| Tacrolimus,which is used mainly after allogeneic organ transplant to lower the risk of organ rejection,is a macrolide lactone that was exacted from the fermentation broth of a Japanese soil sample that contained the bacterium Streptomyces tsukubaensis.Organ transplant patients need long-term use of immunosuppressive agents,which increases the risk of patients suffering from various infectious diseases,including fungal infections.Itraconazole is a broad-specrum antifungal drug,which belongs to the triazole family.In clinical,the combined use of tacrolimus and itraconazole is very common.Tacrolimus is a substrate of CYP3A,whereas itraconazole is a strong inhibitor of CYP3A.Because of the narrow therapeutic window of tacrolimus,the large inter-individual variation of blood concentration and many side effects,it is necessary to monitor tacrolimus blood concentration and strictly control its dose when it is coadministered with itraconazle.In the current study,an LC-MS method was developed to determine tacrolimus concentrations in bio-samples.A physiologically based pharmacokinetic(PBPK)model was developed and applied to investigate the influence of itraconzole on the absorption and distribution of tacrolimus in rats and mice.Based on these data,the PBPK model was successfully scaled to human.Moreover,we also used docetaxel as the model drug.A PBPK model was then developed to evaluate the feasibility and applicability of the interspecies scaling method described in our study.The aim of this study was to provide guidance for rational use of tacrolimus,developing new formulation and structural analogue,and applying new therapeutic regimen.1.Influence of itraconazole on tacrolimus absorption and distribution in ratsAn LC-MS method was developed for the determination of tacrolimus in rat whole blood and tissue samples.The classical compartment model,non-compartmental analysis and PBPK model were applied to investigate the influence of itraconazole on tacrolimus absorption and distribution.The final results showed that the blood tacrolimus concentration-time curves fit the first-order absorption,first-order elimination,two-compartment open model.Rats were randomly divided into four groups:namely males following an oral dose of tacrolimus solution only,females following an oral dose of tacrolimus solution only,males following an oral dose of tacrolimus solution with a combination of itraconazole and females following an oral dose of tacrolimus solution with a combination of itraconazole.The main pharmacokinetic parameters of the four group of rats are list as follows:AUC0-∞ were calculated as 85.4 ± 11.2,93.6±32.8,191.9 ± 56.1 and 160.5±36.6 μg·L-1·h;Cmax were calculated as 30.44± 6.86,32.03± 10.93,56.42±15.94 and 43.89± 8.88 μg· L-1;CLc/F were calculated as 6.226± 1.332,6.224±3.512,2.758±0.779 and 3.774 ± 1.192 L·h-1;CLp/F were calculated as 8.45 ± 2.01,7.692± 2.686,3.533± 1.116 and 4.504±0.536 L·h-1;Vc/F were calculated as 5.554±1.656,5.077±2.242,3.628± 0.876 and 4.704± 0.577 L;t1/2(α)were calculated as 0.2595±0.0510,0.3045±0.0686,0.3795± 0.0506 and 0.3578±0.0787,respectively.All these results showed that compared to rats that took tacrolimus only,rats that took tacrolimus and itraconazole simultaneously were characterized by higher AUCs,higher Cmax,lower CLc/F,lower CLp/F,lower Vc/F and longer t1/2(α).Moreover,lower Vp/F was found in males combined with itraconazole than those took tacrolimus only.There was no significant difference in Ka and t1/2(α)among all the four groups,which indicated that neither itraconazole nor sex influenced the absorption rate of tacrolimus significantly.The tissue distribution results showed that compared to rats that took tacrolimus only,rats that took tacrolimus and itraconazole simultaneously were characterized by higher AUCs and Cmax in all nine tissues.Except for 0.083 h after administration,itraconzole increased tacrolimus concentrations in all tissues for all time points,especially for males.Generally,there was no significant gender difference of tacrolimus tissue concentration in all time point.Only the differences of tacrolimus concentrations of 0.5 h in brain,0.083 h in liver,0.083,0.5,8 and 12 h in kidney were significant.In addition,a significant increase of AUC level in kidney was found in males after combing with itraconazole,while this increasment in females was not significant.With respect to sex,there was no significant gender difference in tacrolimus AUCs.The PBPK analysis result showed that CLli(**p<0·01),CLgu(**p<0.01)and Kin,fa(*p<0.05)decreased significantly in rats after combing with itraconazole.In rats that took tacrolimus and itraconazole simultaneously,Kt,ki was significantly higher(*p<0.05)in females than males.Beside,itraconazole slighty(*p<0.05)raised the absorption rate of tacrolimus in females.These results indicated that itraconazole reduced the elimination rate of tacrolimus in rats significantly,leading to a longer retention time in vivo.Consequently,it is necessary to increase the frequency of tacrolimus concentration mornitoring when combining with itraconazole,reducing the amount of drug administration and dosing frequency appropriately.2.Influence of itraconazole on tacrolimus absorption and distribution in miceAn LC-MS method was developed for the determination of tacrolimus in mouse whole blood and tissue samples.The classical compartment model,non-compartmental analysis and PBPK model were applied to investigate the influence of itraconazole on tacrolimus absorption and distribution.The final results showed that the blood tacrolimus concentrationtime curves fit the first-order absorption,first-order elimination,two-compartment open model.Mice were randomly divided into four groups:namely males following an oral dose of tacrolimus solution only,females following an oral dose of tacrolimus solution only,males following an oral dose of tacrolimus solution with a combination of itraconazole and females following an oral dose of tacrolimus solution with a combination of itraconazole.The main pharmacokinetic parameters of the four group of mice are list as follows:AUCo were calculated as 77.05 ± 10.02,96.7± 20.5,140.2 ± 121.8 and 135.2 ± 95.0 μg · L-1·h;Cmax were calculated as 28.43 ± 3.19,32.65 ± 7.11,51.52 ± 21.34 and 43.22 ± 36.74μg· L-1;CLc/F were calculated as 0.867 ± 0.106,0.7306 ± 0.1185,0.5025±0.1812 and 0.5736±0.2103 L·h-1;CLp/F were calculated as 1.126 ± 0.160,0.993 ± 0.203,0.7092± 0.2039 and 0.835 ± 0.401 L·h-1;Vc/F were calculated as 0.7326± 0.1042,0.6472±0.1332,0.4942 ±0.0375 and 0.5442± 0.2622 L;Vp/F were calculated as 2.456 ± 0.580,2.174± 0.551,2.002 ±0.648 and 1.882 ± 0.780 L;t1/2(α)were calculated as 0.2312 ± 0.0071,0.2372± 0.0074,0.2482± 0.1190 and 0.2414 ± 0.0155;t1/2(β)were calculated as 3.812 ± 0.406,3.998 ± 0.437,5.178±0.560 and 4.246± 0.754,respectively.All these results showed that compared to mice that took tacrolimus only,mice that took tacrolimus and itraconazole simultaneously were characterized by higher Cmax,lower CLc/F,lower CLp/F,lower Vc/F,lower Vp/F,longer t1/2(α)and longer t1/2(β).Female mice took tacrolimus and itraconazole simultaneously were characterized by higher AUC compared to females took tacrolimus only.Except for t1/2(β),there was no significant difference in other pharmacokinetic parameters between males and females.The tissue distribution study result showed that compared to mice that took tacrolimus only,mice that took tacrolimus and itraconazole simultaneously were characterized by higher AUCs in all nine tissues.With respect to males,itraconazole significantly raised the AUCs in lung,spleen,liver,gut,kidney and muscle.While for females,itraconazole increased the AUCs in lung,brain,heart,liver,gut,kidney and muscle.Except for 0.083 h after administration,itraconzole increased tacrolimus concentrations in all tissues for all time points,especially for males.Generally,there was no significant gender difference of tacrolimus tissue concentration in all time point.Only the differences of tacrolimus concentrations of 2 h in lung and 8 h in spleen were significant.Moreover,in mice that took tacrolimus only,females were characterized by higher AUCs in brain and spleen than males,while there was no significant gender difference of AUCs in all nine tissues after combination with itraconazole.With respect to sex,there was no gender difference in tacrolimus AUCs of all mice tissues investigated in this study except that females showed higher brain AUC level than males.The PBPK analysis result showed that CLli(**p<0.01)and CLgu(**p<0.01)decreased significantly in mice after combing with itraconazole.Kt,b,was significantly higher(**p<0.01)in females took tacrolimus and itraconazole simultaneously than those took tacrolimus only.Except for Ka,there was no significant difference(p>0.05)between males and females for all PBPK parameters.These results indicated that itraconazole reduced the elimination rate of tacrolimus in mice significantly,leading to a longer retention time in vivo.This indicated that it is necessary to increase the frequency of tacrolimus concentration mornitoring when combining with itraconazole,reducing the amount of drug administration and dosing frequency in the clinical use.3.Simulation of tacrolimus pharmacokinetics in human using rat/mouse-derived PBPK modelDatasets used for developing the physiological model were collected from the current experimental data.The final PBPK model was successfully scaled to human.The estimation result from the final model showed all pharmacokinetic parameters and corresponding IIVs were precisely estimated,with an RSE%of 10.7%or less.The allometric coefficients for interspecies scaling were between 0.67-0.79,which were in agreement with literature values(0.67 or 0.75).The median of the simulated concentrations adequately described the central tendency of the observed data.The final PBPK model simulations acceptably approximated the observed concentration-time profiles in rat and mouse.The predictive performance of the final model was further evaluated by simulating the concentration-time profiles in human by scaling parameters estimated for rats and mice.As a result,the model predicted profiles are in good agreement with the concentration-time profiles described in literature.4.Evaluation of interspecies scaling method using docetaxelIn order to evaluate the feasibility and applicability of the interspecies scaling method in this study,docetaxel was selected as the model drug.The estimation result from the physiological model of docetaxel showed all pharmacokinetic parameters were precisely estimated,with an RSE%of 43.4%or less.The final PBPK model simulations acceptably approximated the observed concentration-time profiles in rat.The predictive performance of the physiological model of docetaxel was further evaluated by simulating the concentrationtime profiles in human by scaling parameters using the allometric coefficient(0.67)from literature.As a result,the model predicted profiles are in good agreement with the concentration-time profiles described in literature.These results indicated that the PBPK built in the current study is reasonable and predictive,which can be further used in evaluating the influence of itraconazole on tacrolimus disposition in human,improving the effectiveness and safety of drug combination,developing new formulation and analogues,and applying new therapeutic regimen. |
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