Danielle Roman

Imatinib does not induce cardiotoxicity at clinically relevant concentrations in preclinical studies

Cytotoxic concentrations of imatinib mesylate (10-50 microM) were required to trigger markers of apoptosis and endoplasmic reticulum stress response in neonatal rat ventricular myocytes and fibroblasts, with no significant differences observed between c-Abl silenced and nonsilenced cells. In mice, oral or intraperitoneal imatinib treatment did not induce cardiovascular pathology or heart failure. In rats, high doses of oral imatinib did result in some cardiac hypertrophy. Multi-organ toxicities may have increased the cardiac workload and contributed to the cardiac hypertrophy observed in rats only. These data suggest that imatinib is not cardiotoxic at clinically relevant concentrations (5 microM).

Evaluation of a new procedure for the flow cytometric analysis of in vitro, chemically induced micronuclei in V79 cells.

Measurement of the frequency of micronuclei induced in cells by ionizing radiation or by chemical treatment is widely used to analyze cytogenetic damage. The microscopic scoring of micronuclei is a tedious and time‐consuming procedure. Therefore, attempts have been made to automate micronuclei scoring by means of image analysis or flow cytometry. A new procedure for the flow cytometric analysis of chemically induced micronuclei in V79 Chinese hamster cells has been established in our laboratory. Debris was separated from micronuclei by means of a new gating procedure using area and width fluorescence of the stained suspension of micronuclei and nuclei. In order to test the sensitivity and specificity of this improved method of flow cytometric analysis, five well‐known mutagenic compounds were tested. With the new technique, the frequency of micronuclei measured and analyzed corresponded well with results obtained by conventional microscopy. In addition, a large series of negative compounds, and weak, middle, and strong micronuclei inducers, were tested in order to establish criteria for discrimination between genotoxic and nongenotoxic compounds by flow cytometry. This new procedure for flow cytometric detection of micronuclei represents a quick, reliable, and relatively simple method for in vitro micronucleus testing. Environ. Mol. Mutagen. 32: 387–396, 1998

Combination of Low-Dose Valsartan and Enalapril Improves Endothelial Dysfunction and Coronary Reserve in Nω-Nitro-l-Arginine Methyl Ester–Treated Spontaneously Hypertensive Rats

Combination of nonhypotensive doses of valsartan and enalapril markedly improved survival (+87%) compared with untreated animals (37%) in spontaneously hypertensive rats (SHRs) with endothelial dysfunction. However, the combination had no effect on kidney function, and proteinuria persisted over the 12 weeks of the study. It was hypothesized that the greater survival was due to improvement in endothelial function or coronary vasculature despite blockade of nitric oxide synthase and high blood pressure. Therefore, endothelial function was evaluated in isolated aortic ring and maximal coronary blood flow was studied in isolated perfused SHR hearts (20–24 weeks) treated with Nω-nitro-l-arginine methyl ester (l-NAME) (50 mg/l) for 4 weeks. The animals received vehicle, valsartan 5 mg/kg/d, enalapril 1 mg/kg/d, valsartan 50 mg/kg/d, or the combination valsartan 5 mg/kg/d with enalapril 1 mg/kg/d in drinking water. Normotensive Wistar-Kyoto (WKY) rats were used as control. Blood pressure was measured by telemetry. Histopathology was performed on heart, kidney (hematoxylin-eosin), and aorta (Masson trichrome). l-NAME elevated blood pressure by 50 mm Hg after vehicle (199 ± 5 mm Hg). Valsartan 50 mg/kg/d completely abolished this increase (150 ± 4 mm Hg) whereas the valsartan-enalapril combination synergistically decreased blood pressure (−37 mm Hg at 162 ± 7 mm Hg) compared with monotherapy (valsartan 5 mg/kg/d −10 mm Hg; enalapril 1 mg/kg/d −15 mm Hg). All treatments improved the histopathology, most markedly in those receiving the valsartan-enalapril combination. The severity mean grades for lesions were 2.1, 1.9, 1.7, 1.1, and 0.9 in vehicle-treated SHRs, enalapril 1 mg/kg/d, valsartan 5 mg/kg/d, valsartan 5 or 50 mg/kg/d, and the valsartan-enalapril combination, respectively, compared with 0.02 in WKY rats. Acetylcholine-induced relaxation was significantly greater in treated SHRs than after vehicle (−40% at 0.1 mmol acetylcholine) but the combination induced the maximal relaxation (−85%). The ratio of maximal tension induced by serotonin in rings with and without endothelium was 1.4 and 1.3 in vehicle and valsartan 5 mg/kg/d–treated rats whereas it did not differ from 1 in WKY rats and all other treated groups. The cardiac hypertrophy (+27%) was prevented by valsartan 50 mg/kg/d and the valsartan-enalapril combination. Coronary reserve was significantly increased by valsartan 50 mg/kg/d (+85% at 7.8 ± 0.7 ml/min/g) and the valsartan-enalapril combination (+42% at 6.0 ± 0.4 ml/min/g) compared with 4.2 ± 0.5 (vehicle). This was not different of 8.8 ± 0.5 (WKYs). Despite the maintenance of a high blood pressure, low-dose valsartan-enalapril significantly improved endothelial function and histopathology and increased coronary reserve in SHRs chronically receiving l-NAME.

Preclinical evaluation of potential nilotinib cardiotoxicity

In vitro, concentrations ≥ 10 μM of nilotinib were needed to induce markers of cytotoxicity, apoptosis, and endoplasmic reticulum stress in both neonatal rat ventricular myocytes, a putative target tissue, and non-target heart fibroblasts, indicating a lack of cardiomyocyte-specific nilotinib toxicity in vitro. In rats, oral nilotinib treatment at 80 mg/kg for 4 weeks induced increased heart weight; however, this was not associated with relevant histopathological changes or effects on heart function. Thus, nilotinib at and above clinically relevant concentrations (4.27 μM) did not induce overt cardiovascular pathologies or heart failure in vitro or in vivo under study conditions.

LAV694, a new antiproliferative agent showing improved skin tolerability vs. clinical standards for the treatment of actinic keratosis

The skin tolerability of the tubulin polymerisation inhibitor LAV694 was compared to that of 5% 5-fluorouracil (5-FU) and 0.5% podophyllotoxin in vitro using a human reconstructed epidermis (HRE), and in vivo using minipigs. Topical treatment of HRE for 1 or 3 days with a 0.2, 0.6 or 1% LAV694 cream or the placebo showed no signs of irritation in terms of morphology, cell viability (lactate dehydrogenase leakage) or interleukin-8 mRNA expression and release. 5-FU increased interleukin-8 production and induced morphological signs of irritation. The substances were also applied under occlusion to the back of two minipigs, twice daily, for 9 days to allow intraindividual comparison of skin effects and tolerability. Skin reactions were monitored by visual scoring, chromometry, pro-inflammatory activity, cell cycle and apoptosis by RT-PCR, laser scanning cytometry and histopathological examination of biopsies. Application of podophyllotoxin and 5-FU had to be stopped on days 4 and 8, respectively, due to severe skin lesions. LAV694 (1%) induced only moderate skin reddening after 9 days. 5-FU and podophyllotoxin, but not LAV694, increased mRNA expression of pro-inflammatory cytokines. LAV694 arrested keratinocytes in the M phase of the cell cycle and apoptosis was detected histologically in the basal layer. LAV694 increased the expression of pro-apoptotic genes in both experimental models. In conclusion, LAV694 selectively induced apoptosis, rather than necrosis, of growth-arrested keratinocytes, thus avoiding the occurrence of extensive inflammation. This resulted in an improved skin tolerability in comparison with 5-FU and podophyllotoxin.

Effects of latanoprost, timolol and GLC756, a novel dopamine D2 agonist and D1 antagonist on LTC4 release after rat mast cell activation

Purpose:  Mast cells participate in ocular allergic inflammation by releasing biologically active mediators. Leukotrienes are released from activated mast cells via an IgE‐dependent mechanism, and play a crucial role in ocular allergic inflammation. In this study, the effect of three topical antiglaucoma drugs, that is, latanoprost, timolol and GLC756, a novel dopamine D2 agonist and D1 antagonist, on leukotriene C4 (LTC4) release after rat mast cell activation was examined.

Effects of antiglaucoma drugs GLC756, a novel dopamine D2 agonist and D1 antagonist, and timolol on endotoxin-induced TNF-alpha release in serum of rats.

Purpose Anti-inflammatory activity of an antiglaucoma drug may be an advantage for long-term treatment of glaucoma since it may reduce the risk of treatment-related inflammatory processes in outer compartments of the eye and probably also prevent or delay progression of glaucomatous retinal neurodegeneration. In this study, the effect of GLC756, a novel mixed dopamine D2 receptor agonist and dopamine D1 receptor antagonist, and timolol on endotoxin-induced cytokine tumor necrosis factor-α (TNF-α) release in serum was examined. Methods For endotoxin-induced TNF-α release, 8-week-old Lewis rats were intravenously injected with 160 μg lipopolysaccharide (LPS) from Salmonella typhimurium. GLC756, timolol, or betamethasone were either systemically (1 mg/kg SC for 5 days) or topically (0.4%, 0.5%, and 0.1%, respectively, 20 μL eye drops given 16 times over 48 hours in left and right eye) administered. TNF-α was measured in serum 2 and 48 hours after LPS induction. Results A marked TNF-α increase in serum was found 2 hours after LPS induction. Administration of GLC756 and betamethasone, systemically and topically, decreased TNF-α release. However, due to large scattering of mean values only the effect of systemically administered GLC756 was statistically significant. In contrast, timolol increased TNF-α values stronger than LPS alone. Conclusions The significant suppression of LPS-induced TNF-α increase by GLC756 suggests an additional anti-inflammatory potential of the dopaminergic compound in the treatment of glaucoma.

Antiglaucoma drug GLC756 and its effect on cellular cAMP and tumor necrosis factor α release in vitro of activated human monocytic leukemia cells

PurposeGLC756, a putative antiglaucoma drug with dopamine D2 agonist and D1 antagonist properties, significantly decreases tumor necrosis factor α (TNF-α) levels in lipopolysaccharide (LPS)-induced rats. The present study describes the effects of GLC756 on cellular adenosine 3′, 5′-cyclic monophosphate (cAMP) in relation to TNF-α production on LPS-stimulated human acute monocytic leukemia cells.MethodsA human peripheral blood acute monocytic leukemia cell line (THP-1) was activated via LPS. THP-1 cells were incubated with GLC756 or betamethasone (positive control) at concentrations of 1, 10, and 30 μM. The TNF-α concentration in supernatant and cAMP levels in cellular extract were measured by enzyme-linked immunosorbent assay 0,1, 2.5, 4.5, 7, and 24 h post-activation.ResultsCompared with LPS controls, both GLC756 at 30 μM and betamethasone at ≥1 μM had a significant inhibitory effect on TNF-α release from THP-1 cells 2.5 to 24 h post-activation. Parallel to the TNF-α decrease, GLC756 induced significant increases of cellular cAMP 2.5 and 7 h post-activation. Betamethasone had no effect on the cellular cAMP level.ConclusionIntracellular signaling pathway leading to inhibition of the production of the proinflammatory cytokine TNF-α after GLC756 treatment might be mediated through the second messenger cAMP.

Ocular toxicity of AUY922 in pigmented and albino rats.

AUY922, a heat shock protein 90 inhibitor is associated with ocular adverse events (AEs). To provide a better understanding of ocular AEs in patients, 4 investigative studies were performed in a step-wise approach to assess retinal structure and function in pigmented (Brown Norway) and albino (Wistar) rats. In rats administered 30mg/kg of AUY922, the AUC0-24h and Cmax are comparable to that in patients at 70mg/m(2). AUY922 at ≥30mg/kg was poorly tolerated by rats with morbidity or mortality generally after the third weekly treatment. Electroretinography (ERG) changes were observed at doses ≥30mg/kg. The ERG changes were dose dependent, consistent with an effect on the photoreceptors, and fully reversible. The ERG effects could not be minimized by decreasing the Cmax while maintaining AUC. Histopathological changes were seen mainly when rats were administered AUY922 at 100mg/kg. The 2-hour infusion of AUY922 at 100mg/kg caused disorganization of the outer segment photoreceptor morphology in male Brown Norway rats; the severity of the disorganization increased with the number of administrations, but was reversible during a 4-week posttreatment period. There was no major difference in ocular response between Brown Norway and Wistar rats. No changes in serum iron levels, and no changes in rhodopsin, PDE6α, β-transducin concentrations, or retinal pigment epithelium-specific protein RPE65 expression were observed after single and multiple infusions of AUY922 at 100mg/kg compared to vehicle-treated controls. AUY922 retinal toxicity in rats recapitulates and further characterizes that reported in patients and is shown to be reversible, while a precise molecular mechanism for the effect was not determined.