Zhuona Wu

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.

Development and validation of a sensitive HPLC-MS/MS method for determination of chidamide (epidaza), a new benzamide class of selective histone deacetylase inhibitor, in human plasma and its clinical application.

Chidamide (epidaza), a new oral isotype-selective histone deacetylase inhibitor (HDACi), which is just approved in China for the treatment of recurrent or refractory peripheral T-cell lymphoma (PTCL) in December 2014, is the first listed benzamide class of HDACi in the world, and is currently undergoing global clinical trials for solid tumor treatments. Here, we report a sensitive, rapid and robust HPLC-MS/MS method for determination of chidamide in human plasma. Plasma sample was subjected to a simple acetonitrile protein precipitation containing MS-275 used as an internal standard (IS). Chromatography was performed on a Hypersil GOLD C18 analytical column, using a gradient methanol/water mobile phase containing 0.1% formic acid. A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the positive-ion mode. Selected reaction monitoring (SRM) using the precursor/ product transitions (m/z) of 391.1/265.1 for chidamide and 377.1/359.2 for IS were used for quantification, respectively. Good linearity was obtained in the range of 1-1000ng/mL. The method gave R.S.D.% values for precision always lower than 13.8% and R.E.% values for accuracy between -3.7 and 9.1%. In addition, the specificity, recovery, stability and matrix effect were satisfactory too. The method is now being successfully applied to plasma samples as part of an ongoing chidamide phase Ib clinical trial in patients with solid tumors, and had demonstrated consistent AUClast and t1/2 results with the published phase I pharmacokinetic data, which was also analyzed by this method, thus further confirming the reproducibility and accuracy during its clinical application. Considering the excellent performance of this method, it will continue being utilized for future clinical developments of chidamide and for routine monitoring of plasma exposure of chidamide during its clinical therapy.

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.

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.

An optimized LC-MS/MS method for determination of HYNIC-3PRGD2, a new promising imaging agent for tumor targeting, in rat plasma and its application

HYNIC-3PRGD2 is used to prepare a new 99mTc-radiolabeled tracer. HYNIC-3PRGD2, which has a high binding affinity for the integrin αvβ3 due to its special structure, has become a promising tumor imaging agent for diagnosis and monitor of the clinical response to therapeutic effects of anti-tumor agents. Here, we developed and validated a method for determination of HYNIC-3PRGD2 concentration in rat plasma using ultra-high performance liquid chromatography-tandem mass spectrometry system. Following sample extraction by methanol precipitation, satisfactory separation through chromatography was achieved on an hydrophilic reverse-phase C18 column AQ (2.1 mm × 100 mm, 2.7 μm) at a flow rate of 0.2 mL·min-1 with an gradient elution using mobile phase consisting of ultrapure water and acetonitrile fortified with 0.1% formic acid respectively. The calibration curve was developed over a linear range of 3.125-100 ng·mL-1 with the lower limit of quantification of 3.125 ng·mL-1. The HYNIC-3PRGD2 and its internal standard c(RGDfK)(RK5) were detected and quantified with the multiple reaction monitoring (MRM) mode on a triple-quadrupole tandem mass spectrometer. This method was successfully validated and applied for pharmacokinetic evaluation of HYNIC-3PRGD2 during pre-clinical experiments.

A LC-MS/MS method to monitor the concentration of HYD-PEP06, a RGD-modified Endostar mimetic peptide in rat blood

HYD-PEP06 is a novel RGD-modified Endostar mimetic peptide with 30 amino acids that is intended to suppress the formation of neoplasm vessels. This assay was developed and validated to monitor the level of the peptide HYD-PEP06 in rat blood, using liquid chromatography tandem mass spectrometry (LC-MS/MS). HYD-PEP10, another peptide similar to the analyte, was used as an internal standard (IS). A triple quadrupole mass spectrometry in Multiple Reaction Monitoring (MRM) mode and an electrospray interface (ESI) in the positive mode were used for MS analysis. The analysis was optimized with addition of 0.3% formic acid (FA) into the mobile phase as well as with a needle washing solution to overcome the carryover effect. In addition, the carryover was reduced by optimizing the mobile phase gradient. Methanol was used as a diluent of working solutions to avoid any adsorption. Methanol:acetonitrile (1:1, v:v) containing 0.3% FA was employed to precipitate the blood samples. Unknown blood samples must be placed in ice bath immediately, and precipitating agents should be added within 30 min to ensure the stability of blood samples. The assay was established and validated. This method showed a good linear relationship for the HYD-PEP06 in the range of 10 ng·mL-1 to 2000 ng·mL-1, with R > 0.99. HYD-PEP06 was determined with accuracy values (RE%) of -5.06%-8.54%, intra- and inter-day precisions (RSD%) of 3.13%-4.87% and 4.81%-9.42%. The method was successfully in monitoring the concentration of HYD-PEP06 in rat blood.

Preparation and characterization of a new type of porous starch microspheres (PSM) and effect of physicochemical properties on water uptake rate

Porous starch (PS) is a multifunctional biomaterial and has been widely applied in both pharmaceutical and food industry. This study was carried out to develop a new type of porous starch microspheres (PSM) through the dual-modification of the alcohol-alkaline treatment and inverse crosslinking-emulsion method, which could rapidly uptake water. The chemical and physical characteristics of the PSMs were determined by FTIR, SEM, XRD, DSC, ESD (equilibrium swelling ratio) and WS (water solubility). The results showed that PSMs could reach its saturated water uptake volume (about 1 mL/100 mg) in 60 s. The PSMs also revealed rough surface with observable pores and lower crystallinity degree after the dual-modification according to SEM and XRD. The use of epichlorohydrin (ECH) as a crosslinking agent could introduce crosslinks between starch molecular chains, which was determined by ESD, WS and the thermal properties. These results indicated that the dual-modification could be successfully used for preparation of rapid water uptake PSMs with enhanced structure stability.

A sharp, robust, and quantitative method by liquid chromatography tandem mass spectrometry for the measurement of EAD for acute radiation syndrome and its application

17-Ethinyl-3,17-dihydroxyandrost-5-ene (EAD) is an agent designed for the treatment of acute radiation syndrome (ARS). Given its vital role played in the prevention and mitigation of ARS, the development of a sharp, sensitive and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor the metabolism of EAD in vivo was crucial. A new method was constructed and validated for the determination of EAD with the internal standard of androst-5-ene-3β,17β-diol (5-AED). The blood samples were precipitated with methanol, centrifuged, from which the supernatant was separated on UPLC with C18 column and eluted in gradient with acetonitrile and Milli-Q water both containing 0.1% formic acid (FA). Quantification was performed by a triple quadrupole mass spectrometer with electro spray ionization (ESI) in multiple reactive monitoring (MRM) positive mode. A good linearity was obtained with R>0.99 for EAD within its calibration range from 5 to 1000ngmL-1 with a lowest limit of quantification (LLOQ) of 5ngmL-1. Inter- and intra-day accuracy and precision of three levels of quality control (QC) samples were within the range of 15%, while the LLOQ was within 20%. Samples were stable under the circumstances of the experiments. The method was simple, accurate and robust applied to determine the concentrations of EAD in Wistar rat after a single administration of EAD orally at the dose of 100mgkg-1.