| The increasing prevalence of methicillin-resistant Staphylococcus aureus(MRSA) infection represents a serious threat to public health. Furthermore, some diseases caused by MRSA have been associated with high mortality rates, even after appropriate antibiotic therapy. In the face of escalating problems with pathogen control, the development of proper formulations and combined administration of existing antibiotics is as important as the development of novel antibiotics. The present study was aimed at evaluating the synergy effect of two antibiotics, daptomycin and clarithromycin, against methicillin-resistant Staphylococcus aureus (MRSA) infections.In the present study, the formulation of PEGylated liposomal co-delivery of daptomycin and clarithromycin (PL[CD]) was prepared and optimized, and its efficacy against methicillin-resistant Staphylococcus aureus MRSA252strains was investigated. The obtained PL[CD] had a mean vesicle diameter of (98.2±2.21) nm, a polydispersity index of0.251, entrapment efficiency of liposomal co-delivery of daptomycin and clarithromycin (PL[CD]) was (92.94±1.21)%and (94.71±1.37)%, and a mean percent drug loading of PL[CD] was (8.45±0.11)%and (0.29±0.043)%with uniform particle size and high storage stability. Potent activity of PEGylated liposomal co-delivery of daptomycin and clarithromycin (PL[CD]) against MRSA was demonstrated in vitro with a more sustained effect than PEGylated liposomal of daptomycin (PL[D]) and PEGylated liposomal of clarithromycin (PL[C]). In addition, intravenous administration of the clinical doses of PL[CD] significantly increased the survival mice in a MRSA systemic infection model compared with other formulations.Taken together, PL[CD] may represent a promising approach to reduce MRSA infections, especially those involving bloodstream dissemination, such as hematogenous pulmonary infection. Therefore, the above theory can provide relevant basis for future clinical application.The main research topics are following:(1) Daptomycin and clarithromycin collaborative optimization scheme of in vitroThe dynamic checkerboard method was performed in96-well plates containing daptomycin and clarithromycin at two-fold dilutions, determination of the fractional inhibitory concentration (FIC) was0.5, combined of daptomycin and clarithromycin and preliminary determination have synergy in vitro.(2) Single drug liposome preparation and collaborative optimization scheme of in vitroClarithromycin detection by HPLC was carried out using a C18column for chromatographic separation with a flow rate of0.7mL/min, maintained at a temperature of45℃. The mobile phase was composed of organic phase/aqueous phase(40/60, v/v) that acetonitrile containing0.1%TFA (trifluoroacetic acid) and0.033mol/L KH2PO4with the pH adjusted to3.5±0.2using3.5mM HC1. The eluate was monitored at a wavelength of205nm. For HPLC detection of daptomycin, the mobile phase was composed of organic phase/aqueous phase (36/64, v/v) that acetonitrile and0.5%NH4H2PO4. The mobile phase was delivered at a flow rate of1mL/min and maintained at a temperature of35℃. Column eluate was monitored at a wavelength of221nmSeveral factors affect the preparation of daptomycin and clarithromycin by examining liposomes:the molar ratio of hydrogenated lecithin (HSPC) and cholesterol, fat mass ratio. Eventually get the optimal prescription daptomycin and clarithromycin preparation for liposomes, you can get more stable single drug liposome daptomycin and clarithromycin, no obvious drug leakage was detected after storage at4℃for2months. Successful preparation of daptomycin and clarithromycin single drug liposome that the determination of the fractional inhibitory concentration (FIC) with the dynamic checkerboard method. From the FIC data and it was determined that the optimal drug mass ratio was PL[C]:PL[D] of32:1, which was therefore used for subsequent construction of co-delivery liposomes. (3) Preparation and evaluation of co-delivery liposomes containing daptomycin and clarithromycinThe obtained PL[CD] had a mean vesicle diameter of (98.2±2.21) nm, a polydispersity index of0.251, entrapment efficiency of liposomal co-delivery of daptomycin and clarithromycin (PL[CD]) was (92.94±1.21)%and (94.71±1.37)%, and a mean percent drug loading of PL[CD] was (8.45±0.11)%and (0.29±0.043)%with uniform particle size and high storage stability.In vitro pharmacodynamic study that study on the growth of MRSA252followed influenced by three formulations (PL[C], PL[D], PL[CD]), results showed that, PL[CD] inhibition of MRSA252more lasting effect. The qualitative and quantitative results of biofilm susceptibility assays demonstrated that PL[CD] was more potent than formulations containing daptomycin or clarithromycin only.By comparing the PL[C], PL[D], PL[CD] for MRSA252infected mice induced systemic therapeutic effect compared the results showed that PL[CD] intravenously administered can make the survival of infected mice increased to90%in clinical doses. Safety evaluation results showed that liposomal co-delivery of daptomycin and clarithromycin had no obvious toxicity in mice. |
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