Three-dimensional in vitro cell systems are a promising alternative to animals to study cardiac biology and disease. We have generated three-dimensional in vitro models of the human heart ("cardiac ...spheroids", CSs) by co-culturing human primary or iPSC-derived cardiomyocytes, endothelial cells and fibroblasts at ratios approximating those present in vivo. The cellular organisation, extracellular matrix and microvascular network mimic human heart tissue. These spheroids have been employed to investigate the dose-limiting cardiotoxicity of the common anti-cancer drug doxorubicin. Viability/cytotoxicity assays indicate dose-dependent cytotoxic effects, which are inhibited by the nitric oxide synthase (NOS) inhibitor L-NIO, and genetic inhibition of endothelial NOS, implicating peroxynitrous acid as a key damaging agent. These data indicate that CSs mimic important features of human heart morphology, biochemistry and pharmacology in vitro, offering a promising alternative to animals and standard cell cultures with regard to mechanistic insights and prediction of toxic effects in human heart tissue.
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•RG7834 is a novel orally bioavailable HBV viral gene expression inhibitor.•The effect of RG7834 on HBV expression levels is highly virus-specific.•The unique antiviral profile is ...differentiated from that of nucleos(t)ide analogues.•Combination with entecavir and/or PegIFNα improves the antiviral profile and potency.
The hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834).
RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir.
Unlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09 log10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice.
We have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues.
We discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses.
Toxicogenomics held great promise as an approach to enable early detection of toxicities induced by xenobiotics; however, there remain questions regarding the impact of the discipline on ...pharmaceutical nonclinical safety assessment. To understand the current state of toxicogenomics in the sector, an industry group surveyed companies to determine the frequency of toxicogenomics use in in vivo studies at various stages of drug discovery and development and to assess how toxicogenomics use has evolved over time. Survey data were compiled during 2016 from thirteen pharmaceutical companies. Toxicogenomic analyses were infrequently conducted in the development phase and when performed were done to address specific mechanistic questions. Prior to development, toxicogenomics use was more frequent; however, there were significant differences in approaches among companies. Across all phases, gaining mechanistic insight was the most frequent reason cited for pursing toxicogenomics with few companies using toxicogenomics to predict toxicities. These data were consistent with the commentary submitted in response to survey questions asking companies to describe the evolution of their toxicogenomics strategy. Overall, these survey data indicate that toxicogenomics is not widely used as a predictive tool in the pharmaceutical industry but is used regularly by some companies and serves a broader role in mechanistic investigations and as a complement to other technologies.
•Thirteen companies contributed survey data to understand how toxicogenomic approaches are used in drug discovery and development.•Gathering mechanistic insight was the most frequently cited reason to conduct a toxicogenomic analysis.•Toxicogenomic analysis was conducted more frequently early in drug discovery rather than during clinical development.•Toxicogenomics is not widely used to predict toxic responses, but can be a powerful tool for mechanistic research.
Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition ...using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.
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•Diabetic cardiomyopathy can be induced in vitro by environmental or genetic means•Diabetic patient-specific cardiomyocytes show baseline cardiomyopathy•The extent of patient-specific cardiomyopathy is clinically correlated•Phenotypic screening identifies drugs that rescue the disease phenotype
Diabetes causes pathological remodeling of cardiac muscle, which impairs heart function. Drawnel et al. use induced-pluripotent-stem-cell-derived cardiomyocytes to develop environmental and patient-specific in vitro models recapitulating the condition. These models are harnessed in a phenotypic screening assay that identifies candidate protective molecules.
The chemically induced accumulation of α2u-globulin protein in male rats causes specific renal lesions and subsequent nephropathy. Herein, we report additional parallel findings in the kidney of male ...rats consistent with obstructive and retrograde nephropathy. Kidney and urinary bladder samples were evaluated from Wistar rats treated with RG7129 for 2 week and 8 week and from an 8-week mechanistic study using females, intact and castrated males. Histopathological findings were present in intact males in all studies, including hyaline droplet accumulation and granular casts consistent with α2u-globulin nephropathy. In addition, tubular degeneration and regeneration, tubular changes extending from papilla to cortex, tubular dilation, and interstitial and luminal inflammation were observed consistent with retrograde and obstructive nephropathy. Renal and urinary lesions and their severity increased in a time- and dose-dependent manner. Urinalysis findings, including increases in leukocytes, protein, and in kidney biomarkers, kidney injury molecule 1 and clusterin, were present only in intact males. No treatment-related changes were observed in female rats or in castrated males. These results indicate that RG7129 induces α2u-globulin nephropathy, associated with retrograde and obstructive nephropathy secondary to precipitation in intact male rats only, constituting a species- and sex-specific syndrome that is not expected to occur in humans or other species.
Abstract
Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional ...evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.
The use of tubulin binders (TBs) in the treatment of cancer often is associated with cardiotoxicity, the mechanism of which has not been elucidated. To test the hypothesis that interstitial cells of ...the myocardium are the primary target of TBs, we evaluated the acute effects of a single iv administration of three reference TBs: colchicine (0.2 and 2 mg/kg), vinblastine (0.5 and 3 mg/kg), and vincristine (0.1 and 1 mg/kg) 6 and 24 h after dosing. Mitotic arrest was identified at 24 h in all high-dose groups based on an increase in the number of mitotic figures in the interstitium coupled with a decrease in the number of Ki67-positive interstitial cells. Analysis of the myocardial transcriptomic data further supported G2/M cell cycle arrest 6 h after dosing with the high-dose groups of all three compounds. Apoptotic figures and an increase in the number of cleaved caspase 3–positive cells were identified at 6 and 24 h at the highest dose of each compound predominantly in interstitial cells, whereas a few cardiomyocytes were affected as well. Transcriptomic profiling of the myocardium further suggested that some of the affected interstitial cells were endothelial cells based on the upregulation of genes typically associated with vascular damage and downregulation of endothelial cell-specific molecule 1 and apelin. Taken together, these data identify endothelial cells of the myocardium as the primary target of the cardiotoxicity of TBs and identify cell cycle arrest as the mechanism of this toxicity.
The patient's medical history included intrauterine growth restriction, severe growth failure (-4 SD), lymphocytic enteropathy with total villous atrophy, pseudoceliac disease, hepatosplenomegaly, ...dental abnormalities, and atopic dermatitis. ...several publications have suggested that aberrant STAT3 signaling plays a critical role in the pathogenesis of interstitial and fibrotic lung disease (5). Author Contributions: A.F., S.M., L.R.F., and L.G.-C. drafted the initial manuscript, participated in the conceptualization and design of the case report, contributed to the acquisition of data, performed a complete review of the current bibliography, critically revised the manuscript, and approved the final manuscript as submitted; T.P.V. participated in the conceptualization and design of the case report, performed the functional assessment of the mutation with luciferase reporter assay, critically revised the manuscript, and approved the final manuscript as submitted; V.B., V.T., P.-S.R., E. Berard, C.S., M.H., E. Bosdure., M. Baravalle, A.C., and J.-C.D. were involved in clinical follow-up of the patient, participated in the conceptualization and design of the case report, contributed to the acquisition of data, critically revised the manuscript, and approved the final manuscript as submitted; M. Baque participated in the conceptualization and design of the case report, contributed to the acquisition of the computed tomography scan, critically revised the manuscript, and approved the final manuscript as submitted; D.A. participated in the conceptualization and design of the case report, contributed to the acquisition of accessory salivary gland biopsy data, critically revised the manuscript, and approved the final manuscript as submitted; and D.F. participated in the conceptualization and design of the case report, analyzed BAL cytospins, critically revised the manuscript, and approved the final manuscript as submitted. *These authors contributed equally to this work.
OBJECTIVEThe increased mortality observed with the cholesteryl ester transfer protein inhibitor torcetrapib is partly due to increased aldosterone production and blood pressure. The mechanisms ...underlying these effects were investigated.
METHODSCytochrome P450 subunit 11B2 (aldosterone synthase), extracellular signal-regulated kinase (p44/42) and voltage-gated Cachannel alpha subunit mRNA profiling, aldosterone production, cytosolic calcium and RNA interference were assessed in adrenocarcinoma human cells (H295R). Telemetry was conducted in spontaneously hypertensive rats.
RESULTSTorcetrapib and angiotensin II (Ang II) but not dalcetrapib (a structurally different cholesteryl ester transfer protein inhibitor) elevated both cytochrome P450 subunit 11B2 mRNA and aldosterone production in H295R cells at 6 h. At days 1–5, torcetrapib produced a sustained increase of cytochrome P450 subunit 11B2 mRNA, unlike Ang II. Although torcetrapib and Ang II potentiated the effect of 25-OH cholesterol and raised pregnenolone levels, torcetrapib increased neither cytosolic Ca at 5 min nor extracellular signal-regulated kinase1/2 phosphorylation, suggesting initially divergent pathways. Unlike Ang II, torcetrapib steroidogenesis was not affected by Ang II type 1 receptor antagonism or voltage-gated T-type Ca channel antagonism, but was blocked by several L-type Cachannel antagonists. In unbiased genome-wide screening, Ang II and torcetrapib modulated an overlapping but distinct set of genes in H295R cells. Torcetrapib, but not Ang II, upregulated mRNA levels of the L-type Ca channel alpha 1C subunit. In spontaneously hypertensive rat, torcetrapib had a potent hypertensive effect mediated by the L-type Ca channel.
CONCLUSIONThe unique steroidogenic and hypertensive side effects of torcetrapib may be linked and involve voltage-gated L-type Ca channels. Structurally unrelated cholesteryl ester transfer protein inhibitors such as dalcetrapib do not share this effect.
Kidney fibrosis, a scarring of the tubulo-interstitial space, is due to activation of interstitial myofibroblasts recruited locally or systemically with consecutive extracellular matrix deposition. ...Newly published clinical studies correlating acute kidney injury (AKI) to chronic kidney disease (CKD) challenge this pathological concept putting tubular epithelial cells into the spotlight. In this work we investigated the role of epithelial cells in fibrosis using a simple controlled in vitro system. An epithelial/mesenchymal 3D cell culture model composed of human proximal renal tubular cells and fibroblasts was challenged with toxic doses of Cisplatin, thus injuring epithelial cells. RT-PCR for classical fibrotic markers was performed on fibroblasts to assess their modulation toward an activated myofibroblast phenotype in presence or absence of that stimulus. Epithelial cell lesion triggered a phenotypical modulation of fibroblasts toward activated myofibroblasts as assessed by main fibrotic marker analysis. Uninjured 3D cell culture as well as fibroblasts alone treated with toxic stimulus in the absence of epithelial cells were used as control. Our results, with the caveats due to the limited, but highly controllable and reproducible in vitro approach, suggest that epithelial cells can control and regulate fibroblast phenotype. Therefore they emerge as relevant target cells for the development of new preventive anti-fibrotic therapeutic approaches.
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