The reversible P2Y12 antagonist ACT-246475 causes significantly less blood loss than ticagrelor at equivalent antithrombotic efficacy in rat.

The P2Y12 receptor is a validated target for prevention of major adverse cardiovascular events in patients with acute coronary syndrome. The aim of this study was to compare two direct-acting, reversible P2Y12 antagonists, ACT-246475 and ticagrelor, in a rat thrombosis model by simultaneous quantification of their antithrombotic efficacy and surgery-induced blood loss. Blood flow velocity was assessed in the carotid artery after FeCl3 -induced thrombus formation using a Doppler flow probe. At the same time, blood loss after surgical wounding of the spleen was quantified. Continuous infusions of ACT-246475 and ticagrelor prevented the injury-induced reduction of blood flow in a dose-dependent manner. High doses of both antagonists normalized blood flow and completely abolished thrombus formation as confirmed by histology. Intermediate doses restored baseline blood flow to ≥65%. However, ACT-246475 caused significantly less increase of blood loss than ticagrelor; the difference in blood loss was 2.6-fold (P < 0.01) at high doses and 2.7-fold (P < 0.05) at intermediate doses. Potential reasons for this unexpected difference were explored by measuring the effects of ACT-246475 and ticagrelor on vascular tone. At concentrations needed to achieve maximal antithrombotic efficacy, ticagrelor compared with ACT-246475 significantly increased carotid blood flow velocity in vivo (P = 0.003), induced vasorelaxation of precontracted rat femoral arteries, and inhibited contraction of femoral artery induced by electrical field stimulation or by phenylephrine. Overall, ACT-246475 showed a significantly wider therapeutic window than ticagrelor. The absence of vasodilatory effects due to high selectivity of ACT-246475 for P2Y12 provides potential arguments for the observed safety advantage of ACT-246475 over ticagrelor.

Comparison of Macitentan and Bosentan on Right Ventricular Remodeling in a Rat Model of Non-vasoreactive Pulmonary Hypertension.

AIMS: We compared the efficacy of macitentan, a novel dual endothelin A/endothelin B receptor antagonist, with that of another dual endothelin receptor antagonist, bosentan, in a rat model of non-vasoreactive pulmonary hypertension (PH) with particular emphasis on right ventricular (RV) remodeling. METHODS AND RESULTS: Unlike monocrotaline or hypoxic/sugen rats, bleomycin-treated rats presented a non-vasoreactive PH characterized by the absence of pulmonary dilatation to adenosine. We therefore chose the bleomycin rat model to compare the effects of the maximally effective doses of macitentan and bosentan on pulmonary vascular and RV remodeling. Macitentan (100 mg·kg(-1)·d(-1)), but not bosentan (300 mg·kg(-1)·d(-1)), significantly prevented pulmonary vascular remodeling, RV hypertrophy, and cardiomyocyte diameter increase. Cardiac protection by macitentan was associated with a significant attenuation of genes related to cell hypertrophy and extracellular matrix remodeling. Microautoradiography and high performance liquid chromatography analysis showed greater distribution of macitentan than bosentan in the RV and pulmonary tissue. CONCLUSIONS: Macitentan was more efficacious than bosentan in preventing the development of pulmonary and RV hypertrophies in a model of non-vasoreactive PH. Greater ability to distribute into the tissue could contribute to the greater structural improvement by macitentan compared with bosentan.

A novel genomic signature with translational significance for human idiopathic pulmonary fibrosis.

The bleomycin-induced rodent lung fibrosis model is commonly used to study mechanisms of lung fibrosis and to test potential therapeutic interventions, despite the well recognized dissimilarities to human idiopathic pulmonary fibrosis (IPF). Therefore, in this study, we sought to identify genomic commonalities between the gene expression profiles from 100 IPF lungs and 108 control lungs that were obtained from the Lung Tissue Research Consortium, and rat lungs harvested at Days 3, 7, 14, 21, 28, 42, and 56 after bleomycin instillation. Surprisingly, the highest gene expression similarity between bleomycin-treated rat and IPF lungs was observed at Day 7. At this point of maximal rat-human commonality, we identified a novel set of 12 disease-relevant translational gene markers (C6, CTHRC1, CTSE, FHL2, GAL, GREM1, LCN2, MMP7, NELL1, PCSK1, PLA2G2A, and SLC2A5) that was able to separate almost all patients with IPF from control subjects in our cohort and in two additional IPF/control cohorts (GSE10667 and GSE24206). Furthermore, in combination with diffusing capacity of carbon monoxide measurements, four members of the translational gene marker set contributed to stratify patients with IPF according to disease severity. Significantly, pirfenidone attenuated the expression change of one (CTHRC1) translational gene marker in the bleomycin-induced lung fibrosis model, in transforming growth factor-ß1-treated primary human lung fibroblasts and transforming growth factor-ß1-treated human epithelial A549 cells. Our results suggest that a strategy focused on rodent model-human disease commonalities may identify genes that could be used to predict the pharmacological impact of therapeutic interventions, and thus facilitate the development of novel treatments for this devastating lung disease.

Simultaneous pulmonary and systemic blood pressure and ECG Interval measurement in conscious, freely moving rats.

Here we evaluated the ability of a new, dual blood-pressure telemetry transmitter to simultaneously measure pulmonary and systemic blood pressure and the electrocardiogram in rats. The transmitter was implanted in normotensive and monocrotaline-induced pulmonary hypertensive Wistar rats, with sensing catheters placed in the pulmonary artery (channel 1) and descending aorta (channel 2). Biopotential electrodes were positioned to record an apex-based lead II electrocardiogram. Pulmonary and systemic arterial blood pressure and electrocardiographic waveforms were recorded between 2 and 12 wk after implantation of the transmitter. During this period, pulmonary arterial pressure progressively increased in monocrotaline-treated compared with saline-treated rats. The pharmacologic response of rats to reference compounds was measured by using the transmitter to validate the technique and to evaluate the ability of the device to transmit changes in blood pressure and the electrocardiogram. Validation against 2 Millar high-fidelity blood-pressure catheters confirmed the accuracy of the blood pressure data recorded with the transmitter. In addition, local tolerance of the associated catheters was confirmed by histologic examination.

Novel retinoblastoma binding protein RBBP9 modulates sex-specific radiation responses in vivo.

Retinoblastoma (RB) tumor suppressor is a key regulator of apoptosis, a central mediator of the proliferative block induced by ionizing radiation (IR) and a binding target for a variety of proteins that regulate its activity. One of the recently discovered and the least investigated of these is the novel Rb-binding protein RBBP9/BOG. We studied the effects of acute and chronic low dose radiation (LDR) exposure on the induction of RBBP9 and RB signaling pathway in vivo in mouse spleen and found that RBBP9 played a pivotal role in IR responses in vivo. We observed that chronic LDR exposure led to a significant increase of RBBP9 expression in males and a significant decrease in females. Elevated RBBP9 expression in males was paralleled by a pronounced dephosphorylation of RB and a significant drop of PCNA and cyclin A expression. On the contrary, chronic exposure in females led to decreased levels of RBBP9 and increased levels of hyperphosphorylated RB (ppRB) in spleen. Decreased levels of ppRB in spleen of chronically exposed males were correlated with strongly elevated apoptotic rates. In females, the radiation-induced increase of apoptotic index was much less pronounced. Quite surprisingly, the observed sex-specific signaling changes did not result in the sex-specificity of cellular proliferation. The molecular mechanisms and possible repercussions of the radiation-induced sex differences in cellular proliferation and apoptosis are discussed.

Standardized assessment of cell proliferation: the approach of the RITA-CEPA working group.

Since 1985, quantitative data on cell proliferation increasingly form an integral part in the risk assessment of many pharmaceutical, chemical, and agrochemical compounds. Technical guides for the conduct of cell proliferation studies are not readily available. The detection of cell proliferation relies almost exclusively on immunohistochemistry, which is rarely standardized between laboratories. Against this background pathologists formed the RITA-CEPA working group with the aim of elaborating standard guides for the conduct of cell proliferation studies. We present here, as examples, the general BrdU guide as well as the organ-specific guides for hepatocytes, thyroid gland follicular cells, and renal tubular epithelial cells in the rat. Today, RITA-CEPA has available guides for BrdU cell proliferation studies in 20 organs as well as for studies with PCNA as proliferation marker and for apoptosis studies with the TUNEL technique. The relevant information from more than 500 selected publications is organized in a database structure to make it easily traceable. New hardware and software used for image analysis are being assessed and are included in the information exchange practiced in the CEPA working group. Further fields of major relevance for cell proliferation studies, such as statistics and the validation of image analysis equipment in a GLP environment are investigated. A guide on the use of statistics in cell proliferation studies is in preparation. CEPA-group members meet regularly to exchange information. They use the electronic database for planning and interpretation of cell proliferation studies. CEPA will continue with the preparation of organ guides and expand the series for other proliferation markers, e.g. Ki-67. It promotes standardization of techniques applied in proliferation studies, especially immunohistochemistry and image analysis. Members of the CEPA-group have access to a comprehensive and steadily increasing knowledge base on all aspects of cell proliferation. Membership of the CEPA group is open for all industrial organizations who develop chemical, agrochemical, or pharmaceutical products.

Thiamethoxam induced mouse liver tumors and their relevance to humans. Part 2: species differences in response.

Thiamethoxam is a neonicotinoid insecticide that is not a mutagen, but it did cause a significant increase in liver cancer in mice, but not rats, in chronic dietary feeding studies. Previous studies in mice have characterized a carcinogenicity mode of action that involved depletion of plasma cholesterol, cell death, both as single cell necrosis and as apoptosis, and sustained increases in cell replication rates. In a study reported in this article, female rats have been exposed to thiamethoxam in their diet at concentrations of 0, 1000, and 3000 ppm for 50 weeks, a study design directly comparable to the mouse study in which the mode of action changes were characterized. In rats, thiamethoxam had no adverse effects on either the biochemistry or histopathology of the liver at any time point during the study. Cell replication rates were not increased, in fact they were significantly decreased at several time points. The lack of effect on the rat liver is entirely consistent with the lack of liver tumor formation in the two-year cancer bioassay. Comparisons of the metabolism of thiamethoxam in rats and mice have shown that concentrations of the parent chemical were either similar or higher in rat blood than in mouse blood in both single dose and the dietary studies strongly indicating that thiamethoxam itself is unlikely to play a role in the development of liver tumors. In contrast, the concentrations of the two metabolites, CGA265307 and CGA330050, shown to play a role in the development of liver damage in the mouse, were 140- (CGA265307) and 15- (CGA330050) fold lower in rats than in mice following either a single oral dose, or dietary administration of thiamethoxam for up to 50 weeks. Comparisons of the major metabolic pathways of thiamethoxam in vitro using mouse, rat, and human liver fractions have shown that metabolic rates in humans are lower than those in the rat suggesting that thiamethoxam is unlikely to pose a hazard to humans exposed to this chemical at the low concentrations found in the environment or during its use as an insecticide.

Thiamethoxam induced mouse liver tumors and their relevance to humans. Part 1: mode of action studies in the mouse.

Thiamethoxam, a neonicotinoid insecticide, which is not mutagenic either in vitro or in vivo, caused an increased incidence of liver tumors in mice when fed in the diet for 18 months at concentrations in the range 500 to 2500 ppm. A number of dietary studies of up to 50 weeks duration have been conducted in order to identify the mode of action for the development of the liver tumors seen at the end of the cancer bioassay. Both thiamethoxam and its major metabolites have been tested in these studies. Over the duration of a 50-week thiamethoxam dietary feeding study in mice, the earliest change, within one week, is a marked reduction (by up to 40%) in plasma cholesterol. This was followed 10 weeks later by evidence of liver toxicity including single cell necrosis and an increase in apoptosis. After 20 weeks there was a significant increase in hepatic cell replication rates. All of these changes persisted from the time they were first observed until the end of the study at 50 weeks. They occurred in a dose-dependent manner and were only observed at doses (500, 1250, 2500 ppm) where liver tumors were increased in the cancer bioassay. There was a clear no-effect level of 200 ppm. The changes seen in this study are consistent with the development of liver cancer in mice and form the basis of the mode of action. When the major metabolites of thiamethoxam, CGA322704, CGA265307, and CGA330050 were tested in dietary feeding studies of up to 20 weeks duration, only metabolite CGA330050 induced the same changes as those seen in the liver in the thiamethoxam feeding study. It was concluded that thiamethoxam is hepatotoxic and hepatocarcinogenic as a result of its metabolism to CGA330050. Metabolite CGA265307 was also shown to be an inhibitor of inducible nitric oxide synthase and to increase the hepatotoxicity of carbon tetrachloride. It is proposed that CGA265307, through its effects on nitric oxide synthase, exacerbates the toxicity of CGA330050 in thiamethoxam treated mice.