Activation of PXR-Cytochrome P450s axis: A Possible Reason for the Enhanced Accelerated Blood Clearance Phenomenon of PEGylated Liposomes in vivo

: Recently, we reported that repeated injection of PEGylated liposomes (PEG-L) at certain intervals to the same rat lead to the disappearance of their long-circulating properties, referred to as “accelerated blood clearance (ABC)” phenomenon. Evidence from our recent studies suggest cytochrome P450s (CYPs) partly contribute to the induction of the ABC phenomenon, which had previously been ignored. However, little is known about the details of the mechanism for the induction of CYPs. This study was undertaken to investigate the roles of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in the ABC phenomenon, which are the major upstream transcriptional regulators of the CYP genes including CYP3A1, CYP2C6 and CYP1A2. The results demonstrated that the expression of rat PXR and CAR were significantly increased in such 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 (RXRα) transcriptionally active heterodimer, indicating that nuclear translocation of PXR was induced in the ABC phenomenon, whereas nuclear translocation of CAR was not observed. Notably, pretreatment with the specific PXR inducer-dexamethasone significantly induced accelerated systemic clearance of the subsequent injection of PEG-L, associating with increased nuclear co-localization of PXR-RXRα. These results revealed that the induction of CYPs in the ABC phenomenon may be largely attributable to the activation of PXR induced by sequential injections of PEG-L, thus confirming the crucial involvement of PXR-CYPs axis in promoting ABC phenomenon. Significance statement: The results of this study revealed that the induction of CYPs in the ABC phenomenon may be largely attributable to the activation of PXR induced by sequential injections of PEG-L, thus confirming the crucial involvement of PXR-CYPs axis in promoting ABC phenomenon. The data may help to extend our insights into 1) the role of CYPs, which are regulated by the liver-enriched nuclear receptor PXR, in the ABC phenomenon, and 2) the therapeutic potential of targeting the PXR-CYP axis for reducing the magnitude of the ABC phenomenon in clinical practice. This study was undertaken to ascertain the molecular mechanism of the induction of rat PXR and/or CAR-CYPs axis on the ABC phenomenon upon repeated injection of PEG-L. We are currently determining the pharmacokinetic parameters of PEG-DTX-L to evaluate the magnitude of the ABC phenomenon. Our previous studies have shown that the induction process of the ABC phenomenon is partly due to the induced activities and expression of hepatic CYPs. Here we unraveled whether nuclear translocation of PXR and CAR occurred in the induction phase of the ABC phenomenon. In this context, we thereby firstly detected the expression and localization of PXR and CAR in the hepatocyte nucleus and cytoplasm. Moreover, dexamethasone (DEX), a prototypical inducer of rat PXR through transactivation without effect on CAR (Moore et al., 2000; Shi et al., 2010), was used to characterize the effect of PXR-mediated induction of CYP3A1 signaling axis in rats. We assessed the impact of DEX on the pharmacokinetic profile of the subsequent injection of PEG-DTX-L. In addition, the gene and protein expression of hepatic PXR and CYPs, as well as alteration of expression and localization of PXR protein in nucleus and cytoplasm, were measured. These data may help to extend our insights into the role of the CYPs, which are regulated by liver-enriched PXR and CAR, in the induction of the ABC phenomenon. Each by CAR and RXRα were analyzed by western blotting. Bar graphs show quantitative evaluation of PXR, CAR and RXRα bands by densitometry from triplicate separate experiments. Each band density was evaluated by ImageJ software and these