Synthesis Of A Kind Of PH-Responsive Amphotericin B Derivative For Treatment Of C.Neoformans Infection

Background:Amphotericin B（AMB）is a polyene macrolide antibiotic and the preferred drug for the treatment of a wide array of invasive fungal infections.However,the clinical application of AMB is greatly limited by its serious organ toxicity,particularly hepatotoxicity and nephrotoxicity.Lots of studies have shown that the toxicity of AMB can be significantly reduced by modifying its chemical structure or encapsulating it with drug carriers.The structure of AMB molecule is complex due to the presence of a large macrolactone ring containing seven conjugated double bonds and a mycosamine unit,so the chemical structural modifications of AMB easily lead to a significant decrease or even loss of antifungal activity.Therefore,it is important to study how to reduce the toxicity of AMB and maintain its antifungal activity by rational chemical structural modifications.Objective:Based on the slightly acidic microenvironment of the fungal infection sites,one kind of pH-responsive AMB derivative was prepared by modifying the chemical structure of AMB with pH-responsive degradable groups.The reactions of maleic anhydride（MAH）or citraconic anhydride（CIT）with amino compounds give maleamic acid derivatives withβ-carboxylic acid amide bond that can protect amino group.The amide bond can be hydrolyzed under acidic conditions,exposing the free amino compounds.Based on this feature,the amide bond can be used for the acid-sensitive release of prodrugs.Consequently,selectively using maleic anhydride and citraconic anhydride as two acylating agents,one kind of pH-responsive amphotericin B derivative will be synthesized to reduce the toxicity of AMB and maintain its antifungal activity.Methods and Results:The research mainly includes the following three aspects:（1）Synthesis,characterization,and in vitro biological activity evaluation of one kind of pH-responsive amphotericin B derivative;（2）Preparation,characterization,and in vitro biological activity evaluation of one kind of pH-responsive amphotericin B derivative liposomes;（3）In vivo evaluation of one kind of pH-responsive amphotericin B derivative liposomes.（1）Synthesis,characterization,and in vitro biological activity evaluation of one kind of pH-responsive amphotericin B derivative.In this chapter,using maleic anhydride and citraconic anhydride as acylating agents,two amphotericin B derivatives withβ-carboxylic acid amide bond were obtained upon chemical modification of the amino group of the amphotericin B.The chemical structure of derivatives was characterized by¹H-NMR,UV-vis spectroscopy,FT-IR,and LC-MS.The results showed that the synthesis of amphotericin B derivatives was successful.A reversed-phase HPLC method was established to determine the AMB and its derivatives.The pH-responsive degradation behaviors of derivatives were tested in phosphate solutions with different pH values.The results showed that AMB-CIT,a kind of AMB derivative based on citraconic anhydride,displayed 30.6%degradation in 24 h at pH 7.4,but 46.1%and 73.2%degradation at pH6.5 and pH 5.5,respectively,showing an apparent acid-responsive degradation property.In addition,the in vitro hemolytic testing and antifungal activity of derivatives were investigated.The results showed that AMB-CIT exhibited lower hemolytic toxicity than AMB and had good antifungal activity when under slightly acidic conditions.（2）Preparation,characterization,and in vitro biological activity evaluation of one kind of pH-responsive amphotericin B derivative liposomes.In this chapter,egg yolk lecithin was chosen as the drug-carrying material.Amphotericin B derivative liposomes（AMB-MAH-LIP and AMB-CIT-LIP）were prepared by a thin film dispersion method in order to improve the water solubility and evaluate the in vitro and in vivo effects of the derivatives.The mean particle sizes of AMB-MAH-LIP and AMB-CIT-LIP were69.07±1.96 nm and 69.69±1.27 nm,respectively,and zeta potentials were–14.5±1.2 m V and–14.2±0.9 m V.Further,transmission electron microscopy（TEM）showed that the shape of the liposomes was relatively regular.The encapsulation efficiency of AMB-MAH-LIP and AMB-CIT-LIP were 98.2±0.98%and 98.9±0.87%,respectively.and the drug loading of the liposomes were 4.69±0.33%and 4.71±0.44%.Moreover,after placing drug-loaded liposomes at 4°C for 24 h,the in vitro stability results demonstrated that the drug contents of AMB-MAH-LIP and AMB-CIT-LIP did not change significantly.The results of UV-vis absorption spectra suggested that AMB-MAH and AMB-CIT did not exist in the carrier material in a highly aggregated form.The results of in vitro hemolysis test indicated that when the hemolysis rate was more than 5%,the minimum concentration value of amphotericin B liposomes for injection was 5μg/m L,and the values of AMB-LIP,AMB-CIT-LIP and AMB-MAH-LIP were 0.5μg/m L,10μg/m L,and 20μg/m L,respectively.Compared with amphotericin B liposomes for injection and AMB-LIP,the hemolytic toxicity of AMB-CIT-LIP and AMB-MAH-LIP was significantly reduced.（3）In vivo evaluation of one kind of pH-responsive amphotericin B derivative liposomes.In this chapter,with amphotericin B liposomes for injection and AMB-LIP as controls,the acute toxicity,antifungal activity,pharmacokinetic behavior,and distribution of tissues of AMB derivatives liposomes in vivo were investigated.The results of in vivo acute toxicity experiments indicated that when the number of dead mice was more than half,the administered doses of amphotericin B liposomes for injection group and AMB-LIP group were 6 mg/kg and 3 mg/kg,respectively.In contrast,the doses of AMB-MAH-LIP and AMB-CIT-LIP were increased to 34.7 mg/kg and 28.9 mg/kg.The in vivo antifungal activity of AMB derivatives liposomes were investigated in a mouse model of cryptococcosis infection.In the low-dose group（2 mg/kg）,the results showed that the fungal burdens in the lung and brain of the untreated group were（4.32±0.16）log₁₀CFU/g and（4.99±0.08）log₁₀CFU/g,respectively.The fungal burdens of the AMB-LIP group were（2.76±0.23）log₁₀CFU/g and（3.89±0.19）log₁₀CFU/g.Relative to the untreated group,AMB-LIP reduced the fungal burden by 1.5 log units and 1.1 log units.The fungal burdens in the lung and brain of the AMB-MAH-LIP group were（3.83±0.11）log₁₀CFU/g and（4.95±0.07）log₁₀CFU/g,respectively.The fungal burdens of the AMB-CIT-LIP group were（3.35±0.23）log₁₀CFU/g and（4.16±0.09）log₁₀CFU/g.When the administered dose was increased to 5 mg/kg,compared with the untreated group,the lung fungal burden of AMB-MAH-LIP group was reduced by 1.2 log units,and the brain fungal burden was reduced by only 0.3 log unit.The fungal burdens of lung and brain in AMB-CIT-LIP group were reduced by 2 log units and 1 log unit.The results of pharmacokinetic behavior and tissues distribution in infected mice showed that AMB-CIT was stable in blood circulation,and it could maintain the form of derivative for a long time,thus reducing the toxicity caused by amphotericin B.After reaching the infected tissues,AMB-CIT could display rapid degradation to amphotericin B under a slightly acidic environment,and then exhibit high antifungal activity.Conclusion:In summary,one kind of pH-responsive amphotericin B derivative AMB-CIT was synthesized in this study.AMB-CIT had obvious pH-responsive degradation ability under a slightly acidic environment,and it could reduce the hemolytic toxicity of AMB.Compared with AMB,the acute toxicity of AMB-CIT was significantly reduced,and the antifungal activity was slightly reduced,but the efficacy could be improved by increasing the dosage.This study provides a strategy for exploiting amphotericin B derivatives with stimuli-responsive degradation properties.