Study Of Amphotericin B Liposome For Injection Guided By Physiologically Based Pharmacokinetic Model

Objectives:Invasive fungal disease（IFD）is an infectious disease that seriously harms human health and even threatens life.Amphotericin B（AmB）is the classical drug and the last line of defense in the treatment of IFD.At present,the most preparation of AmB is deoxychlate preparation（AmB-DOC）.However,its serious adverse reactions,especially dose-limiting nephrotoxicity,limit the application of AmB.The novel Liposomal amphotericin B for injection(Lipo-AMB,Gilead production,AmBisome^?)is a single-compartment double-layered liposome.Lipo-AmB（AmBisome^?）can significantly reduce renal toxicity and infuse-related adverse reactions.Lipo-AmB has been marketed overseas about three decades,but is still in the research and development stage in domestic.About 90%of dose of amphotericin B was recovered in urine,feces and blood one week after administration of AmB-DOC.In contrast,the excretion rates of urine and feces within one week after administration of Lipo-AmB were only about 5%respectively,and the total amphotericin B in urine,feces and blood circulation was only recovered about 25%of the dose,75%amphotericin B of dose was still up to unexplained,speculating that amphotericin B was retained in deep tissues.These differences indicated that PK and tissue distribution behavior of AmB changed significantly after modification of liposome preparation.Amphotericin B has strong nephrotoxicity,and its nephrotoxicity is closely related to renal drug concentration.Amphotericin B is a concentration-dependent bactericidal agent,and its bactericidal effect is directly related to the concentration of the drug in target tissues.Therefore,the prediction of target tissue concentration is very important for clinical efficacy and safety,and has important guiding significance for the development of safe and effective individualized treatment plan.However,usually we can only obtain the human blood concentration after a certain dose,and the level of drug exposure in human tissues is almost impossible to obtain due to ethical reasons.Based on physiological anatomy,the physiologically based pharmacokinetic（PBPK）model uses each major physiological organ as a compartment connected by blood circulation and establishes differential equations combining with drug-specific kinetic parameters.The temporal concentration of the drug in each compartment was calculated.In this study,preclinical and clinical pharmacokinetics of Lipo-AmB were studied,and physiologically based pharmacokinetics（PBPK）model of amphotericin B liposome for injection was established.Human target tissue concentrations of AmB-LPU,AmB-LP and total AmB were simultaneously described.The dose-exposure-nephrotoxicity relationship of patients was preliminarily explored,and the influencing factors of target tissue exposure level after Lipo-AmB administration were studied,providing reference for the optimization of treatment plan and the control of known risks in Chinese population.Methods:1.Development LC-MS/MS methods for AmB-LP,AmB-LPU and total AmB in human plasma.Amphotericin B mainly existed in liposome form in the blood circulation,three liquid chromatography-tandem mass spectrometry（LC-MS/MS）methods were developed for the determination of AmB-LP,AmB-LPU and total AmB in human plasma,respectively.All three methods were systematically validated according to the relevant guidelines for bioanalysis.2.Pharmacokinetic studies of Lipo-AmB in Chinese population.Pharmacokinetics of single dose in healthy subjects:an open label,single-dose,two cycles,crossover pharmacokinetic study was conducted in 12 healthy volunteers with the dose of 2 mg/kg,obtained the tolerance and PK profile of AmB-LP,AmB-LPU and total AmB in healthy Chinese population.Pharmacokinetic study of patients under multiple doses:36 patients were enrolled and each subject received 2cycles of medication,every cycle was given for 5 days,once a day at dose of 3 mg/kg for2 h.PK parameters such as C_ss,maxand AUC_0-τof AmB-LP,AmB-LPU and total AmB were obtained,and the tolerance and safety of Lipo-AmB were evaluated in Chinese patients.3.Establishment and application of physiologically based pharmacokinetic（PBPK）model.The tissue distribution of Lipo-AmB and AmB-DOC in SD rats were studied to investigate the distribution process of different preparations of amphotericin B in rats.In this study,the PBPK model was established by stepwise method that means the rat PBPK model was established firstly by the preclinical data,which was then verified in the mouse,and finally extrapolated to human.Firstly,collecting physiological parameters of rats reported in the literature and AmB drug dependent parameters such as plasma protein binding rate and clearance rate,etc.,and then combining with PK blood sampling and tissue distribution of rats,finally the rat PBPK model of AmB-DOC was constructed.Model equations were established according to the principle of material conservation.Model coding,parameter fitting,and model running simulations were performed in Berkeley Madonna?（Version 8.3.23;University of California,Berkeley,CA,USA）.The model parameters were compared between species and the PBPK model of AmB-DOC was finally extrapolated to the human level after verification at mouse level.Based on the PBPK model of AmB-DOC,we combined the PK blood sampling and tissue distribution data of Lipo-AmB in rat to construct the rat PBPK model of Lipo-AmB.After verification at the mouse level,we extrapolated to human level.Plasma concentrations of AmB-LP and AmB-LPU in Chinese population and human tissue data in literature were used for the model validation,the exposure levels of AmB-LP and AmB-LPU in human blood and major tissues after Lipo-AmB administration were predicted and described to explore the factors affecting exposure levels.Results:1.Development LC-MS/MS methods for AmB-LP,AmB-LPU and total AmB in human plasma:Adding cryopreservation agent for plasma samples-35%Glucose solution（LPStab07）,AmB-LP and AmB-LPU were stable in human plasma at-80℃under short-term,long-term and freeze-thaw conditions.It was verified that AmB-LP was not transformed into AmB-LPU during the whole sample processing process,and the SPE method could completely separate AmB-LP from AmB-LPU.Methods for determination of AmB-LPU,AmB-LP and total AmB were successfully established,and all three methods were methodological verified.Under the determined chromatographic and mass spectrometry conditions,the peak shapes of the three substances and internal standard were good,and the endogenous impurities did not interfere with the detection.The linear ranges of AmB-LP,AmB-LPU and total AmB were 100～50000 ng/ml,10.0～3000 ng/ml and 100～50000 ng/ml,respectively,and the lower limits of quantification were 100,10.0 and 100 ng/ml,respectively.The precision,accuracy,recovery,matrix effect,residual,dilution reliability and stability of the three methods all meet the requirements of the methodology.2.The pharmacokinetic study of Lipo-AmB in Chinese population.Comparative pharmacokinetic study of single dose of Lipo-AmB in healthy Chinese population:Amphotericin B mainly existed in liposome form,accounting for more than 90%of the total amphotericin B at 72 h after administration,and about 70%one week after administration,and then rapidly decreased to below LLOQ（100 ng/ml）at 216 h after administration.The terminal half-life of AmB-LPU was longer 408.63 h±117.29 h,and C_maxwas about 900 ng/ml,accounting for about 4%of the total AmB;Pharmacokinetic study of patients given multiple doses:patients with persistent neutropenia and fever received intravenous infusion of Lipo-AmB 3 mg?kg^-1/d for 10 consecutive days,C_ss,maxof total AmB was 26.9±14.5μg/ml,AUC_0-τwas 249.6±195.0 h·μg/ml.After infusion of Lipo-AmB for 10 days,the proportion of AmB-LP and AmB-LPU were 89.74%±11.11%and 10.94%±6.04%,respectively.In terms of nephrotoxicity and hepatotoxicity,there were 5 cases of renal damage,with an incidence rate of 13.9%and severity was mild to moderate.There were11 cases of liver injury,the incidence was 30.6%,and the severity was mostly moderate.Based on the results of hepatotoxicity and nephrotoxicity in different populations,the incidence rate in Chinese population was lower than previously reported populations,and the types of adverse reactions of hepatotoxicity and nephrotoxicity in different populations were similar.3.Establishment and application of physiologically based pharmacokinetic（PBPK）model.Preclinical animal studies showed that Lipo-AmB altered the pharmacokinetic characteristics of AmB-DOC in rats.Lipo-AmB was mainly distributed in spleen and liver,accounting for more than 80%of total exposure,and less than 1%in kidney,while AmB-DOC was more distributed in kidney,accounting for about 15%,much higher than Lipo-AmB.According to PBPK model,the AmB-LPU exposure in kidney after administered Lipo-AmB 6 mg·kg^-1/d for five days was equivalent to AmB exposure in kidney after administered AmB-DOC 0.7 mg·kg^-1/d for five days,therefore we speculated that Lipo-AmB 6 mg·kg^-1/d and AmB-DOC 0.7mg·kg^-1/d have similar nephrotoxicity.The result simulated from PBPK model was consistent with the observed effect in clinical trials.Although studies of Lipo-AmB shown that dose-limiting toxicity was not found at 10 mg/kg in children and 15 mg/kg in adults,based on the simulated results of PBPK model and safety consideration,it is recommended that Lipo-AmB dose not exceed 6 mg·kg^-1/d.Sensitivity analysis of PBPK model parameters showed that the AUC of amphotericin B in plasma and kidney were relatively sensitive to FR and rel,suggesting that encapsulation efficiency,composition and structure of liposomes have a certain impact on the efficacy and safety of drugs.Conclusions:1.The established LC-MS/MS methods for the determination of liposome encapsulated,unencapsulated and total amphotericin B in human plasma can be used for the determination of plasma concentration in human.2.About 90%or more of amphotericin B was in the form of liposomes in plasma within 72 h after single dose of Lipo-AmB in healthy Chinese subjects and within 24 h after repetitive dose of Lipo-AmB in patients at steady-state.The C_maxof total amphotericin B was 24.29±5.44μg/ml for single dose 2 mg/kg Lipo-AmB in healthy Chinese,and it was higher than the C_max（17.55±6.94μg/ml）after 5 doses of 3 mg/kg Lipo-AmB,and consistant with the C_max（26.89±14.48μg/ml）after 10 doses of 3 mg/kg Lipo-AmB.After repeated administration,the incidence of TEAE was 97.2%（35/36）,and the severity was mostly moderate and the prognosis was mostly recovery,indicating that the patients could tolerate the dose.3.Based on preclinical and clinical pharmacokinetic studies,the PBPK model of Lipo-AmB was finally established.Human target tissue and plasma concentrations of AmB-LPU,AmB-LP and total AmB were simultaneously described.The dose-exposure-nephrotoxicity relationship of patients was preliminarily explored,and the influencing factors of target tissue exposure level after Lipo-AmB administration were studied.