Cardiovascular toxicity of minoxidil in the marmoset.

The aim of the experiments was to assess the toxicity of minoxidil, a potent vasodilator, in marmosets. The animals were treated either at escalating doses from 2 to 40 mg/kg, escalating doses from 40 to 200 mg/kg or single doses of 150 mg/kg or 200 mg/kg. ECG recording and echocardiographic examination were conducted before and 1h after treatment. Necropsy and histopathology were performed 24h after the last dose. The treatment with minoxidil induced myocardial necrosis, coronary arteriopathy and degeneration of renal tubules in animals treated with 150 mg/kg or 200 mg/kg. Myocardial necrosis associated with fibrosis in some animals was located mainly in the left and right ventricles (including papillary muscles), but also in the right atrium, left atrium and/or interventricular septum. Arteriopathy was observed in small coronary arteries of the right or left atrium. ECG and echocardiographic examinations showed that in animals treated with 150 mg/kg or 200 mg/kg, there were positive chronotropic and inotropic effects that compensated for the hypotensive effect of the drug and were considered to have played a key role in the pathogenesis of the cardiovascular lesions. The cardiotoxicity of minoxidil in marmosets was similar to that described in dogs, but occurred at much higher doses. In conclusion minoxidil produced cardiovascular toxicity in the marmoset, which was probably due to the marked changes in the cardiac function associated with exaggerated pharmacological effects of the compound. The marmosets were found to be less sensitive than dogs to the cardiotoxicity of minoxidil.

Characterisation of the vascular and inflammatory lesions induced by the PDE4 inhibitor CI-1044 in the dog.

Phosphodiesterase 4 (PDE4) inhibitors are potential therapeutic agents but vascular injury and perivascular inflammation occurs frequently during preclinical toxicology testing of these drugs. The lesions induced by PDE4 inhibitors have been described mainly in rats but there is limited data available for monkeys and no data for dogs. Here we present the toxicological profile of CI-1044, a PDE4 inhibitor, administered orally to dogs. Dogs were treated for 4 days with 5, 10, 20 or 50 mg/kg of CI-1044, and a group of dogs was submitted to a 4-week recovery period after treatment with 20 mg/kg. CI-1044 induced disseminated vascular necrosis and inflammation in various organs/tissues from 20 mg/(kg day). The nasal turbinates and the scrotal skin were the most sensitive tissues but lesions were also observed in the stomach, heart, kidneys and, to a lower extent, in the liver, mesenteric lymph nodes, adrenals and lung. The inflammation was mainly characterized by an infiltration of polynuclear neutrophils, oedema and necrosis. The inflammation observed microscopically correlated with marked increases in serum amyloid A and C-reactive protein. Variations in these acute phase response proteins were detected 24 h after the first dose and were further increased over the course of the treatment. The vascular and inflammatory lesions were reversible over 4 weeks. In conclusion, the lesions induced by the PDE4 inhibitor CI-1044 in dogs differed from the haemodynamically mediated coronary arteritis reported with PDE3 inhibitors.

Investigation of the molecular mechanisms preceding PDE4 inhibitor-induced vasculopathy in rats: tissue inhibitor of metalloproteinase 1, a potential predictive biomarker.

Phosphodiesterase (PDE) 4 inhibitors are a class of drugs that can provide novel therapies for asthma and chronic obstructive pulmonary disease. Their development is frequently hampered by the induction of vascular toxicity in rat mesenteric tissue during preclinical studies. Whereas these vascular lesions in rats have been well characterized histologically, little is known about their pathogenesis and in turn, sensitive and specific biomarkers for preclinical and clinical monitoring do not exist. In order to investigate the early molecular mechanisms underlying vascular injury, time-course studies were performed by treating rats for 2-24 h with high doses of the PDE4 inhibitor CI-1044. Transcriptomics analyses in mesenteric tissue were performed using oligonucleotide microarray and real-time RT-PCR technologies and compared to histopathological observations. In addition, protein measurements were performed in serum samples to identify soluble biomarkers of vascular injury. Our results indicate that molecular alterations preceded the histological observations of inflammatory and necrotic lesions in mesenteric arteries. Some gene expression changes suggest that the development of the lesions could follow a primary modulation of the vascular tone in response to the pharmacological effect of the compound. Activation of genes coding for pro- and antioxidant enzymes, cytokines, adhesion molecules, and tissue inhibitor of metalloproteinase 1 (TIMP-1) indicates that biomechanical stimuli may contribute to vascular oxidant stress, inflammation, and tissue remodeling. TIMP-1 appeared to be an early and sensitive predictive biomarker of the inflammatory and the tissue remodeling components of PDE4 inhibitor-induced vascular injury.

Altered gene expression in rat mesenteric tissue following in vivo exposure to a phosphodiesterase 4 inhibitor.

Vascular injury is a relatively common finding during the pre-clinical toxicity testing of drugs. The mechanisms of the injury are poorly understood and in turn, sensitive and specific biomarkers for pre-clinical and clinical monitoring do not exist. The present study was undertaken to investigate the molecular mechanisms of drug-induced vascular injury in mesenteric tissue of rats treated with the selective phosphodiesterase 4 (PDE4) inhibitor CI-1044. In a time-course study, male Sprague Dawley rats were given daily doses of 40 or 80 mg/kg for 1, 2 or 3 successive days and were euthanized the following day. Gene expression profiles in mesenteric tissue were determined using Affymetrix RG_U34A microarrays and fibrinogen and cytokine measurements were performed in blood samples. Hierarchical clustering analysis produced a clear pattern separation of the animals with inflammation, animal with inflammation and necrosis and animals without any lesion. Genes associated with inflammation, procoagulation, extracellular matrix remodeling were up-regulated. An altered expression of genes involved in vascular tone regulation, lipid and glucose metabolism was also observed. Selected genes expression changes were confirmed by TaqMan real-time RT-PCR. The inflammatory process was also detected in the bloodstream at the protein level since fibrinogen, IL6 and IL1beta concentrations were increased in treated animals. Overall, the present study reveals several molecular changes supporting the hypothesis by which PDE4 inhibitor-induced vascular lesions in rats are triggered by an inflammatory mechanism and/or a vascular tone dysregulation.

Exploration of global gene expression in human liver steatosis by high-density oligonucleotide microarray.

Understanding the molecular mechanisms underlying fatty liver disease (FLD) in humans is of major importance. We used high-density oligonucleotide microarrays (22.3 K) to assess the mechanisms responsible for the development of human liver steatosis. We compared global gene expression in normal (n=9) and steatotic (n=9) livers without histological signs of inflammation or fibrosis. A total of 34 additional human samples including normal (n=11), steatosis (n=11), HCV-related steatosis (n=4) or steatohepatitis associated with alcohol consumption (n=4) or obesity (n=4) were used for immunohistochemistry or quantitative real-time PCR studies. With unsupervised classification (no gene selection), all steatotic liver samples clustered together. Using step-down maxT multiple testing procedure for controlling the Family-Wise Error-Rate at level 5%, 110 cDNAs (100 over- and 10 underexpressed) were found to be differentially expressed in steatotic and normal livers. Of them were genes involved in mitochondrial phosphorylative and oxidative metabolism. The mean ratio of mitochondrial DNA to nuclear DNA content was higher in liver steatosis compared to normal liver biopsies (1.12+/-0.14 vs 0.67+/-0.10; P=0.01). An increased expression of genes involved in inflammation (IL-1R family, TGFB) was also observed and confirmed by quantitative RT-PCR or immunochemistry. In steatohepatitis, an increase of the protein expression of mitochondrial antigens, IL-1R1, IGF2 and TGFB1 was also observed, interleukin 1 receptor being always strongly expressed in steatohepatitis linked to alcohol or obesity. In conclusion, mitochondrial alterations play a major role in the development of steatosis per se. Activation of inflammatory pathways is present at a very early stage of steatosis, even if no morphological sign of inflammation is observed.