Contribution Of PXR-mediated CYP3A1 Induction In The Enhanced Accelerated Blood Clearance Phenomenon Induced By PEGylated Liposomal Docetaxel

Polyethylene glycol?PEG?is recognized as an attractive excipient to modify liposomes due to their extended-circulation properties.Nevertheless,intravenous injection of PEGylated liposomes?PEG-L?usually triggers a rapid systemic clearance of the subsequent dose from blood circulation that is referred to as"accelerated blood clearance?ABC?”phenomenon.Since the dramatic induction of cytochrome P450?P450?activities may lead to accelerated drug elimination,it therefore motivates us to explore whether P450s are involved in the induction of the ABC phenomenon.In this study,PEGylated liposomal docetaxel?PEG-DTX-L?was prepared and used to evaluate the magnitude of the ABC phenomenon in rats induced by repeated injection of PEG-modified liposomes.We first evaluated the magnitude of the ABC phenomenon induced by PEG-L in rats.Then we developed a P450 activity cocktail assay using model substrates for assessing P450 activities when repeated injection of PEG-L,and mRNA and protein levels of these P450s were also measured.Moreover,P450s selective inhibitors were employed to confirm the role of hepatic P450s in induction process of the ABC phenomenon.In addition,the mRNA and protein expression of nuclear receptors?NRs?,ligand-activated transcription factors of P450genes invovled in the ABC phenomenon,were measured.The specific NR inducer was used as a tool to demonstrate a crucial involvement of NRs-P450s signaling pathway in the ABC phenomenon.OBJECTIVE In the presence of ABC phenomenon induced by repeated injection of PEG-L to rats,this work was attempted to investigate the effects of the activities and expression of P450s in the induction process of the ABC phenomenon via comparing pharmacokinetics and accumulation of DTX in liver and spleen.Additionally,specific inhibitors of P450s were employed to unravel the contributions of P450s in the systemic clearance of the second dose of PEG-L.Moreover,the expression and nuclear translocation of the upstream NRs of P450s were investigated in the liver of rats,and the specific NR inducer was employed to unveil the contribution of PXR-CYP3A1 signaling pathway activation in the ABC phenomenon.METHODS We first prepared PEG-DTX-L formulation and the optimal drug formulation was determined by a single factor analysis and orthogonal test,and the physiochemical properties of the colloidal systems including the mean particle size,PDI and zeta potential were evaluated.TEM image and in vitro release kinetics of PEG-DTX-L were observed.DSC studies were performed to measure the phase transition temperatures of PEG-DTX-L formulation along with its individual components.Then,the sensitivity,reliability and specificity of the HPLC-MS/MS method was applied to investigate the pharmacokinetics and biodistribution of PEG-DTX-L upon repeated administration,and the effects of the presence of encapsulated DTX in the first dose as well as various time intervals between two injections on the ABC phenomenon were investigated.Subseqently,the sensitivity,reliability and specificity of the HPLC-MS/MS method was applied for the pharmacokinetic study of cocktail probes in rat plasma,the pharmacokinetic parameters including area under the concentration-time curve?AUC??mean residence time?MRT?,plasma clearance?CLz?and half-life?t1/2?were compared between the single dose of PEG-L and repeated injection of PEG-L.We further investigated the effect of repeated injection of PEG-L with different time intervals on the expression of P450s mRNA and protein levels in liver.By pretreating with P450 inhibitors before the administration of repeated injection of PEG-L with a time interval of 3 days,the contribution of P450s in the induction of the ABC phenomenon was evaluated by observing the magnitude of the ABC phenomenon.Lastly,the expression and nuclear translocation of pregnane X receptor?PXR?and constitutive androstane receptor?CAR?were investigated in liver induced by repeated injection of PEG-L.The effect of the presence of specific NR inducer on the pharmacokinetics of the subsequent dose of PEG-DTX-L was compared with that of repeated injection of PEG-L.Meanwhile,the nuclear and cytoplasmic protein expression of hepatic NRs was also measured in the presence of NR inducer.RESULTS1.Characterization of PEGylated liposomesThe mean particle size of PEG-DTX-L prepared by ethanol injection method was about 118 nm with a narrow size distribution?PDI<0.2?,this formulation was negatively charged?-32.40±1.04 mV?and displayed a high EE value of 96.62%.The liposomes exhibit not only well-characterized size distribution but also stable structure with good sustained release profile of the drug.All these results met the requirements of Chinese Pharmacopeia.2.Effects of PEGylated liposomes with different factors on the induction of the ABC phenomenon upon repeated injection2.1 Effects of presence of DTX and first injection of PEG₂₀₀₀-DSPE doseThe pharmacokinetic parameter ratios of the control groups to different test groups,such as AUC_C/T?ratio of AUC of the control group to the test group?and t_1/2-C/T(ratio of t_1/2 of the control group to the test group),were used as key markers to assess the pharmacokinetic profiles of these test groups.A significant decrease in AUC_0-t and t_1/2/2 as well as increase in CLz was found in the repeated injection groups when compared with the control group,indicating a rapid clearance of the second dose from the circulatory system occurred both in PEG-DTX-L and PEG-B-L pretreated rats.It must be noted that a similar circulating characteristics and ABC phenomenon magnitude were observed in the two test PEG-L groups,indicating that the drug itself is not required for the induction of the enhanced clearance effect.In addition,the first dose of PEG₂₀₀₀-DSPE range from 0.025?mol/kg to 0.05?mol/kg exhibited no significant variation on the magnitude of the ABC phenomenon.2.2 Effects of different time intervals between two injectionsThe pharmacokinetic profiles of the five test groups were dramatically different compared with control group.The ABC phenomenon did occur after repeated injection of PEG-L with different time intervals?1 day,3 days,5 days,and 7 days?.The values of AUC_0-t and MRT_0-t in the test groups were significantly decreased compared to control level,particularly for group 3 d,the values of AUC_0-t,MRT_0-t and t_1/2 were distinctly lower?p<0.05?,and the CLz?0.74±0.16 L/hr/kg?was markedly higher?p<0.01?than those of the control.Moreover,a gradual decrease of the magnitude of the ABC phenomenon was observed with time interval extended from 3 days to 7 days.Therefore,repeated injection of PEG-L with a time interval of 3 days could trigger the strongest magnitude of the ABC phenomenon,and the prolongation of time interval between two injections alleviated the magnitude of the ABC phenomenon.Compared to the control group,the lowest concentration of DTX accumulated in the liver of group 3 d at 12 hr with an obvious ABC effect,while the accumulation of DTX in spleen was higher than that of the control and other test groups.Interestingly,gradually increased accumulation of the second dose in liver and decreased accumulation in spleen were observed together with the prolonged time interval.3.The activities and expression of P450s in plasma induced by single injection or repeated injection of PEGylated liposomesDetermination of P450 activities.A marked difference of pharmacokinetic profiles between single injection and repeated injection of PEG-L were observed using probe cocktail assay in rats.As for midazolam?probe for CYP3A1?,the CLz value of repeated injection group was increased significantly?P<0.01?,compared with single injection group,the AUC_0-t and AUC_0-?values of repeated PEG-L injection group were decreased significantly?P<0.01?,the t1/2 value was also decreased?P<0.05?.As for diclofenac?probe for CYP2C6?,the CLz value of repeated injection group was increased apparently?P<0.01?and the AUC_0-t,AUC_0-?,t_1/2,MRT_0-t and MRT_0-?of repeated injection group were decreased substantially?P<0.01?,compared with single dose group.As for phenacetin?probe for CYP1A2?,the CLz value of repeated injection group was increased significantly?P<0.01?and the AUC_0-t and AUC_0-?values of repeated injection group were decreased significantly?P<0.01?,compared with single dose group.In addition,the t1/2 of repeated PEG-L injection group was also decreased compared to single PEG-L injection group but not reached significant difference.However,for the remaining selected probe substrates,no significant difference was exaimed in the pharmacokinetics of CYP2D1 probe dextromethorphan CYP2C11 probe omeprazole,CYP2C7 probe amodiaquine,and CYP2B1 probe bupropion.Determination of the mRNA and protein expression of P450s.Significant increase of mRNA and protein expression of CYP3A1,CYP2C6 and CYP1A2 were observed in the repeated injection groups compared to single dose group.It is worth mentioning that the mRNA and protein expression of the three P450 isoforms increased from 1 d interval and reached a peak in 3 d interval before gradually decreasing with the extended time interval.4.Effects of the presence of P450 inhibitors on the ABC phenomenonIn comparison with repeated injection of PEG-L group,the value of t_1/2-C/T for pretreated with CYP3A1 inhibitor group was reduced by 1.88 times whereas the value of AUC_C/T/T was not decreased apparently.Surprisingly,the accumulation of DTX in liver was upregulated dramatically?P<0.01?in pretreated with CYP3A1 inhibitor group compared to non-inhibitor group,while a comparable results of spleen was found in the two test groups.Neither AUC_C/T nor t_1/2-C/T was decreased in the group that pretreated with CYP2C6 inhibitor.Likewise,the accumulation of DTX in liver and spleen was aslo not changed in the case of CYP2C6 inhibition.5.The contribution of the activation of PXR-CYP3A1 signaling pathway in the ABC phenomenon induced by PEGylated liposomes in vivoThe expression of rat PXR and CAR were significantly increased in the ABC phenomenon,accompanied by elevated CYP3A1,CYP2C6 and CYP1A2 levels.Further findings revealed that PXR but not CAR protein was substantially upregulated in the hepatocyte nucleus,together with marked nuclear co-localization of PXR-retinoid X receptor alpha?RXR??transcriptionally active heterodimer,indicating that nuclear translocation of PXR was induced in the ABC phenomenon,whereas a failed nuclear translocation of CAR was observed.Notably,pretreating with specific PXR inducer-DEX could significantly induce accelerated systemic clearance of the subsequent injection of PEG-L,associating with increased nuclear co-localization of PXR-RXR?,suggesting that the activation of PXR in response to sequential injections of PEG-L induce the increased P450s production,which may be partly responsible for the rapid clearance of the second dose of PEG-L.CONCLUSIONS1.The model drug-DTX is mainly metabolized by CYP3A1.Thus,inhibiting rats CYP3A1 activity could suppress the oxidative metabolism of PEG-DTX-L from plasma and liver,and thereby slightly attenuating the magnitude of the ABC phenomenon.In this context,it cannot be ruled out that P450s may be involved in the induction of the ABC phenomenon.3.PXR is activated by repeated injection of PEG-L,which probably responsible for the increased expression and activity of inducible CYP3A1 involved in the ABC phenomenon.Accordingly,the activation of PXR-CYP3A1 signaling pathway may play a crucial role in the ABC phenomenon.