| Fungal infection is one of the most serious zoontic diseases,it is known that approximately 100 of fungus are pathogenic to animals and man.Opportunistic and invasive fungal infections have emerged as a major cause of hospital related morbidity and mortality in immuno-compromised individuals such as HIV,cancer and organ transplant patients[1-4]The incidence of fungal infections has increased at a staggering rate in the past few decades,and the reported mortality rates of systemic infection are greater than 30%[5-7].The most common antifungals currently used for invasive fungal infections are azole antifungal agents and Amphotericin B.However,ongoing emergence of drug-resistance in primary,opportunistic fungal pathogens and problems of host toxicity have prompted the search for the development of new antifungal agents[8-12].Echinocandins are lipopeptide antifungal agents that are potent noncompetitive inhibitors of(1,3)-β-D-glucan synthase,an enzyme essential to the structural integrity of the fungal cell wall[13].The absence of cell wall in mammals helps make the echinocandins very attractive in terms of low toxicity and reduced side effects.Also,the low incidence of resistance and few drug-drug interactions give the echinocandinsadvantages over other classes of antifungal drugs[14].Echinocandins are now considered as important agents for treatment of serious fungal infections[15].Anidulafungin,the most recently developed echinocandin,has fungicidal activity against a broad spectrum of Candida spp,including those resistant to azoles and polyenes,and also has fungistatic activity against Aspergillus spp[16,17].Nevertheless,Anidulafungin is a poorly water-soluble semi-sythetic echinocandin,solubility enhancement using toxic surfactant such as Tween 80 for the formulation and the requirement for daily parenteral administration associated with high cost limited its clinical use[18].Various strategies to improve solubility and extend the elimination half-life were used in modifying anidulafungin.BG40018 is a semisynthetic echinocandin derived from anidulafungin with much better solubility,and exerts its antifungal activity via inhibition of fungal(1,3)-β-D-glucan synthase.This study investigated the in vitro antifungal activities,in vivo protency,pharmacokinetics properties and safety of BG40018 to provide the theoretical basis for potential druggability and further development.1.In vitro antifungal susceptibility testing of BG40018 and anidulafunginTo investigate the in vitro antifungal activities of BG40018 againest yeasts and Aspergillus species.The MICs of BG40018 were determined against each isolate according to the methods proposed by Clinical and Laboratory Standards Institute(M38-A2,M27-A3),and anidulafungin was used for positive control.BG40018 and anidulafungin were diluted two fold to the designated concentration range,and MICs were determined in Rosewell Park Memorial Institute(RPMI)1640 buffered to a pH of 7.0 with MOPS.The starting inoculum was approximately 0.5 ×103 to 2.5 ×103 CFU/mL.Microtiter trays were incubated at 35 ℃ in a moist,dark chamber,and the MICs were recorded after 24 h of incubation for all Candida spp.and after 72 h for C.neoformans.MIC values were defined as the lowest concentration of antifungal which inhibited 50%of visible growth.The MICs of BG40018 were<0.125μg/mL for all tested Candida species.BG40018 was approximately as active as anidulafungin with the MIC values within two gradients against all strains.C.neoformans and Aspergillus species were also tested,both of BG40018 and anidulafungin had less activity against C.neoformans and Aspergillus species.The in vitro susceptibility studies demonstrated potency of BG40018 against fluconazole-sensitive and-resistant Candida species as well as Candida glabrata and Candida krusei,variable but overall comparable to anidulafungin.2.Time-killing test of BG40018 and anidulafunginTo confirm the fungicidal effect of BG40018 by time-killing test.C.albicans SC5314,C.tropicalis ATCC20026,C.krusei ATCC2340,C.neoformans H99 were prepared at the starting inoculum of 105 cells/mL.The concentrations were 2 μg/mL for BG40018 and anidulafungin.At predetermined time points(0,4,8,12,16 and 24 h)after incubation with agitation at 35 ℃,a 100 μL of aliquot was removed from every solution and serially diluted by sterile water.A 100 μL of aliquot from each dilution was spread on the sabouraud dextrose agar plate.Colony counts were determined after incubation at 35 ℃ for 48 h.Against C.albicans SC5314,C.tropicalis ATCC20026 and C.krusei ATCC2340,both BG40018 and anidulafungin resulted in fungistatic activity.The tested agents resulting in approximately a 0.7-2.2 log10 CFU/mL decrease from the starting inoculum at 24h.Compared to anidulafungin,BG40018 yielded approximately a 0.8-1.1 log10 CFU/ml decrease,and appeared to be slightly more active.Against C.neoformans H99,both BG40018 and anidulafungin showed a modest growth inhibition activity.BG40018 also presented a slightly better activity than anidulafungin,yielding approximately a 0.4 log10 CFU/mL decrease.The in vitro pharmacodynamics findings by time kill curves revealed that BG40018 fungicidal rate was slightly better than anidulafungin.3.The effect of of BG40018 and anidulafungin in systemic fungal infected miceTo compare the in vivo anitifungal protency of BG40018 and anidulafungin in the mice systemic infected with C.albicans SC5314.The mice were rendered neutropenic by injecting cyclophosphamide intraperitoneally(150 mg/kg)on 4 days and 1 day before infection.Disseminated infection with C.albicans SC5314 organisms were produced by injection of 1×104 blastoconidia in 0.1 mL of saline via the lateral tail vein 2 h prior to drug therapy.BG40018 and anidulafungin were single administrated to the infected animals at 0.5,1.5 and 4.5 mg/kg intravenously.The animals were euthanatized by CO2 asphyxiation at 24 h post dose.After sacrifice,the kidneys of each mouse were immediately removed and placed insterile water.The right kidney was homogenized in 0.5 mL PBS for fungal burdens measurement,and the left one was fixed in 10%neutral formalin for H&E and PAS staining.The homogenized kidney aliquots were plated onto SDA,and colony counts were performed after incubation for 48 h at 35 ℃.In another study,BG40018 and anidulafungin were single administrated to the infected animals at 1.5 mg/kg intravenously,and control group was injected with normal saline.Survival status was monitored up to 25 days,survival rate and the median survival time were calculated.At 24 h post dose,BG40018 and anidulafungin was slightly effective at 0.5 mg/kg,while the kidney fungal burdens of mice intravenously infected with C.albicans SC5314 organisms significant decreased at 1.5 and 4.5 mg/kg.BG40018 treated mice had significantly lower fungal burdens in the kidneys as compared to those treated with anidulafungin at 1.5 and 4.5 mg/kg.Moreover,H&E staining revealed that inflammatory infiltration and tissue necrosis of the kidneys from mice received BG40018 and anidulafungin treatment were significantly improved compared to mice without treatment.PAS staining also identified fewer hyphae in the kidneys of from mice received BG40018 and anidulafungin treatment compared to without treatment.Furthermore,both H&E and PAS staining demonstrated the pathological damage of kidney from BG40018 treatment mice were slightly improved compared to anidulafungin treatment.All mice intravenously infected with C.albicans SC5314 organisms without treatment died within 3 days.By contrast,few mice treated with BG40018 died within over a 25-day observation period.The mice treated with BG40018 had a much higher survival rate than those treated with anidulafungin.The median survival time of control group was 2 days,while BG40018 and anidulafungin group were>25 and 24.5 days,repectively.BG40018 displayed the potential efficacy against infections by reduction of fungal burdens in the kidneys and improvement of pathological damage compared to anidulafungin.The superiority of BG40018 was mainly demonstrated by higher survival rate in mice infected with C.albicans compared to anidulafungin.4.Pharmacokinetics study of of BG40018 and anidulafungin in beagle dogs To investigate the pharmacokinetics profile of BG40018 and anidulafungin after intravenous administration in beagle dogs.The dogs received BG40018 or anidulafungin as a 10-minute intravenous infusion at 1 mg/kg.Whole blood samples(K2 EDTA anticoagulant)were collected via jugular vein up to 72 h after dosing.The blood samples were centrifuged at 2000×g for 10 min at 4 ℃ to obtain plasma,and plasma was stored below-70 ℃ until analysis.Plasma samples were assayed for BG40018 or anidulafungin using liquid chromatography-tandem mass spectrometry(LC-MS/MS)methods.BG40018 or anidulafungin from dog plasma samples were extracted using protein precipitation with acetonitrile followed by liquid-liquid extraction with deionized water.LC-MS/MS used an Agilent ZORBAX Eclipse XDB-C18(50×2.1 mm,3.5 μm)or SB-C18(50×2.1 mm,5 μm)column,operated at RT,at a flow rate of 0.4 mL/min with a gradient consisting of 0.1%formic acid in water and 0.1%formic acid in 95:5 acetonitrile/water coupled to an API 4000(Applied Biosystems).The positive ionization mode with MRM transitions:m/z 1217.2→104.1 for BG40018(IS:m/z 455.2→165.1)and m/z 1140.7→343.3 for anidulafungin(IS:m/z 494.2→369.1).Pharmacokinetic parameters were calculated from the plasma concentration-time data using standard noncompartmental methods and utilizing WinNonlin analysis software.After administration by 10-minute intravenous infusion,BG40018 exhibited a slow clearance(0.208 mL/min/kg)and a long half-life(51 h),the AUC0-of and Vdss were 80730 ng h/mL and 0.794 L/kg,respectively.Anidulafungin exhibited a faster clearance(0.748 mL/min/kg)and a shorter half-life(17 h)compared to BG40018,the AUC0-inf and Vdss were 22396 ng h/mL and 0.835 L/kg,respectively.These results suggested BG40018 exhibited longer elimination half-life which helps to enhance systemic exposure compared to anidulafungin.5.Repeated intravenous administration toxicity of BG40018 and anidulafunginTo evaluate the toxicity of BG40018 afetr repeated intravenous administartion in rats.SD rats(5/sex/group)were administered BG40018(0,10,20,and 40 mg/kg)or anidulafungin(40 mg/kg)by Ⅳ injection via the tail vein for 14 days.Clinical signs,body weight,chemistries,hematology,coagulation,urinalysis gross necropsy,major organ weights and histopathology were studied in all survial rats.The rats treated with BG40018 did not exhibit effects on clinical signs,body weight,hematology,coagulation or urinalysis.No gross lesions,organ weight change and microscopic findings were o’bserved for BG40018 at all doses.At 40 mg/kg,anidulafungin treatment resulted in reduced body weight,decreases in RBC、HGB、HCT、MCV、MCHC、PLT、MPV and RET#.Elevated ALT and AST,enlarged liver,single cell hepatocyte necrosis and hepatocellular hypertrophy were also observed for anidulafungin.According to the reference,the maximum non-lethal dose of anidulafungin in rats was 50 mg/kg,a dose which is equivalent to 10 times the recommended daily dose for esophageal candidiasis(50mg/day)or equivalent to 5 times the recommended daily dose for candidemia and other Candida infections(100mg/day),based on relative body surface area comparison.Liver toxicity,including single cell hepatocellular necrosis,hepatocellular hypertrophy and increased liver weights were observed in rat toxicity study.We hypothesized that hepatotoxicity may be due to the inherent chemical lability of anidulafiungin generating potentially reactive intermediates.In this study,the toxicity profile of anidulafungin was consistant with the reference.The study results demonstrated BG40018 was devoid of hepatotoxicity at doses up to 40 mg/kg when dosed in rats for 2 weeks.In conclusion,BG40018 exhibited comparable in vitro activities,but more potent in vivo antifungal activities with anidulafungin.In addition,BG40018 confers both superior pharmacokinetics(PK)properties and the potential for an improved safety profile relative to anidulafungin.All the data above support the characterization of BG40018 as a promising long-acting drug candidate for the treatment of serious,life-threatening,invasive fungal infections,and it will be investigated further for future clinical use. |
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