In Vitro And In Vivo Study Of An Amphotericin B Formulation With Guaternized Bioreducible Lipidoids

Background and PurposeInvasive fungal infections are a major cause of morbidity and mortality in immunodeficient individuals and in transplant recipients or tumor patients undergoing immunosuppressive chemotherapy caused by Candida albicans(C.albicans),Aspergillus fumigatus and Cryptococcus.neoformans,etc.Additionally,the increased number of fungal isolates resistant to azoles and echinocandins represent another antifungal therapeutic challenge.Amphotericin B(AmB)is polyene antifungal agent which was first isolated in 1955 from Streptomyces nodosus.It is a golden standard in the treatment of severe systemic fungal infections because of the high affinity for ergosterol in the fungal cell membrane.Due to two characteristically physicochemical properties:amphipilic and amphoteric behavior,AmB presents unique chemotherapeutic properties such as high activity,broad antifungal spectrum,fungicidal action,and ability to overcome multi-drug resistance.However,its usefulness is limited due to poor solubility and significant systemic toxicities,including infusion-related reactions,nephrotoxicity,hypokalemia and hepatotoxicity due to binding with less selectivity to the cholesterol of mammalian cellsespecially in kidney cells.Currently,there are already several commercial AmB formulations including micellar dispersion(Fungizone(?)),lipid complexes(Abelcet(?)),colloidal dispersions(Amphocil(?))and liposomes(Ambisome(?))[7-9].However,the nephrotoxicity of Fungizone(?) limits its use in clinical mediated by vasoconstriction and direct tubular toxicity.It is estimated that the serum creatinine concentration increased in 80%of patient administrated with Fungizone(?).Abelcet(?) and Amphocil(?) are characterized by larger particles and taken up by reticuloendothelial system(RES)resulted in fast decline of AmB levels from plasma.Ambisome(?) is the least toxic of the commercial AmB formulation,but its major limitation is the high cost due the complex manufacturing process.Another limitation of Ambisome(?) is the component of cholesterol which support the parasite bind to macrophages.Thus,new AmB formulation need developed to overcome these challenges especially in developing countries.Recently,the combinatorial library of lipidoids become more attractive delivery vectors because ofthe economic and easy combinatorial synthesis,as well as the high delivery efficiencies.Based on the accumulated data of the relationship between structure and function of delivery system,thequaternized lipidoids(QLDs)with disulfide bond(S-S)intail were screened to be the optimized AmB delivery vector.Because the QLDs is characterized by biodegradableatreductive environment and increasefluidityofassembledcomplexes due to the disulfide bond(S-S)intail and high solubility and stability due to the quaternary amino group.The QLDs were synthesized by reaction of a variety of amine with 1,2-exoyhexadecane,followed by the quaternization of ternary amines.Moreover,polyetheylene glycol(PEG)is widely recognized by its biocompatibility to form adequate coating on nanoparticle surface.We found that DSPE-PEG2000 alone not effective for AmB delivery system.However,when DSPE-PEG2000 was formulated with QLDs,the formulations exhibited increased AmB solubility,stability,optimized particle sizes and effective antifungal activity.They also demonstrated decreased hemolysis to RBCs and high cell viabilities to HEK cells.After in vitro evaluations,the Q78-O14B-PEG AmB formulation(Q78-P-AmB)was screened to further explore pharmacokinetic profiles,tissue distribution and the toxicity evaluation in vivo.Q78-P-AmB showed optimized and characterized plasma pharmacokinetic profiles,tissues distribution and significantly superior to Fungizone(?).Methods(1)Exploring the preparation of amphotericin B liposomes and evaluating liposome stability,in vitro toxicity and antifungality through a series of in vitro tests to screen out the best performance liposome preparations.(2)The pharmacological kinetics,tissue distribution and toxicity of the selected Amphotericin B liposomes were evaluated by in vivo.(3)Further establish and optimize the liposome library according to the synergistic antifungal properties of AmB and liposome in the novel amphotericin B liposome.Results(1)QLDs efficiently enhanced AmB solubility and stability with homogenous transparent yellow solutions of significantly decreased particle sizes.When further modified with DSPE-PEG2000,the AmB encapsulates exhibited optimal particle size of 70-100 nm.Moreover,QLDs increased AmB solubility and decreased the amount of DSPE-PEG2000 by intercalating AmB within the lipid bilayer.Another superiority of AmB encapsulates described here is the low-cost excipients and easier preparation as compared to Ambisome(?).(2)Apart from the formation of stable and small AmB encapsulates with condensed structures,QLDs and DSPE-PEG2000 also dramatically decreased the toxicity of AmB encapsulates showing decreased cytotoxicity and did not cause hemolysis of RBCs in vitro.After encapsulate with QLDs and DSPE-PEG2000,the DLC efficiency were also enhanced which depended on the polarity and partition coefficient determining AmB localization in the liposomal membrane.16 Finally,after comparison of evaluation in vitro,AmB/Q78-014B-P demonstrated minimal toxicity,lowest MIC and most stability among these AmB encapsulates and was subjected to further analysis in vivo.Moreover,the AmB/Q78-O14B-P exhibited superiority in antifungal evaluation in vitro.The high loading efficiency and structural characteristic of the quaternary amino group may contribute to higher antifungal efficacy by increasing AmB concentrations in fungal cell membranes and exerting a synergistic antifungal effect with AmB.In future experiments,different fungal infection animal models will be used to further confirm the superior antifungal efficacy of this encapsulate.(3)In pharmacokinetic evaluation,the behavior of AmB/Q78-O14B-P exhibiting high Cmax,AUC,slow clearance(CL)and small Vd.Fungizone(?) displayed low AUC and Cmax,large CL and wide Vd.AmB/Q78-014B-P also presented attractive tissue distribution features yielding higher concentrations in lung,lower concentrations in kidney and comparable concentrations in liver and spleen when compared with Fungizone(?) at the equivalent 2 mg AmB/kg,suggesting that it would have higher antifungal activity and lower nephrotoxicity in vivo.The targeting of AmB to a lung site is expected to be beneficial for treatment of pulmonary fungal infections such as invasive aspergillosis and fungal pneumonia.Moreover,the long-term tissue retention suggests that the drug could be given intermittently,instead of daily,without losing efficacy and this would reduce the cost and possible toxic side-effects.AmB/Q78-O14B-P didn’t show any hepatotoxicity or nephrotoxicity at compared dose of Fungizone(?).The optimized particle size contributes to a reduction of AmB in glomerular filtration and drug renal excretion.(4)According to the anti-fungal properties of the quaternized liposome structure,the liposome library of this structure was established in this laboratory,and the stronger antifungal liposome Q87-EC12 and Q87-EC14 was screened out,and a new antifungal agent was expected to be developed.ConclusionQuaternized bioreducible lipidoids efficiently enhanced AmB solubility and stability with homogenous transparent yellow solutionsand significantly decreased particle sizes.When further modified with DSPE-PEG2000,the AmB encapsulates exhibited decreased cytotoxicity and did not cause hemolysis of RBCs in vitro.AmB/Q78-O14B-P manifested the most optimal particle size between 70-1 00nm,best efficiency in vitro antifungal efficacy,lowest toxicities to RBC and HEK cells and highest AmB loading amount,all of which were significantly superior to Fungizone(?).In vivo evaluation showed that AmB/Q78-O14B-P produced improved pharmacokinetic parameters such as higher AUC,Cmax,T1/2 and lower Vd in comparison to Fungizone(?).AmB/Q78-O14B-P also presented attractive tissue distribution features yielding higher concentrations in lung,lower concentrations in kidney and comparable concentrations in liver and spleen when compared with Fungizone(?) at the equivalent AmB dose of 2 mg/kg,suggesting that it would have higher antifungal activity and lower nephrotoxicity in vivo.Meanwhile,AmB/Q78-O14B-P exhibited reduced toxicity at higher doses and retained antifungal effects at low doses.Therefore,AmB/Q78-O14B-P shows great promise for the development of novel antifungal agents that will be more therapeutically efficacious and less toxic for the treatment of systemic fungal infections.It is also an affordable formulation due to its easier preparation and low-cost excipients.In future experiments,different fungal infection animal models will be used to further confirm the superior antifungal efficacy and safety characterizations of this encapsulate.