Guifang Dou

The performance of a fly-larva shell-derived chitosan sponge as an absorbable surgical hemostatic agent.

Chitosan is a versatile biomaterial lately used as a new generation of local hemostatic agent approved to date for external use only. Here we introduced a fly-larva shell-derived chitosan sponge (CS) and its feasibility for internal use as an absorbable surgical hemostatic agent was evaluated in a rat hepatic hemorrhage model. CS was a better implantable hemostatic material than gelatin sponge (GS) or oxidized cellulose (OC) in both the acute and chronic bleeding models. The better efficacy of CS may be due to its greater ability to enhance platelet activation, erythrocyte aggregation and morphological alteration, and thrombin generation at sites it is applied than GS or OC. Moreover, preliminary safety evaluations have demonstrated excellent blood and cell compatibility in hemorheological measurements, blood coagulation analysis, histological evaluations and hepatocytes culture experiments. None of CS, GS, or OC induced acute inflammation or other adverse effects while normal tissue growth and vascularization were observed in each case 4 weeks after each hemostatic agent had been implanted. Thus, CS has promising properties as an absorbable, implantable agent for promoting perioperative hemostasis and this material warrants further study.

Effects of genipin cross-linking of chitosan hydrogels on cellular adhesion and viability.

PURPOSE The aim of the present study was to investigate the effects of genipin (Gp) cross-linking of chitosan (CHI) hydrogels on the cell adhesion and viability. METHOD Series of Gp crosslinked CHI hydrogels were prepared by incubation of solutions containing a mixture of Gp and CHI in different ratios. The resulting hydrogels were characterized by scanning electron microscopy (SEM), parallel plate rheometer, contact angle and swelling ratio measurement. The in vitro cytocompatibility of hydrogels was evaluated with L929 fibroblasts by MTT method. The cell adhesion morphology on gel surface was characterized by SEM, and the cell viability was assessed through cell count and flow cytometry analysis. RESULTS It was found that macroporous structure of the CHI hydrogels could be tailored by varying Gp or CHI amount. Gp cross-linking of hydrogels enhanced their storage modulus significantly, and also altered their hydrophilicity and swell properties. The MTT results revealed that the cross-linked hydrogels did not induce cytotoxic effects. Cell count and flow cytometry analysis demonstrated that denser surface milieu of hydrogels could facilitate better cell adhesion and viability. CONCLUSIONS It could be concluded that increased cross-linking density significantly improved the cell adhesion and viability on hydrogel surface. This research provides prospective biocompatible approaches by making gel stiffness modifications to hydrogel scaffolds for the purpose of different tissue engineering.

Enhanced oral bioavailability and anti-tumour effect of paclitaxel by 20(s)-ginsenoside Rg3 in vivo.

The purpose of this study was to investigate the effect of paclitaxel in combination with 20(s)‐ginsenoside Rg3 on its anti‐tumour effect in nude mice. In the Caco‐2 transport assay, the apparent permeability from the apical side to the basal side (Papp) (A‐B) and Papp (B‐A) of paclitaxel were measured when co‐incubated with different concentrations of 20(s)‐ginsenoside Rg3. The results indicated that the penetration of paclitaxel through the Caco‐2 monolayer from the apical side to the basal side was facilitated by 20(s)‐ginsenoside Rg3 in a concentration‐dependent manner. Meanwhile, 20(s)‐ginsenoside Rg3 inhibited P‐glycoprotein (P‐gp), and the maximum inhibition was achieved at 80 µ m (p < 0.05). The pharmacokinetic parameters of paclitaxel after oral co‐administration of paclitaxel (40 mg/kg) with various doses of 20(s)‐ginsenoside Rg3 in rats were investigated by an in vivo pharmacokinetic experiment. The results showed that the AUC of paclitaxel co‐administered with 20(s)‐ginsenoside Rg3 was significantly higher (p < 0.001 at 10 mg/kg) compared with the control. The relative bioavailability (RB) % of paclitaxel with 20(s)‐ginsenoside Rg3 was 3.4‐fold (10 mg/kg) higher than that of the control. The effect of paclitaxel orally co‐administered with 20(s)‐ginsenoside Rg3 against human tumour MCF‐7 xenografts in nude mice was also evaluated. Paclitaxel (20 mg/kg) co‐administered with 20(s)‐ginsenoside Rg3 (10 mg/kg) exhibited an effective anti‐tumour activity with the relative tumor growth rate (T/C) values of 39.36% (p <0.05). The results showed that 20(s)‐ginsenoside Rg3 enhanced the oral bioavailability of paclitaxel in rats and improved the anti‐tumour activity in nude mice, indicating that oral co‐administration of paclitaxel with 20(s)‐ginsenoside Rg3 could provide an effective strategy in addition to the established i.v. route. Copyright

Evaluation of genipin-crosslinked chitosan hydrogels as a potential carrier for silver sulfadiazine nanocrystals

In the present study genipin crosslinked chitosan (CHI) hydrogels, which had been constructed and reported in our previous studies (Gao et al., 2014 [22]), were further evaluated for their advantage as a carrier for silver sulfadiazine (AgSD) nanocrystal systems. Firstly, AgSD nanocrystals with a mean particle size of 289nm were prepared by wet milling method and encapsulated into genipin crosslinked CHI hydrogels. AgSD nanocrystals displayed a uniform distribution and very good physical stability in the hydrogel network. Swelling-dependent release pattern was found for AgSD nanocrystals from hydrogels and the release profile could be well fitted with Peppas equation. When AgSD nanocrystals were encapsulated in hydrogels their fibroblast cytotoxicity decreased markedly, and their antibacterial effects against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were still comparable to unencapsulated AgSD nanocrystals. In vivo evaluation in excision and burn cutaneous wound models in mice showed that AgSD nanocrystal hydrogels markedly decreased the expression of inflammatory cytokine IL-6, but increased the levels of growth factors VEGF-A and TGF-β1. Histopathologically, the wounds treated by hydrogels containing AgSD nanocrystals showed the best healing state compared with commercial AgSD cream, hydrogels containing AgSD bulk powders and blank hydrogels. The wounds treated by AgSD nanocrystal hydrogels were dominated by marked fibroblast proliferation, new blood vessels and thick regenerated epithelial layer. Sirius Red staining assay indicated that AgSD nanocrystal hydrogels resulted in more collagen deposition characterized by a large proportion of type I fibers. Our study suggested that genipin-crosslinked CHI hydrogel was a potential carrier for local antibacterial nanomedicines.

A novel exendin-4 human serum albumin fusion protein, E2HSA, with an extended half-life and good glucoregulatory effect in healthy rhesus monkeys.

Glucagon-like peptide-1 (GLP-1) has attracted considerable research interest in terms of the treatment of type 2 diabetes due to their multiple glucoregulatory functions. However, the short half-life, rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) and excretion, limits the therapeutic potential of the native incretin hormone. Therefore, efforts are being made to develop the long-acting incretin mimetics via modifying its structure. Here we report a novel recombinant exendin-4 human serum albumin fusion protein E2HSA with HSA molecule extends their circulatory half-life in vivo while still retaining exendin-4 biological activity and therapeutic properties. In vitro comparisons of E2HSA and exendin-4 showed similar insulinotropic activity on rat pancreatic islets and GLP-1R-dependent biological activity on RIN-m5F cells, although E2HSA was less potent than exendin-4. E2HSA had a terminal elimation half-life of approximate 54 h in healthy rhesus monkeys. Furthermore, E2HSA could reduce postprandial glucose excursion and control fasting glucose level, dose-dependent suppress food intake. Improvement in glucose-dependent insulin secretion and control serum glucose excursions were observed during hyperglycemic clamp test (18 h) and oral glucose tolerance test (42 h) respectively. Thus the improved physiological characterization of E2HSA make it a new potent anti-diabetic drug for type 2 diabetes therapy.

In vivo and in vitro metabolism of lefucoxib in rats

The in vivo and in vitro biotransformation study of lefucoxib, 5-(3,4-dimethyl-phenyl)-1-methanesulfonyl-3-trifluoromethol-pyrazole, a new cyclooxygenase-2 (COX-2) inhibitor, was investigated in rats. To conduct the in vivo metabolism study, Wistar rats received lefucoxib in an oral dose, then their plasma and excreta were collected and analyzed. Through HPLC coupled with fluorescence detector and LC-MSn analysis, hydroxylation was found to be the primary metabolism pathway of lefucoxib in rats. The chemical structure of the di-hydroxy metabolite was identified by MSn spectra, and it was detected in rat plasma, urine and feces after an oral dose. However, the chemical structure of mono-hydroxy metabolites could not be identified by MSn analysis due to the existence of two similar methyls on the phenyl ring of rofecoxib. To solve this problem, in vitro metabolism studies with liver microsome incubation helped accumulate enough metabolites for 1H NMR analysis, which was employed and proved to be successful. Through further analysis of 1H-(1)H correlated spectroscopy (1H-(1)H COSY), chemical structures of two isomeric metabolites (mono-hydroxy metabolites) which had the same retention time in chromatograms were identified. The quantitative ratio of the two isomeric metabolites was also clarified to be 1:2 after analysis of the integrating height of 1H NMR signals. Additionally, the present study illustrated the co-application of in vivo and in vitro metabolism on drug metabolite identification.

An improved LC-MS/MS method for simultaneous determination of 1,5-dicaffeoylquinic acid and its active metabolites in human plasma and its application to a pharmacokinetic study in patients.

In this study, a sensitive, selective and reproducible liquid chromatography-tandem mass spectrometry method for the simultaneous determination of 1,5-dicaffeoylquinic acid (1,5-DCQA) and its active metabolites, 1-caffeoyl-5-feruoylquinic acid and 1,5-O-diferuoylquinic acid, in human plasma, using puerarin as internal standard, was developed and validated. Analytes were extracted from plasma samples by liquid-liquid extraction with ethyl acetate, separated on a C(18) reversed-phase column with water containing 5 mM ammonium acetate and acetonitrile as the mobile phase and detected by electrospray ionization mass spectrometry in negative selected reaction monitoring mode. The accuracy and precision of the method were acceptable and linearity was good over the range 1-200 ng/mL for each analyte. In addition, the selectivity, extraction recovery and matrix effect were satisfactory too. The validated LC-MS/MS method was successfully applied to phase II clinical pharmacokinetic study of 1,5-DCQA in patients.

Quantitative determination of β,β-dimethylacrylshikonin (DASK) in rat whole blood by liquid chromatography–tandem mass spectrometry with pre-column derivation and its pharmacokinetic application

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of beta,beta-dimethylacrylshikonin (DASK) in rat whole blood. DASK was pretreated using pre-column derivatization with 2-mercaptoethanol followed by liquid-liquid extraction with cyclohexane. Detection was performed on Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer by selected reaction monitoring mode via electrospray ionization source. The linear range for the determination of DASK spiked in rat whole blood (0.25 mL) was 3-3000 ng/mL. The accuracy was within 9%. Intra- and inter-day precisions were no more than 16.1 and 13.3%, respectively. The validated LC-MS/MS method was successfully applied to the preliminary pharmacokinetic study in rats. After DASK administration (60 mg/kg, p.o.) in rats, pharmacokinetic parameters were obtained, where the area under the drug concentration-time curve was 2393.7 +/- 224.4 ng h/mL and the elimination half-life was 27.6 +/- 5.3 h.

Application of ultra high-performance liquid chromatography tandem mass spectrometry to investigate the regioselective glucuronidation of icaritin in vitro

HIGHLIGHTSWe studied the glucuronidation metabolic profile, enzyme kinetics and reaction phenotyping of icaritin in HLMs, RLMs, HIMs and UGTs.Three conjugated metabolites of icaritin were detected simultaneously in vitro.We evaluated regioselective glucuronidation of icaritin by UGT isoforms, and UGT1A1 is the principle contributing enzyme.Time‐dependent experiment was conducted to elucidate the conversion pathway for icaritin elimination. ABSTRACT Icaritin is one of the Epimedium products with various biological activities. In the present study, we developed a rapid, reliable and robust UHPLC–MS/MS method to simultaneously determine unconjugated icaritin and its multiple glucuronides (icaritin‐3‐glucuronide, icaritin‐7‐glucuronide and icaritin‐3,7‐diglucuronide) in microsomal incubation systems, and applied it to study icaritin regioselective glucuronidation in vitro. We identified the involvement of human UDP‐glucuronosyltransferase (UGT) isoforms in icaritin metabolism and further studied the kinetic profiles of icaritin glucuronidation using pooled human liver microsomes (HLMs), pooled rat liver microsomes (RLMs), pooled human intestine microsomes (HIMs) and UGTs, respectively. We also evaluated regioselective glucuronidation of icaritin by UGT isoforms and conducted time‐dependent experiment to elucidate the metabolic pathways for icaritin clearance. Catalytic efficiency of microsomes is determined according to rank orders of total intrinsic clearance (CLint): CLint,HLM (24.19mL/mg/min)>CLint,RLM (13.15mL/mg/min)>CLint,HIM (6.43mL/mg/min). Besides, icaritin glucuronidation is mediated by multiple enzymes, with UGT1A1 the principal metabolizing enzyme (total CLint,UGT1A1=6.38mL/mg/min). As for the regioselectivity, except for UGT1A8 and UGT2B7, most UGT isoforms exhibit preference for the position of 3‐OH on icaritin structure. Moreover, time‐dependent conversion from monoglucuronides to diglucuronide indicate that icaritin‐3,7‐diglucuronide may be the final metabolite from icaritin elimination.

Quantitative determination of acetylshikonin in macaque monkey blood by LC-ESI-MS/MS after precolumn derivatization with 2-mercaptoethanol and its application in pharmacokinetic study

AbstractAim:To develop and validate a novel precolumn derivatization method for the quantitative determination and pharmacokinetic application of acetylshikonin in macaque monkeys by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS).Methods:2-Mercaptoethanol was added to the blood sample as the derivatization reagent. The derivatization reaction formed 1 major derivation product, which was well correlated with acetylshikonin. The acetylshikonin concentrations in the biological samples were calculated by quantitative determination of the major derivation product using LC-ESI-MS/MS. Separation was achieved using a C18 column (2 mm×50 mm, 5 μm) at room temperature and a linear gradient elution with a mobile phase containing methanol (1.96% acetic acid) and 10% methanol in water (1.96% acetic acid and 10 mmol/L ammonium acetate) at a flow rate of 0.2 mL/min. In addition, the major derivative, named derivative III, was identified by UV spectra, MS, and the 1H-NMR and 13C-NMR spectra.Results:Good linearity was obtained within the range of 5 and 2000 ng/mL (r>0.99 using a linear regression model with 1/x2 weighting) for acetylshikonin. The interday and intraday precisions were found to be less than 12.3%, with the exception of the lowest concentration, which was less than 17.2%. The interday and intraday accuracies, which were between –3% and 0.6%, were also observed. After the administration of acetylshikonin (80 mg/kg, po) in macaque monkeys, the pharmacokinetic parameters were obtained through the non-compartmental analysis, where the area under the concentration-time curve to the last measurable concentration, the terminal elimination half-life, and the mean residual time were 615.4±206.5 ng h/mL,12.3±1.6 h, and 10.2±0.7 h, respectively.Conclusion:The method was validated and applied to the quantitative determination and pharmacokinetic study of acetylshikonin in the blood samples of macaque monkeys.