the following results: 1 The ratio of enzyme activities between test and control groups were all below than 1 (varied from 0.662 to 0.928); 2 The induction potential of Y101 were lower than forty percent compared with that of positive groups. The above results suggest that Y101 has little activity in the regulation of metabolic drug-drug interactions based on the CYP isoform changes following co-administration of drugs.

Effects of Drug Transporters on Pharmacological Responses and Safety.

Recently, it is realized that transporters, apart from enzymes, play a key role in drug metabolism and pharmacokinetics. More and more pharmaceutical researchers focused on transporter study and found that drug transporters not only involved in pharmacokinetics including absorption, distribution, metabolism and excretion (ADME). but also in Drug-Drug interactions (DDIs). DDIs induced by drug transporters are the important safety evaluation factors which have to be taken into account at stage of drug discovery and development. Therefore, it should pay more attention to the studies on step of preclinical and clinical trial. In this review, the author focused on the effects of drug transporters on pharmacological and safety responses, such as the effects on plasma elimination half-lives, on drug accumulation in body after repeated dosing, on potentiating either pharmacological or adverse effects and molecular mechanisms of transporter-mediated DDIs. Present studies showed that DDIs involving the drug transporters including ABC transporters, organic anion and cation transporters, peptide transporters, monocarboxylate transporters, nucleoside transporters and folate transporters, and the possible side effects derived from clinical combination therapy must pay attention. The author also discussed the molecular mechanisms of transporter-mediated DDIs by the data obtained from preclinical and clinical studies, and inferred the available curative effects and the potential risk of the drug combination involving these drug transporters, which provides a reference for the safety of clinical medication and a consideration for a successful drug discovery. This article carefully reviewed transporter-based DDIs and highlighted areas that DDIs were poorly predicted through transporters or areas are still confronted with challenges in future.

Quantitation of bentysrepinine (Y101) in rat plasma by liquid chromatography tandem mass spectrometry: application to pharmacokinetic study.

A simple, accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of bentysrepinine (Y101) in rat plasma. After the addition of diphenhydramine (internal standard, IS), plasma samples were pretreated by protein precipitation. Chromatographic separation was carried out on an Atlantis(®) analytical column (4.6 mm × 100 mm, 5 µm, Waters) with methanol: 20 mM ammonium formate consisting of 1.0% formic acid (65:35, v/v) as the mobile phase at an isocratic flow rate of 0.4 mL/min for 7.5 min. The multiple reaction monitoring (MRM) transitions were performed at m/z 490.2→339.5 for Y101 and m/z 256.0→167.0 for IS on a SCIEX API 4000 mass spectrometer in the positive ion mode with electrospray ionization (ESI) source. Good linearity was achieved over the concentration range of 1-2500 ng/mL. The intra- and inter-day precisions were less than 8.3%, and the accuracy ranged from -4.0% to 2.8%. Y101 was stable during the analysis and the storage period. The pharmacokinetic profiles of Y101 at three dose levels were successfully studied for the first time in rats by this method. After single intra-gastric administration of Y101 at the doses of 25, 50 and 100 mg/kg, C(max) and AUC(0-t) were proportional to the doses given.