Single-cell ATAC and RNA sequencing reveal pre-existing and persistent cells associated with prostate cancer relapse.

Prostate cancer is heterogeneous and patients would benefit from methods that stratify those who are likely to respond to systemic therapy. Here, we employ single-cell assays for transposase-accessible chromatin (ATAC) and RNA sequencing in models of early treatment response and resistance to enzalutamide. In doing so, we identify pre-existing and treatment-persistent cell subpopulations that possess regenerative potential when subjected to treatment. We find distinct chromatin landscapes associated with enzalutamide treatment and resistance that are linked to alternative transcriptional programs. Transcriptional profiles characteristic of persistent cells are able to stratify the treatment response of patients. Ultimately, we show that defining changes in chromatin and gene expression in single-cell populations from pre-clinical models can reveal as yet unrecognized molecular predictors of treatment response. This suggests that the application of single-cell methods with high analytical resolution in pre-clinical models may powerfully inform clinical decision-making.

A high-content, in vitro cardiac fibrosis assay for high-throughput, phenotypic identification of compounds with anti-fibrotic activity.

A key feature in the pathogenesis of heart failure is cardiac fibrosis, but effective treatments that specifically target cardiac fibrosis are currently not available. A major impediment to progress has been the lack of reliable in vitro models with sufficient throughput to screen for activity against cardiac fibrosis. Here, we established cell culture conditions in micro-well format that support extracellular deposition of mature collagen from primary human cardiac fibroblasts - a hallmark of cardiac fibrosis. Based on robust biochemical characterization we developed a high-content phenotypic screening platform, that allows for high-throughput identification of compounds with activity against cardiac fibrosis. Our platform correctly identifies compounds acting on known cardiac fibrosis pathways. Moreover, it can detect anti-fibrotic activity for compounds acting on targets that have not previously been reported in in vitro cardiac fibrosis assays. Taken together, our experimental approach provides a powerful platform for high-throughput screening of anti-fibrotic compounds as well as discovery of novel targets to develop new therapeutic strategies for heart failure.

IGFBP2 potentiates nuclear EGFR-STAT3 signaling.

Insulin-like growth factor binding protein 2 (IGFBP2) is a pleiotropic oncogenic protein that has both extracellular and intracellular functions. Despite a clear causal role in cancer development, the tumor-promoting mechanisms of IGFBP2 are poorly understood. The contributions of intracellular IGFBP2 to tumor development and progression are also unclear. Here we present evidence that both exogenous IGFBP2 treatment and cellular IGFBP2 overexpression lead to aberrant activation of epidermal growth factor receptor (EGFR), which subsequently activates signal transducer and activator of transcription factor 3 (STAT3) signaling. Furthermore, we demonstrate that IGFBP2 augments the nuclear accumulation of EGFR to potentiate STAT3 transactivation activities, via activation of the nuclear EGFR signaling pathway. Nuclear IGFBP2 directly influences the invasive and migratory capacities of human glioblastoma cells, providing a direct link between intracellular (and particularly nuclear) IGFBP2 and cancer hallmarks. These activities are also consistent with the strong association between IGFBP2 and STAT3-activated genes derived from The Cancer Genome Atlas database for human glioma. A high level of all three proteins (IGFBP2, EGFR and STAT3) was strongly correlated with poorer survival in an independent patient data set. These results identify a novel tumor-promoting function for IGFBP2 of activating EGFR/STAT3 signaling and facilitating EGFR accumulation in the nucleus, thereby deregulating EGFR signaling by two distinct mechanisms. As targeting EGFR in glioma has been relatively unsuccessful, this study suggests that IGFBP2 may be a novel therapeutic target.

Absence of interaction between erythromycin and a single dose of clozapine.

OBJECTIVE: To study the suggested pharmacokinetic interaction between erythromycin, a strong inhibitor of CYP3A4, and clozapine. METHODS: Twelve healthy male volunteers received a single dose of 12.5 mg of clozapine alone or in combination with a daily dose of 1500 mg erythromycin in a randomised crossover study. Clozapine and its metabolites clozapine-N-oxide and desmethyl-clozapine were measured in serum samples which were collected during a 48 h period and in a sample of the urine secreted over the interval 0-12 h. RESULTS: There were no significant differences in mean area under the serum concentration time curves (1348 (633) nmol h x l(-1) in the control phase and 1180 (659) nmol h x l(-1) in the erythromycin phase), terminal halflives (19 (13) h and 15 (6) h, respectively), peak serum concentrations (92 (53) nmol x l(-1) and 77 (40) nmol x l(-1), respectively), time to peak serum concentrations (1.4 (0.7) h and 1.5 (1.0) h, respectively) or apparent oral clearances of clozapine (34 (15) l x h(-1) and 46 (37) l x h(-1), respectively). There were no significant differences in partial metabolic clearances to clozapine-N-oxide (5.1 (3.6) l x h(-1) and 7.8 (9.4) l x h(-1), respectively) or to desmethyl-clozapine (1.5 (1.3) l x h(-1) and 1.8 (1.7) l x h(-1), respectively) or in renal clearances of clozapine (0.8 (0.5) l x h(-1) and 1.0 (0.7) l x h(-1), respectively) between the two phases. CONCLUSION: These results demonstrate that erythromycin at a clinically relevant dosage does not inhibit the metabolism of clozapine. Hence, CYP3A4 seems to be of minor importance in the disposition of clozapine in humans at least when clozapine is taken at a low single dose.

Nuclear magnetic resonance spectroscopy study of muscle growth, mdx dystrophy and glucocorticoid treatments: correlation with repair.

Proton nuclear magnetic resonance spectroscopy (1H NMR) can be used to study skeletal muscle metabolism. The mdx mouse is a unique animal for studies of muscle regeneration, and models the disease of Duchenne muscular dystrophy (DMD). The goals of this study were to determine the potential of 1H NMR spectroscopy as an alternative to conventional histology in monitoring: (1) normal growth in control muscle and the progression of dystrophy in mdx muscle, and (2) beneficial treatments (glucocorticoids) on mdx dystrophy. Ex vivo 1H NMR spectra of limb and diaphragm muscles were obtained from different ages of control and mdx mice, and from mice which were treated with prednisone or deflazacort. Peaks with contributions from creatine, taurine and lipids were examined. Lower levels of taurine and creatine characterized predystrophy and active dystrophy intervals in mdx muscle compared to control. Levels of taurine increased with stabilization of the disease by repair. A measure of accumulated muscle repair, fiber centronucleation and many spectral peaks were highly and significantly correlated. Greater amounts of lipids were found in the diaphragm compared to limb spectra. Treatment of dystrophy, which improved muscle phenotype, resulted in greater levels of taurine and creatine, especially in the limb muscle. Therefore, 1H NMR differentially discriminates: (1) control and mdx muscle; (2) the progression of mdx dystrophy and developmental stages in normal growth; (3) mild and severe dystrophic phenotypes (diaphragm vs limb); and (4) changes associated with improved muscle phenotype and regeneration (due to treatment or injury). The results focus on monitoring muscle repair, not degeneration. We conclude that 1H NMR is a reliable tool in the objective investigation of muscle repair status during muscular dystrophy.

Non-linear fluvoxamine disposition.

AIMS: To study the pharmacokinetics of fluvoxamine when given in increasing doses to healthy volunteers. METHODS: Ten healthy, non-smoking men were given maintenance treatment with fluvoxamine for 4 weeks. Eight subjects were CYP2D6 extensive metabolisers (EMs) and two were CYP2D6 poor metabolisers (PMs). As a measure of the CYP1A2 phenotype, the paraxanthine/caffeine ratio in saliva after intake of caffeine was studied. The fluvoxamine doses given were 25 mg day(-1) the first week, 50 mg day(-1) the second week, 100 mg day(-1) the third week and 200 mg day(-1) the fourth week, divided in two daily doses. On the seventh day every week, serum concentrations of fluvoxamine were followed for a dose interval of 12 h. After discontinuation of treatment, fluvoxamine concentrations were followed for 1 week. RESULTS: For each of the three two-fold increases in given dose, the mean AUC increased 3.25-fold, 3.17-fold and 3.14-fold, respectively (P < 0.0001), indicating a decrease in oral clearance with increasing dose. The elimination half-life based upon the serum concentrations 12-48 h after discontinuation of fluvoxamine was 32.1 +/- 11.0 h whereas the half-life based upon the concentrations 3-7 days after discontinuation was significantly shorter, 15.8 +/- 4.2h (means +/- s.d.; P < 0.001). There were no significant correlations between the CYP1A2 phenotype and fluvoxamine AUCs at different doses (r = -0.56; P = 0.095 for the correlation between the paraxanthine/caffeine ratio in saliva and fluvoxamine AUC at a dose of 50 mg day[-1]). The two CYP2D6 PMs had AUC values in the same range as the EMs. CONCLUSIONS: The present study conclusively demonstrates that fluvoxamine exhibits non-linear kinetics within the therapeutic dose interval. The reason for non-linearity is not Michaelis-Menten saturation kinetics of a single metabolic pathway, but rather a complex involvement of multiple parallel pathways.

Relationship between fluvoxamine pharmacokinetics and CYP2D6/CYP2C19 phenotype polymorphisms.

OBJECTIVE: The purpose of this study was to investigate whether the disposition of fluvoxamine is associated with the CYP2D6 and CYP2C19 phenotype polymorphisms. METHODS: The serum concentration of fluvoxamine was followed for 48 h after oral administration of a single dose of 50 mg fluvoxamine to five poor metabolizers of the CYP2D6 test drug dextromethorphan, five poor metabolizers of the CYP2C19 test drug mephenytoin, and five extensive metabolizers of both test drugs. RESULTS: Poor metabolizers of dextromethorphan had significantly higher areas under the serum concentration-time curve than extensive metabolizers of dextromethorphan (mean 1.31 vs 1.00 mumol.h.l-1). There were no differences between poor and extensive metabolizers of mephenytoin (mean, 1.00 vs 1.15 mumol.h.l-1). CONCLUSION: The results are consistent with a possible minor to moderate role of CYP2D6, but not CYP2C19, in fluvoxamine metabolism.

Urinary excretion of codeine, ethylmorphine, and their metabolites: relation to the CYP2D6 activity.

The formation of morphine from codeine and ethylmorphine is mainly mediated by the polymorphic enzyme CYP2D6. The objective of this study was to investigate whether CYP2D6 poor metabolizers (PM) and CYP2D6 extensive metabolizers (EM) would respond differently during testing for opiate drugs of abuse in urine after intake of these drugs. Five PM and five EM of dextromethorphan were administered single oral doses of codeine (25 mg) and ethylmorphine (25 mg), and the urinary excretion of parent compounds and selected metabolites was observed for 72 hours. Analysis was performed with GC-MS after hydrolysis of the glucuronide conjugates. Selected urine samples were screened for the presence of opiates by the Abbott ADx immunoassay method. The results from one PM and one EM were excluded because of technical analytical problems. EM excreted significantly more morphine than PM after intake of both codeine (6.5% vs. 1.1% of the dose; p < 0.05) and ethylmorphine (11.0% vs. 3.0% of the dose; p < 0.05). Screening results were positive significantly longer for EM than for PM after codeine intake (mean, 33 hours vs. 17 hours; p < 0.05), and the same trend, albeit nonsignificantly, was noted for ethylmorphine (mean, 33 hours vs. 24 hours). Regardless of CYP2D6 phenotype, significantly more morphine was formed after intake of ethylmorphine than after intake of codeine (7.0% vs. 3.8% of the dose; p < 0.05). There were high correlations between dextromethorphan metabolic ratios and the ratios of codeine to morphine, ethylmorphine to morphine, norcodeine to normorphine, and norethylmorphine to normorphine (r = 0.80 to 0.92; p = 0.030 to 0.001). Although this study should be interpreted with caution because of the few subjects included and the single-dose design, it demonstrates that the CYP2D6 phenotype clearly affects the results when testing for opiates in urine after intake of codeine and ethylmorphine.

Ferrous sulfate does not reduce serum levels of famotidine or cimetidine after concurrent ingestion.

A series of randomized crossover studies were performed to determine whether there was a reduction in serum levels of cimetidine and famotidine when coingested with ferrous sulfate (300 mg). Coingestion of a ferrous sulfate tablet with cimetidine (300 mg) was associated with little reduction in serum cimetidine area under the curve (AUC) (mean versus mean, 20.8 versus 23.4 mumol.hr/L; mean percentage difference, -11%; 95% confidence interval [CI] of percentage difference, -26% to 4.2%) or peak concentration (Cmax) (mean versus mean, 5.1 versus 6.1 mumol/L; mean percentage difference, -16%; CI of percentage difference, -36% to 4%). Similarly, ferrous sulfate solution coingested with cimetidine caused little change in cimetidine AUC (mean versus mean, 19.9 versus 23.0 mumol.hr/L; mean percentage difference, -13%; CI of percentage difference, -34% to 7%) or Cmax (mean versus mean, 5.0 versus 5.0 mumol/L; mean percentage difference, 1%; CI of percentage difference, -18% to 20%). Concurrent ingestion of famotidine (40 mg) with a ferrous sulfate tablet did not result in significant reductions in serum famotidine AUC (mean versus mean, 1.78 versus 1.99 mumol.hr/L; mean percentage difference, -10%; CI of percentage difference, -34% to 13%) or Cmax (mean versus mean, 0.31 versus 0.32 mumol/L; mean percentage difference, -3%; CI of percentage difference, -27% to 22%). The formation of famotidine:iron(III) complexes was shown in methanol but was not observed in an aqueous buffer at pH 6.5. Ranitidine did not bind iron in an aqueous buffer and only weakly bound iron in methanol. Coingestion of ferrous sulfate with either cimetidine or famotidine does not cause a clinically relevant reduction in serum histamine H2-receptor blocker levels and, on the basis of in vitro binding experiments, iron is unlikely to interact with ranitidine.

Ferrous sulfate reduces cimetidine absorption.

A variety of drugs that bind to iron have significant reductions in absorption when coadministered with iron compounds. Cimetidine has a structure that would suggest strong binding to iron ions. In vitro experiments were performed to examine a variety of characteristics of the binding of iron to cimetidine. Further studies were conducted to determine the effect of concurrent administration of ferrous sulfate on cimetidine absorption in an in vivo isolated perfused rat jejunal model of drug absorption. The dose of cimetidine was chosen to represent a human dose of 300 mg, while the ferrous sulfate doses were chosen to represent 150- and 300-mg doses. The higher ferrous sulfate dose completely inhibited cimetidine absorption (P < 0.01), while the lower dose of ferrous sulfate caused a 63% reduction in cimetidine absorption (P < 0.05). In vitro iron in its ferrous from rapidly oxidizes to the ferric form. The ferric form of iron binds to cimetidine and may be the cause of the decreased cimetidine absorption. Care should be taken in prescribing iron supplements with cimetidine.