Pregnane X receptor activation induces liver enlargement and regeneration and simultaneously promotes the metabolic activity of CYP3A1/2 and CYP2C6/11 in rats.

Human pregnane X receptor (PXR) is critical for regulating the expression of key drug-metabolizing enzymes such as CYP3A and CYP2C. Our recent study revealed that treatment with rodent-specific PXR agonist pregnenolone-16α-carbonitrile (PCN) significantly induced hepatomegaly and promoted liver regeneration after two-thirds partial hepatectomy (PHx) in mice. However, it remains unclear whether PXR activation induces hepatomegaly and liver regeneration and simultaneously promotes metabolic function of the liver. Here, we investigated the metabolism activity of CYP1A2, CYP3A1/2 and CYP2C6/11 during PXR activation-induced liver enlargement and regeneration in rats after cocktail dosing of CYP probe drugs. For PCN-induced hepatomegaly, a notable increase in the metabolic activity of CYP3A1/2 and CYP2C6/11, as evidenced by the plasma exposure of probe substrates and the AUC ratios of the characteristic metabolites to its corresponding probe substrates. The metabolic activity of CYP1A2, CYP3A1/2 and CYP2C6/11 decreased significantly after PHx. However, PCN treatment obviously enhanced the metabolic activity of CYP2C6/11 and CYP3A1/2 in PHx rats. Furthermore, the protein expression levels of CYP3A1/2 and CYP2C6/11 in liver were up-regulated. Taken together, this study demonstrates that PXR activation not only induces hepatomegaly and liver regeneration in rats, but also promotes the protein expression and metabolic activity of the PXR downstream metabolizing enzymes such as CYP3A1/2 and CYP2C6/11 in the body.

Gut Microbiota Affects Mouse Pregnane X Receptor Agonist Pregnenolone 16α-Carbonitrile-Induced Hepatomegaly by Regulating Pregnane X Receptor and Yes-Associated Protein Activation.

Pregnane X receptor (PXR) is essential in the regulation of liver homeostasis, and the gut microbiota is closely linked to liver physiologic and pathologic status. We previously found that activation of PXR significantly promotes liver enlargement through interaction with yes-associated protein (YAP). However, whether gut microbiota contributes to PXR-induced hepatomegaly and the involved mechanisms remain unclear. In this study, C57BL/6 mice were administered the mouse-specific agonist pregnenolone 16α-carbonitrile (PCN) for 5 days. Depletion of gut microbiota was achieved using broad-spectrum antibiotics (ABX) and fecal microbiota transplantation (FMT) was performed to restore the gut microbia. The composition of gut microbiota was analyzed by 16S rRNA sequencing, while the expression of PXR, YAP, and their downstream target genes and proteins were assessed. The results indicated that PCN treatment altered the composition and abundance of specific bacterial taxa. Furthermore, depletion of gut microbiota using ABX significantly attenuated PCN-induced hepatomegaly. FMT experiments further demonstrated that the fecal microbiota from PCN-treated mice could induce liver enlargement. Mechanistic studies revealed that ABX treatment impeded the PXR and YAP activation induced by PCN, as evidenced by decreased expression of PXR, YAP, and their downstream targets. Moreover, alterations in PXR and YAP activation were likely contributing to hepatomegaly in recipient mice following FMT from PCN-treated mice. Collectively, the current study demonstrated that gut microbiota is involved in PCN-induced hepatomegaly via regulating PXR and YAP activation, providing potential novel insights into the involvement of gut microbiota in PXR-mediated hepatomegaly. SIGNIFICANCE STATEMENT: This work describes that the composition of gut microbiota is altered in mouse pregnane X receptor (PXR) agonist pregnenolone 16α-carbonitrile (PCN)-induced hepatomegaly. Treatment with an antibiotic cocktail depletes the intestinal microbiota, leading to the impairment of liver enlargement caused by PCN. Additionally, fecal microbiota transplantation from PCN-treated mice induces liver enlargement. Further study revealed that gut microbiota is involved in hepatomegaly via regulating PXR and yes-associated protein activation.

PXR triggers YAP-TEAD binding and Sirt2-driven YAP deacetylation and polyubiquitination to promote liver enlargement and regeneration in mice.

Pregnane X receptor (PXR) plays an important role in the regulation of metabolic homeostasis. Yes-associated protein (YAP) is a critical regulator of liver size and liver regeneration. Recently, we reported that PXR-induced liver enlargement and regeneration depend on YAP signalling, but the underlying mechanisms remain unclear. This study aimed to reveal how PXR regulates or interacts with YAP signalling during PXR-induced hepatomegaly and liver regeneration. Immunoprecipitation (IP), Co-IP and GST pull-down assays were performed in vitro to reveal the regulatory mechanisms involved in the PXR-YAP interaction. The roles of YAP-TEAD binding and Sirt2-driven deacetylation and polyubiquitination of YAP were further investigated in vitro and in vivo. The results showed that the ligand-binding domain (LBD) of PXR and the WW domain of YAP were critical for the PXR-YAP interaction. Furthermore, disruption of the YAP-TEAD interaction using the binding inhibitor verteporfin significantly decreased PXR-induced liver enlargement and regeneration after 70 % partial hepatectomy (PHx). Mechanistically, PXR activation significantly decreased YAP acetylation, which was interrupted by the sirtuin inhibitor nicotinamide (NAM). In addition, p300-induced YAP acetylation contributed to K48-linked YAP ubiquitination. Interestingly, PXR activation remarkably inhibited K48-linked YAP ubiquitination while inducing K63-linked YAP polyubiquitination. Sirt2 interference abolished the deacetylation and K63-linked polyubiquitination of YAP, suggesting that the PXR-induced deacetylation and polyubiquitination of YAP are Sirt2 dependent. Taken together, this study demonstrates that PXR induce liver enlargement and regeneration via the regulation of YAP acetylation and ubiquitination and YAP-TEAD binding, providing evidences for using PXR as potential target to promote hepatic development and liver repair.

Time-Dependent Distribution of Hydroxychloroquine in Cynomolgus Macaques Using Population Pharmacokinetic Modeling Method.

To evaluate the biodistribution of hydroxychloroquine (HCQ) in cynomolgus macaques and receive dynamic quantitative relationship between plasma, blood, and lung tissue concentration using the population pharmacokinetic modeling method, seventeen cynomolgus macaques were divided into six groups according to different HCQ dosing regimens over 5 days. The monkeys were euthanized, and blood, plasma, urine, feces and ten tissues were collected. All the samples were prepared by protein precipitation and analyzed by HPLC-MS/MS detection. The population pharmacokinetics of HCQ in the plasma, red blood cells, and lung tissue was conducted and simulated via ADAPT program. Results demonstrated that the maximum concentration (C max) of HCQ was 292.33 ng/mL in blood and 36.90 ng/mL in plasma after single dose of 3 mg/kg. The value of area under curve (AUC0-∞) was determined as 5,978.94 and 363.31 h* ng/mL for the blood and plasma, respectively. The descending order of the tissue-to-plasma concentration ratio was liver > spleen > kidney > lung > heart > subcutaneous fat > brain. The tissue-to-plasma concentration ratio and the tissue-to-blood concentration ratio for lung were found to be time-dependent with 267.38 and 5.55 at 120 h postdose, respectively. A five-compartment model with first-order oral absorption and elimination best described the plasma, blood, and lung tissue pharmacokinetics. The estimated elimination rate constant (ke) for a typical monkey was 0.236 h-1. The volume of distribution in central (Vc/F) and other two peripheral compartments (Vb/F and Vl/F) were 114, 2.68, and 5.55 L, respectively. Model-based simulation with PK parameters from cynomolgus macaques showed that the ratio of the blood or plasma to lung tissue was a dynamic change course, which suggested that the rate of HCQ concentration decrease in the blood or plasma was faster than that in the lung tissue. HCQ was found to be accumulated in tissues, especially in the liver, kidney, lung, and spleen. Also, the tissue-to-plasma concentration ratio increased over time. The population pharmacokinetic model developed could allow for the assessment of pharmacokinetics-pharmacodynamics relationships, especially relevant tissue concentration-response for HCQ. Determining appropriate treatment regimens in animals allows translation of these to clinical studies.

Association analysis of SNPs present in plasma with adverse events and population pharmacokinetics in Chinese sunitinib treated patients with renal cell carcinoma.

BACKGROUND: Sunitinib is a tyrosine kinase inhibitor with effective therapeutic outcomes in patients with renal-cell carcinoma. The study were to analyze the association of single-nucleotide polymorphisms present in cell-free DNA and pharmacokinetics with sunitinib treatment-emergent adverse events in Chinese patients with renal-cell carcinoma. MATERIALS AND METHODS: We genotyped 8 keys SNPs in 6 candidate genes. The plasma concentrations of sunitinib and N-desethyl sunitinib were measured using a high performance liquid chromatography-tandam mass spectrometry method. Correlations between the single-nucleotide polymorphisms and adverse events were investigated by univariate and multivariate logistic regression and we quantitatively evaluated the effect of single-nucleotide polymorphisms on the pharmacokinetics of sunitinib by using a population PK model. RESULTS: Necessary dose reductions of sunitinib were significantly correlated with SNP rs1933437 in FLT3. A higher severity of AEs were collected with SNP rs2032582 in ABCB1 and rs1800812 in PDGFRα. Thrombocytopenia was collected with rs1800812 in PDGFRα. Our study provides a population PK model of sunitinib with the ABCB1 genotype as a predictive covariate for apparent oral clearance. CONCLUSIONS: Our study preliminarily confirmed the hypothesis that the pharmacokinetics of sunitinib is affected by the SNPs of enzyme in Chinese renal-cell carcinoma patients, and this affects the different distribution and severity of adverse events of sunitinib.

Therapeutic effect of transmembrane TAT-tCNTF via Erk and Akt activation using in vitro and in vivo models of Alzheimer's disease.

Suppressing Alzheimer's disease (AD) progression via its pathological characteristics, namely senile plaques and neurofibrillary tangles, is an efficient treatment approach. Numerous studies have indicated that ciliary neurotrophic factor (CNTF) not only promotes neuronal growth and maintains cell survival but also significantly reduces amyloid beta (Aß) aggregation and deposition. In this study, transactivator of transcription (TAT) was linked to truncated ciliary neurotrophic factor (tCNTF) and expressed as a fusion protein, TAT-tCNTF, to overcome the transmembrane inability of CNTF. Accordingly, TAT-tCNTF was shown to automatically transport across biomembranes and enter cells mainly by macropinocytosis. Furthermore, TAT-tCNTF increased cell viability in hippocampal neurons treated with Aß. After intracerebroventricular Aß injection, mice exhibited amyloid deposits, which were significantly reduced after intraperitoneal TAT-tCNTF injection. Indeed, TAT-tCNTF significantly reduced Aß-induced tau hyperphosphorylation, and yet barely affected amyloid precursor protein. Accordingly, it was possible to elucidate its potential pharmacological mechanism, with the working effect of TAT-tCNTF shown to be performed by specifically binding to its receptor, CNTFRα, and then activating the Extracellular regulated protein kinases (Erk) and Protein kinase B/Akt pathways exclusive of the Signal transducers and activators of transcription 3 (Stat3) pathway.