Development Of Drug Delivery Systems For Platensimycin And Their Antibacterial Activities

Purpose: The rapid development of bacterial resistance has posed a serious threat to human health and a major challenge to the development of new antibiotics.Most of the antibiotics developed and approved in the past three decades are based on the molecular backbone of existing antibiotics,and there is a lack of antibiotics with new molecular backbones and antibacterial mechanisms for clinical practice.Platensimycin(PTM)is a natural small molecule compound that selectively inhibits bacterial fatty acid synthase(FASII),which was screened from the fermentation products of Streptomyces platensis in the soil of South Africa.PTM exhibits potent antibacterial activity against common Gram-positive bacteria including drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus(MRSA)and vancomycin-resistant Enterococcus(VRE)(MIC = 0.1 ～ 1μg/m L),and no cross-resistance was detected.Moreover,based on the structural difference between FASII and mammalian fatty acid synthase(FASI),PTM can selectively act on bacteria and show low toxicity to mammalian cells.Therefore,PTM is expected to be developed into a novel antibiotic for clinical practice.However,the low solubility and poor pharmacokinetic properties of PTM limit its in vivo efficacy evaluation and clinical development.Addressing these limitations through multiple approaches will accelerate the development of PTM antibiotics.In this thesis,relying on the high-yielding strain of PTM constructed by our research group,as well as the optimized fermentation process and efficient separation and purification method,the PTM was isolated from the fermentation product.Through the exploration of different drug delivery systems for PTM,we hope to improve its pharmacodynamics and pharmacokinetic properties in vitro and in vivo,and provide a new and effective way to deal with the crisis of drug-resistant bacterial infection such as MRSA.Methods and Results: Liposomes and micelles with clinical application prospects were selected as drug delivery vehicles to construct PTM-loaded liposomes(Lip/PTM)and micelles(PLGA-PEG/PTM).In order to deal with the problem of insufficient treatment and infection recurrence caused by the persistence of bacteria in macrophages,mannosemodified liposome(M-Lip/PTM)targeting mannose-receptor on macrophage surface and p H-responsive micelle(PLGA-PEOz/PTM)responding to the microacid environment of lysosome or phagolysosome in macrophage were designed accordingly.All constructed PTM-loaded liposomes and micelles were used in the treatment of MRSA-infected macrophages and MRSA-infected mice with peritonitis,and the in vivo pharmacokinetic properties were also investigated.The results showed that compared with free PTM,all PTM-loaded liposomes and micelles enhanced the anti-intracellular infection effect,and mannose-modified MLip/PTM and p H-sensitive PLGA-PEOz/PTM exhibited better effects.In the MRSA-infected mouse peritonitis models,the survival rates of the PTM-loaded nano-delivery treatment groups were all higher than those of the free PTM treatment groups.Among them,the M-Lip/PTM and PLGAPEOz/PTM exhibited the best survival effect and enhanced the pharmacokinetic properties of PTM in vivo.In order to further expand the application of PTM in skin and soft tissue infections,we selected the PTM-loaded dendrimer(PAMMA)nanodelivery system(PAMAM/PTM)with the best encapsulation efficiency and in vivo activity from the above studies and other delivery systems we constructed.Then,PAMAM/PTM nanoparticles were encapsulated in a polyacrylamide hydrogel with good biosafety to prepare a PTM-loaded nanoparticle-hydrogel delivery system(NP-gel(PTM)).Compared with free PTM and PAMAM/PTM nanoparticles,NP-gel(PTM)exhibited sustained drug release properties during the in vitro drug release process.The in vitro anti-MRSA activity and the inhibition of MRSA biofilm formation were investigated under the condition of simulating drug loss.Among them,the NP-gel(PTM)group exhibited the best effect.In addition,the drug retention time of the nanoparticle-hydrogel system was longer than that of free drug and drug-loaded PAMAM after subcutaneous injection in vivo.In the treatment of MRSA-infected skin wounds and subcutaneous abscesses,NP-gel(PTM)showed superior antibacterial and tissue recovery effects than free PTM.To explore the possibility of developing oral dosage forms that could improve the in vivo pharmacokinetic properties of PTM.Co-amorphous drug delivery system with advantages such as improved drug solubility and in vivo pharmacokinetic behavior was selected to prepare co-amorphous PTM-BCL drug delivery system by combining PTM with clinically used berberine chloride(BCL).The prepared samples were characterized by powder X-ray diffraction,modulated differential scanning calorimetry,and scanning electron microscopy,and confirmed the successful preparation of co-amorphous PTM-BCL and amorphous PTM.Both the equilibrium solubility and in vitro dissolution rate of PTM and BCL in amorphous PTM-BCL were reduced relative to crystalline PTM,crystalline BCL,and amorphous PTM.The potential interactions between PTM and BCL molecules were investigated by infrared spectroscopy,X-ray photoelectron spectroscopy and hydrogen nuclear magnetic resonance spectroscopy.The results showed that no further inter-molecular-skeletal interactions were observed between PTM and BCL except for the proton signal fluctuation in the relevant region,which may be due to the strong hydrogen bond interaction between PTM and BCL rather than the strong ionic bond or electrostatic interaction.The co-amorphous PTM-BCL exhibited good physical stability at 25°C or 40°C/dry conditions.After administration by gavage,the half-life of PTM in the co-amorphous PTM-BCL group was 2-fold and 3-fold longer than that of crystalline PTM and amorphous PTM,respectively.Therefore,the co-amorphous PTM-BCL form is expected to enhance the therapeutic activity of PTM against MRSA and other resistant bacterial infections in vivo by oral administration.Conclusions: PTM is a selective inhibitor of bacterial FASII with the advantages of novel structure,excellent antibacterial activity and low toxicity.In this work,liposome or micelle drug delivery system,nanoparticle-hydrogel drug delivery system and co-amorphous drug delivery system were constructed for the delivery of PTM.The antibacterial and therapeutic effects of PTM on MRSA-infected macrophages,MRSA-infected peritonitis mice,and MRSA-infected subcutaneous abscesses and skin wounds were significantly improved.In addition,the in vivo pharmacokinetic properties of PTM were also improved.These provide strategies for the development of PTM and alleviating the crisis of drug-resistant bacterial infection such as MRSA,and also serves as a reference for the reseach of drug candidates with excellent biological activity and potential development prospects.