Molecular biomarkers of phospholipidosis in rat blood and heart after amiodarone treatment.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and concurrent development of concentric lamellar bodies. It is induced in humans and in animals by drugs with a cationic amphiphilic structure. The purpose of the present study was to identify a set of molecular biomarkers of PLD in rat blood and heart, hypotheticallya pplicable in preclinical screens within the drug development process. A toxicological study was set up in rats orally treated up to 11 days with 300 mg kg(­1) per day(­1) amiodarone (AMD). Light and transmission electron microscopy investigations were performed to confirm the presence of lamellar bodies indicative of phospholipid accumulation. The effects of AMD upon the transcriptome of these tissues were estimated using DNA microarray technology. Microarray data analysis showed that a total of 545 and 8218 genes were modulated by AMD treatment in heart and blood, respectively. Some genes implicated in the phospholipid accumulation in cells, such as phospholipase A2, showed similar alterations of gene expression. After transcriptome criteria of analysis and target selection, including also the involvement in the onset of PLD, 7 genes (Pla2g2a, Pla2g7, Gal, Il1b, Cebpb, Fcgr2b, Acer 2) were selected as candidate biomarkers of PLD in heart and blood tissues, and their potential usefulness as a sensitive screening test was screened and confirmed by quantitative Real-Time PCR analysis. Collectively, these data underscore the importance of transcriptional profiling in drug discovery and development, and suggest blood as a surrogate tissue for possible phospholipid accumulation in cardiomyocytes.

Atrial natriuretic peptides in Han Wistar, Sprague-Dawley and spontaneously hypertensive rats.

The atrial natriuretic peptide (ANP) and its precursor (N-terminal fragment of atrial natriuretic peptide, NT-proANP) are natriuretic peptides released into the circulation as a consequence of an acute atrial stretch. As for the brain natriuretic peptide and its N-terminal fragment, the biological significance of ANP and NT-proANP has been widely studied in humans, but the literature is lacking information about the determination of these biomarkers in veterinary medicine and, in particular, in the toxicological species used in preclinical pharmaceutical drug development. This paper describes the evaluation of ANP and NT-proANP levels in a healthy population of Han Wistar and Sprague-Dawley rats, as well as in a rodent model of hypertension (Spontaneously Hypertensive rats). Both biomarkers were measured by mean of two commercially available enzyme immunoassays and serum levels were correlated with heart weight and histopathological findings in the heart, with the aim of building an integrated assessment of the significance of these biomarkers. Results obtained demonstrated that NT-proANP and ANP can be accurately measured in the different rat strains, with NT-proANP concentrations higher than those of ANP, as expected because of its longer half-life. In addition, both correlated well with cardiac hypertrophy evaluated by means of heart weight and histopathological examination. NT-proANP and ANP represent reliable markers of cardiac hypertrophy in the rat.

NT-proBNP as a biomarker for the assessment of a potential cardiovascular drug-induced liability in beagle dogs.

The amino-terminal pro-brain natriuretic peptide (NT-proBNP) is released into the plasma predominantly from ventricular cardiomyocytes, particularly in patients with chronic cardiac diseases, although small amounts are detectable in the plasma of healthy subjects. While NT-proBNP has been widely exploited in human medicine, limited literature is available related to its characterization in veterinary medicine (e.g., correlation with damage and specificity) and, particularly, in the context of preclinical drug safety assessment. This paper describes the analytical performance characteristics and the biological variability of NT-proBNP in male beagle dogs by using a commercially available enzyme-linked immunosorbent assay. Male beagle dogs were treated with Casopitant, an NK1 receptor antagonist under development for depression and anxiety, which, when administered chronically to dogs, caused cardiac toxicity. Heart weight increase, myocardial necrosis, degeneration, and inflammation associated with high serum levels of cardiac troponin I characterized the end stage pathology observed in dogs treated orally at 40 mg/kg for 39 weeks. Based on these data, ad hoc studies were designed in order to evaluate the possible relationship between NT-proBNP serum levels and both standard toxicology endpoints, such as the organ weight and histology, as well as nonstandard endpoints such as macroscopic morphometry and echocardiography. Early changes of NT-proBNP serum levels were observed following 2 weeks of treatment onward, preceding most, if not all of the anatomical and functional changes. The results obtained demonstrate that NT-proBNP acts as an early biomarker of cardiac changes, representing a sensitive and predictive marker of drug-induced cardiac liability.

Localization of Helicobacter spp. in the fundic mucosa of laboratory Beagle dogs: an ultrastructural study.

In dogs Helicobacter spp. are found in all gastric regions usually localized in the surface mucus, gastric glands and parietal cells. The aim of this study was to detail the distribution of Helicobacter spp. in the fundic mucosa of asymptomatic Beagle dogs and their intracellular localization within parietal cells, in order to evaluate species-specific pathogenetic effects on gastric cells. The presence of Helicobacter spp. was investigated by immunohistochemistry, TEM, and PCR in the fundic mucosa of six Beagle dogs. Helicobacter spp. were found in all dogs examined, and H. bizzozeronii and H. felis were identified by PCR and confirmed by TEM. In the lumen of the fundic glands, co-localization was common. H. bizzozeronii was present in larger numbers than H. felis in both intraluminal and intraparietal localization. The amounts of H. bizzozeronii were similar in superficial and basal portions of the glands. H. felis was predominantly localized in the superficial portions of gastric glands but almost absent from the base. Within parietal cells, most Helicobacter organisms were intracanalicular, but intact and degenerate Helicobacter organisms were also visualized free in the cytoplasm or in secondary lysosomes. No specific degenerative lesions were found in infected parietal cells. Helicobacter organisms were also observed within macrophages in the lamina propria. In conclusion, there is a differential distribution of H. bizzozeronii and H. felis in the fundic mucosa of Beagle dogs, and their intracellular localization in parietal cells and macrophages suggests novel pathogenic scenarios for the development of immune response and maintenance of chronic gastritis in dogs.

A novel and integrated approach for the identification and characterization of drug-induced cardiac toxicity in the dog.

Cardiovascular toxicity represents one of the major reasons for the termination of the development of drugs, even in late development phases. This growing issue is often not restricted to specific therapeutic areas, and it is gaining critical importance, in particular for chronically administered drugs, highlighting the limitations in terms of sensitivity of the current investigational paradigms. Furthermore, drug-related changes may become evident after long-term administration for different reasons, including accumulation of the drug in the heart. This article describes how the integrated use of investigational tools represents a powerful approach for the early identification and characterization of cardiotoxicity in preclinical development. Cardiac changes were observed in the dog after long-term oral administration of casopitant, a neurokinin 1 receptor antagonist, developed for the treatment of depression and anxiety. Different approaches and sensitive biomarkers were used in a time-course study to investigate the onset, progression, and reversibility of the lesion. The integrated evaluation of cardiovascular parameters, electron microscopy, troponin I, and natriuretic peptide results highlighted any minimal early changes, allowing the full and deep characterization of the lesion. The outcome of this study was the driver for drug development decision making on casopitant and backup drugs.

Gold(III)-dithiocarbamato anticancer agents: activity, toxicology and histopathological studies in rodents.

Gold(III)-dithiocarbamato complexes have recently gained increasing attention as potential anticancer agents because of their strong tumor cell growth--inhibitory effects, generally achieved by exploiting non-cisplatin-like mechanisms of action. The rationale of our research work is to combine the antitumor properties of the gold(III) metal center with the potential chemoprotective function of coordinated dithiocarbamates in order to reduce toxic side effects (in particular nephrotoxicity) induced by clinically established platinum-based drugs. In this context, [Au(III) Br(2) (ESDT)] (AUL12) was proved to exert promising and outstanding antitumor activity in vitro and to overcome both acquired and intrinsic resistance showed by some types of tumors toward cisplatin. As a subsequent extension of our previous work, we here report on detailed in vivo studies in rodents, including antitumor activity toward three transplantable murine tumor models, toxicity, nephrotoxicity and histopathological investigations. Remarkably, the gold(III) complex AUL12 stands out for higher anticancer activity than cisplatin toward all the murine tumor models examined, inducing up to 80% inhibition of tumor growth. In addition, it shows low acute toxicity levels (lethal dose, LD(50) = 30 mg kg(-1) ) and reduced nephrotoxicity. Altogether, these results confirm the reliability of our drug design strategy and support the validation of this gold(III)-dithiocarbamato derivative as a suitable candidate for clinical trials.

Flow cytometry, with double staining with Nile red and anti-CD3 antibody, to detect phospholipidosis in peripheral blood lymphocytes of rats treated with amiodarone.

A flow cytometry method, to monitor peripheral lymphocytes phospholipidosis, has been set up using a single staining with Nile red and double staining with Nile red and anti-CD3 monoclonal antibody. Blood has been collected from rats treated with amiodarone (phospholipidogenic antiarrhythmic drug). By flow cytometer, it is possible to detect phospholipids, using Nile red, a probe for intracellular lipids staining, changing its fluorescence on the stained lipid basis. CD3 antigen has been selected to focus on T cells, to evaluate whether these cells are the target of phospholipidosis amiodarone-dependent. In the study A, Sprague-Dawley rats were treated with three different doses (75, 150, and 300 mg kg(-1) day(-1)) of amiodarone or vehicle alone, for 14 days, followed by 14 days of recovery: Data obtained show that by flow cytometry, with Nile red alone, it is possible to detect a dose- and time-related response of phospholipidosis-positive lymphocytes; a partial recovery is also assessed. In the study B, Sprague-Dawley rats were treated with a single dose (300 mg kg(-1) day(-1)) of amiodarone, for 14 days: Data obtained show that animals treated with amiodarone have a significant increase of phospholipidosis-positive lymphocytes (p = 0.008), in particular of CD3+ cells (p = 0.0056). Transmission electron microscopy analysis confirmed data obtained by flow cytometry. This work shows that flow cytometry with Nile red could be a good tool to monitor ex vivo phospholipidosis in lymphocyte cells of animals treated with amiodarone: The phospholipidogenic effect is more evident focusing on CD3+ T lymphocytes, thus suggesting that these cells are probably the target of phospholipidosis.

Early onset of spontaneous renal preneoplastic and neoplastic lesions in young conventional rats in toxicity studies.

Although occurring in aged laboratory rodents, spontaneous renal tumour are unknown in animals younger than 18 weeks. A survey on renal preneoplastic and neoplastic lesions has been performed on Sprague-Dawley rats from general toxicology studies over the period January 2004 - May 2006. Data from 2249 rats necropsied and 1206 rats (688 males and 518 females) examined microscopically from 52 studies, are reported. The age range at necropsy was between 12 and 18 weeks and all the animals were obtained from the same supplier. Three cases of tubular carcinoma, 1 tubular adenoma, and 4 cases of atypical tubular hyperplasia were observed in 5 females and 3 males from both control and treated groups from 6 studies with unrelated test compounds. In treated rats, the lesions were considered spontaneous in nature, rather than related to treatment, because of: (1) their sporadic incidence, (2) the short duration of the studies, and (3) the absence of similar lesions in other rats given the same test compound. These lesions are considered a recently occurring spontaneous finding, and the similarities with the familial renal cancer models, namely the Eker and the Nihon models, strongly suggest genetic factors as responsible for the lesions.

Involvement of the nitric oxide system in the anti-atherosclerotic potential of lacidipine in the ApoE-deficient mouse: a morphological, functional, and electrochemical study.

The present study investigated the anti-atherosclerotic activity of lacidipine, a calcium antagonist with antioxidant properties in apoE-deficient mice. These mice show widespread vascular lesions which closely resemble the inflammatory-fibrous plaques seen in humans in atherosclerosis. Mice were fed a Western-type diet (WTD), and treated for 8 weeks with either vehicle or lacidipine at 3 or 10 mg/kg/day. In parallel with histological studies of atherosclerotic lesions in the aorta, functional studies on vascular acetylcholine (ACh) reactivity and analysis of voltammetric levels of nitric oxide (NO) were performed. Recent work has suggested that dihydropyridines (DHPs) modulate vascular relaxation via an increase in the release of NO. Lacidipine treatment had no effect on the plasma lipid profile. However, a significant (p < 0.01) dose-related reduction of 36.4% and 43.3% of the aortic lesion area in respect to methocel-treated mice was observed. Moreover, the aortic ring from control apoE-deficient mice fed a WTD for 8 weeks showed a lower relaxation in response to ACh in comparison to wild-type C57BL/6J mice; on the contrary, lacidipine-treated apoE-deficient mice lacidipine-treated displayed a response similar to that of wildtype C57BL/6J mice. Voltammetric analyses demonstrated a significant decrease of NO release in apoE-deficient mice, while lacidipine-treated mice showed enhanced activity of the NO system. We conclude that lacidipine reduced the extent of atherosclerotic area in hypercholesterolemic apoE-deficient mice, and this reduction may be associated with the capacity of the drug to maintain endothelial NO levels at concentrations useful to protect against vascular damage.

Reduced progression of atherosclerosis in apolipoprotein E-deficient mice treated with lacidipine is associated with a decreased susceptibility of low-density lipoprotein to oxidation.

A study has been carried out in the apolipoprotein (apo) E-deficient mouse to investigate the activity of lacidipine (a calcium antagonist with antioxidant properties) in inhibiting the development of atherosclerotic lesions; of particular interest were changes in the susceptibility of low-density lipoproteins (LDL) to oxidation. Mice receiving a Western-type diet to accelerate the development of atherosclerosis were treated orally with vehicle or lacidipine at 3 or 10 mg/kg/day for 8 weeks. Lacidipine treatment (at 3 or 10 mg/kg) had no effect on the plasma lipid profile. However, a significant (P < 0.01) dose-related reduction of 43 and 50% of the aortic lesion area in respect to vehicle-treated mice was observed. Moreover, the resistance of mouse plasma LDL to undergo lipid peroxidation was significantly (P < 0.01) increased in apo E-deficient mice treated with lacidipine. The native LDL-like particle, derived from apo E-deficient mice treated with lacidipine, contained significantly lower concentrations of malonyldialdehyde than the vehicle-treated control group (P < 0.01). After exposure to human umbilical vein endothelial cells, LDL-like particle vitamin E levels (expressed as area under the curve; AUC), were significantly higher (P < 0.01) in both the 3 and 10 mg/kg lacidipine-treated groups, in comparison with the vehicle-treated control animals. We conclude that lacidipine reduced the extent of the atherosclerotic area in hypercholesterolaemic apo E-deficient mice, and that this reduction may be associated with the capacity of the drug to decrease the susceptibility of LDL to oxidation.