The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo / 中西医结合学报

OBJECTIVE@#Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy.@*METHODS@#The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.@*RESULTS@#rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.@*CONCLUSION@#Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

GSH-responsive SN38 dimer-loaded shape-transformable nanoparticles with iRGD for enhancing chemo-photodynamic therapy

Accurate tumor targeting, deep penetration and superb retention are still the main pursuit of developing excellent nanomedicine. To achieve these requirements, a stepwise stimuli-responsive strategy was developed through co-administration tumor penetration peptide iRGD with shape-transformable and GSH-responsive SN38-dimer (d-SN38)-loaded nanoparticles (d-SN38@NPs/iRGD). Upon intravenous injection, d-SN38@NPs with high drug loading efficiency (33.92 ± 1.33%) could effectively accumulate and penetrate into the deep region of tumor sites with the assistance of iRGD. The gathered nanoparticles simultaneously transformed into nanofibers upon 650 nm laser irradiation at tumor sites so as to promote their retention in the tumor and burst release of reactive oxygen species for photodynamic therapy. The loaded d-SN38 with disulfide bond responded to the high level of GSH in tumor cytoplasm, which consequently resulted in SN38 release and excellent chemo-photodynamic effect on tumor.

Research progress and consideration of polymeric prodrugs / 中国药科大学学报

@#Currently the available therapies cannot satisfy all the clinical requirements, therefore advanced technologies are urgently demanded. Delivery system the polymeric prodrug based has both advantages of prodrug strategy and nanoparticle drug delivery strategy. The system can improve the drug bioavailability, enhance the drug stability, and make the drug targeting system more effective. The system can reduce the side effects and improve the therapeutic effect of drug. According to the mechanism of this drug system, passive targeting, active targeting, triggered release and co-administration were reviewed. Finally, the research prospects and issues in this field were pointed out.

Effects of clinical P-glycoprotein inhibitors on oral bioavailability and brain penetration of gefitinib / 中国药科大学学报

@#To investigate the effects of clinical P-glycoprotein inhibitors on oral bioavailability and brain penetration of gefitinib, 16 inhibitors and gefitinib were co-administered orally to ICR mice. The suspension of gefitinib and CMC-Na were co-administered to the control group. The suspension of gefitinib and clinical P-glycoprotein inhibitors were co-administered to the control group. Blood samples and brain homogenate samples were extracted by protein precipitation with acetonitrile and determinated by LC-MS/MS. It was found that ritonavir can significantly increase the oral bioavailability of gefitinib, and the area under the plasma concentration-time curves(AUC)of gefitinib was increased by 2 times; while brain exposure was increased, there was no increment in brain penetration. Some other drugs can also increase the plasma AUC of gefitinib, but can not enhance the brain penetration; After we corrected brain concentration with fraction of unbound drug in brain, it was found that the brain concentration of gefitinib in both control group and ritonavir group did not achieve the in vitro IC50 of inhibiting non-small cell lung cancer(NSCLC)cell growth. Our results suggest that clinical doses of the 16 clinical P-glycoprotein inhibitors can not specifically increase brain tissue exposure, more specific and safer P-gp inhibitors are required. After we corrected brain concentrations with fraction of unbound drug in brain, our preclinical studies found that insufficient brain exposure may be one of the reasons for the unsatisfactory efficacy of gefitinib in the treatment of brain metastases.

The Pharmacokinetic and Biopharmaceutical Effect of Ascorbic acid (Vitamin C) on Pefloxacinon Concurrent Administration in Human.

The aim of the study was to assess the pharmacokinetic and biopharmaceutical effect of ascorbic acid (Vitamin C) on pefloxacin on concurrent administration in man through urine excretion data and microbiological evaluation. A two way crossover study was performed in ten healthy male volunteers aged (Mean ± SD) 45 ± 5.5 years and weight (Mean ± SD) 75.4 ± 12.5 Kg recruited and given pefloxacin 400mg as single dose and urine samples collected and pooled up at time intervals. The drug was concurrently given with 500mg vitamin C after a washout period of 4 weeks and urine samples similarly collected. Urine samples collected were analyzed and pefloxacin concentrations were determined with UV spectrophotometer from a validated calibration curve. The pharmacokinetic parameters Cmax, tmax, Ke and t1/2 were determined and compared. Microbial evaluation of the interaction of the drugs was performed through MIC determination using urinary isolate S. aureus (U-11420). The ke for pefloxacin alone was significantly lower than that for pefloxacin concurrently administered with vitamin C (0.1hr-1 and 0.3hr-1) P<0.05. The amount of pefloxacin excreted was significantly lower on single administration of pefloxacin compared to the co-administration with vitamin C, (44.13mg against 141.99mg) at P<0.05. The MIC obtained against S. aureus was 0.025mg/ml for pefloxacin alone while the co-solution with vitamin C at below 2hr and 4hr impregnation period was 0.05mg/ml and 0.1mg/ml respectively. There was significant chemical, microbiological and biopharmaceutical interaction on co-administration of pefloxacin with vitamin C.