Identification of approved drugs as potent inhibitors of pregnane X receptor activation with differential receptor interaction profiles.

Activation of pregnane X receptor (PXR) results in the induction of first-pass metabolism and drug efflux. Hereby, PXR may cause adverse drug reactions or therapeutic failure of drugs. PXR inhibition is thus an attractive option to minimise adverse effects or to improve therapeutic efficiencies; however, only a limited number of antagonists have been identified so far. We performed a cell-based high-throughput screen to identify PXR antagonists, using a library of approved and investigational drugs. Two approved drugs, pimecrolimus and pazopanib, emerged as novel potent antagonists of PXR activation, with IC50 values of 1.2 and 4.1 µM, respectively. We further characterised these with respect to receptor specificity, assembly of the PXR ligand-binding domain (LBD) and interactions with co-factors. In vitro and in silico assays were carried out to identify the site(s) of interaction with the PXR LBD. Primary human hepatocytes were used to investigate antagonism of the induction of endogenous PXR target genes. Pimecrolimus and pazopanib did not affect the transcriptional activity of other nuclear receptors. Both induced the release of co-repressor from PXR and likewise interfered with agonist-induced recruitment of co-activator. Cumulative evidence from cellular and in vitro assays, as well as molecular docking, suggested additional or exclusive binding outside the PXR ligand-binding pocket for both. The compounds differentially antagonised the induction of PXR-regulated genes by rifampicin in primary human hepatocytes. In conclusion, we here have identified two approved drugs as novel potent PXR inhibitors with differential receptor interaction profiles and gene selectivity in primary human hepatocytes.

Adherence, satisfaction and functional health status among patients with multiple sclerosis using the BETACONNECT® autoinjector: a prospective observational cohort study.

BACKGROUND: Maintaining patient adherence to disease modifying drugs in multiple sclerosis is a challenge, which can be improved by autoinjectors. The BETACONNECT® is a fully electronic autoinjector for the injection of interferon beta-1b (IFN beta-1b) automatically recording injections. METHODS: The BETAEVAL study was a prospective, observational, cohort study over 24 weeks among patients with relapsing remitting multiple sclerosis or clinically isolated syndrome treated with IFN beta-1b in Germany using the BETACONNECT®. The primary aim was to investigate treatment adherence, secondary aims included assessing satisfaction and functional health status. Adherence was evaluated from injection data recorded by the device. Patient-related data were obtained from clinical examinations and patient questionnaires. RESULTS: Of the 151 patients enrolled, 143 were available for analysis. Thirty-four patients discontinued the study prematurely. 107/143 (74.8%) patients still used the BETACONNECT® at the end of the study. Injection data from the device at any visit was available for 107 patients. Among those, the percentage of adherent patients injecting ≥80% of doses and still participating in the study was 57.9% at week 24. 29% of patients prematurely stopped the study, 13.1% injected <80%. Among patients with BETACONNECT® data at the respective visit, the proportion of adherent patients was high over the entire study period (week 4: 81.1% [N = 95], week 12: 86.7% [N = 83], week 24: 80.5% [N = 77]). Participants (N = 143) indicated high satisfaction with the BETACONNECT®. At week 24, 98.0% of patients who completed the corresponding questionnaire (strongly) agreed that it was user-friendly, 81.2% felt confident in using it compared to their previous way and 85.5% preferred it to their previous way of injection. Injection-related pain was rated as mild to moderate at all follow-up visits. Whereas 17.2% of patients with corresponding questionnaire indicated using analgesics prior to injection at week 4, only 9.1% did at week 24. Outcomes from questionnaires assessing functional health status, depression, fatigue and cognitive function were very similar throughout the study course. CONCLUSIONS: The majority of patients continued using the BETACONNECT® for IFN beta-1b treatment during the 24-week study period. Adherence was high among participants still using the BETACONNECT® and patients were highly satisfied with the device. Ongoing studies will evaluate long-term adherence and treatment outcomes in patients using the BETACONNECT®. TRIAL REGISTRATION: clinicaltrails.gov NCT02121444 (registered April 22, 2014).

Ligand-dependent and -independent regulation of human hepatic sphingomyelin phosphodiesterase acid-like 3A expression by pregnane X receptor and crosstalk with liver X receptor.

Pregnane X receptor (PXR) mainly regulates xenobiotic metabolism and detoxification. Additionally, it exerts pleiotropic effects on liver physiology, which in large parts depend on transrepression of other liver-enriched transcription factors. Based on the hypothesis that lower expression levels of PXR may reduce the extent of this inhibition, an exploratory genome-wide transcriptomic profiling was performed using HepG2 cell clones with different expression levels of PXR. This screen and confirmatory real-time RT-PCR identified sphingomyelin phosphodiesterase acid-like (SMPDL) 3A, a novel nucleotide phosphodiesterase and phosphoramidase, as being up-regulated by PXR-deficiency. Transient siRNA-mediated knock-down of PXR in HepG2 cells and primary human hepatocytes similarly induced mRNA up-regulation, which translated into increased intracellular and secreted extracellular protein levels. Interestingly, ligand-dependent PXR activation also induced SMPDL3A in HepG2 cells and primary human hepatocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated binding of PXR to the previously identified liver X receptor (LXR)-binding DR4 motif as well as to an adjacent ER8 motif in intron 1 of SMPDL3A. Constitutive binding of the unliganded receptor to the intron 1 chromatin indicated ligand-independent repression of SMPDL3A by PXR. Transient transfection and reporter gene analysis confirmed the specific role of these motifs in PXR- and LXR-dependent activation of the SMPDL3A intronic enhancer. PXR inhibited LXR mainly by competition for binding sites. In conclusion, this study describes that a decrease in PXR expression levels and ligand-dependent activation of PXR and LXR increase hepatic SMPDL3A levels, which possibly connects these receptors to hepatic purinergic signaling and phospholipid metabolism and may result in drug-drug interactions with phosphoramidate pro-drugs.

Human pregnane X receptor is activated by dibenzazepine carbamate-based inhibitors of constitutive androstane receptor.

Unintentional activation of xenosensing nuclear receptors pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR) by clinical drug use is known to produce severe side effects in patients, which may be overcome by co-administering antagonists. However, especially antagonizing CAR is hampered by the lack of specific inhibitors, which do not activate PXR. Recently, compounds based on a dibenzazepine carbamate scaffold were identified as potent CAR inhibitors. However, their potential to activate PXR was not thoroughly investigated, even if the lead compound was named "CAR inhibitor not PXR activator 1" (CINPA1). Thus, we performed a comprehensive analysis of the interaction of CINPA1 and four analogs with PXR. Cellular assays were used to investigate intra- and intermolecular interactions and transactivation activity of PXR as a function of the compounds. Modulation of PXR target gene expression was analyzed in primary human hepatocytes. Ligand binding to PXR was investigated by molecular docking and limited proteolytic digestion. We show here that CINPA1 induced the assembly of the PXR ligand-binding domain, released co-repressors from and recruited co-activators to the receptor. CINPA1 and its analogs induced the PXR-dependent activation of a CYP3A4 reporter gene and CINPA1 induced the expression of endogenous cytochrome P450 genes in primary hepatocytes, while not consistently inhibiting CAR-mediated induction. Molecular docking revealed favorable binding of CINPA1 and analogs to the PXR ligand-binding pocket, which was confirmed in vitro. Altogether, our data provide consistent evidence that compounds with a dibenzazepine carbamate scaffold, such as CINPA1 and its four analogs, bind to and activate PXR.

Expression of chemokine receptors on circulating tumor cells in patients with solid tumors.

BACKGROUND: The study was performed to investigate the expression of chemokine receptors (CR) on circulating tumor cells (CTC), which may be of importance for organ-specific metastases and cancer treatment since CR are potential drug-targets. METHODS: Blood samples from patients with metastatic carcinoma (MC) or melanoma (MM) were enriched for CTC and expression of CR (CXCR4, CCR6, CCR7 and CCR9) was evaluated by flow cytometry. RESULTS: CTC were detected in 49 of 68 patients (72%) [28 MC; 21 MM] with a median number of 3 CTC (range: 1-94)/10 mL of blood. CXCR4 was expressed on CTC in 82% (40/49) of patients [median number 1 CTC/10 mL blood; range 1-14] and CCR6 in 29 patients (59%; median 1, range: 1-14). In MM patients, CCR7 was expressed on CTC in 6 (29%) samples and CCR9 in 12 (57%). A positive correlation between surface expression of CR and organ-specific metastatic pattern was not observed. CONCLUSIONS: CR were expressed on CTC of patients with solid tumors. Along with our findings, the observation that CR could be involved in CTC proliferation and migration of tumor cells appoints CTC as potential CR-antagonist therapeutic target.