Patrizia Cristofori

A cell-based approach for the early assessment of the phospholipidogenic potential in pharmaceutical research and drug development.

Phospholipidosis is a term commonly used to indicate a phospholipid storage disorder; in affected cells, phospholipids accumulate in lysosomes that acquire a multilamellar morphological appearance. Cationic amphiphilic drugs (CADs) are suggested to induce phospholipidosis by direct interaction of xenobiotics with intracellular phospholipids or by the action of xenobiotics on the synthesis and metabolism of phospholipids. To date, electron microscopy (EM) represents the most reliable and the preferred method for the demonstration of phospholipidotic cell damage. Nevertheless, EM has a low throughput, it is expensive, and it is not suitable for screening purposes.We discuss here the assessment of the the phospholipidogenic potential of drugs using a cell culture-based model. In this test, intracellular phospholipids of treated U-937 cells (a human monocyte-derived cell line) were measured using the fluorescent probe Nile red. Eleven CADs reported to induce phospholipidosisin vivo and eight nonphospholipidogenic drugs were tested. Results obtained with the U-937 model confirmed the phospholipidogenic potential of drugs tested as described in the literature. Results have also been correlated with data obtained with a physical-chemical model (chromatographic hydrophobicity index measurement). Good correlation was obtained, confirming that the physical-chemical properties of CADs play a crucial role in the development of phospholipidosis.This work demonstrates that the U-937 model is a rapid and sensitive method for the determination of phospholipidosis-mediated cell damage. The specificity and the predictive potency observed make this method suitable for screening purposes in pharmaceutical development.

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, [AuIIIBr2(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, LD50 = 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.

Qualitative and quantitative analysis of the progressive cerebral damage after middle cerebral artery occlusion in mice

The middle cerebral artery occlusion (MCAO) in mice induces a focal cerebral ischaemia at the level of the tempo-parietal cortex. Histological staining and immunohistochemical markers were used to characterize the temporal progression of the cerebral infarct: both qualitative and quantitative analyses were performed at different days after the MCAO. At 3 days after MCAO, an extensive necrosis of the cerebral parenchyma was accompanied by extravasation and by massive oedema. After 7 days, GFAP marker showed a gliotic reaction with alteration of the astrocytes membrane permeability (S100 marker). Positivity for acid phosphatase staining indicated the presence of macrophages. At Day 14 and 21 following MCAO, the histological profile was essentially similar. Interestingly, at Day 7, 14 and 21, a previously unreported gliosis was observed in the subthalamic area. Quantitative analysis showed a significantly large infarct volume at Day 3 (7.88 +/- 1.95 mm3 +/- S.E.M.) compared to Day 7 (4.28 +/- 0.47 mm3 +/- S.E.M.). At Day 14 and Day 21 the infarct volumes were further decreased to 2.00 +/- 0.52 and 1.43 +/- 0.39 mm3 +/- S.E.M., respectively. These results suggest that it is important to consider the time of evaluation of cerebral ischaemia-induced cerebral infarct, especially in studies which aim to evaluate the neuroprotective effect of putative therapeutic agents.

Kidney injury molecule-1 expression in rat proximal tubule after treatment with segment-specific nephrotoxicants: a tool for early screening of potential kidney toxicity.

Dose-response expression of kidney injury molecule-1 (KIM-1) gene in kidney cortex and its correlation with morphology and traditional biomarkers of nephrotoxicity (plasma creatinine and blood urea nitrogen, BUN) or segment-specific marker of proximal tubule injury (kidney glutamine synthetase, GSK) were studied in male rats treated with proximal tubule segment-specific nephrotoxicants. These included hexachloro-1:3-butadiene (HCBD, S3 segment-specific), potassium dichromate (chromate, S1-S2 segment-specific), and cephaloridine (Cph, S2 segment-specific). Rats were treated with a single intraperitoneal (ip) injection of HCBD 25, 50, and 100 mg/kg, subcutaneous (sc) injection of chromate 8, 12.5, and 25 mg/kg; or ip injection of Cph 250, 500, and 1,000 mg/kg. KIM-1 gene showed a dose-dependent up-regulation induced by all segment-specific nephrotoxicants. Interestingly, magnitude of the up-regulation reflected the severity of microscopic tubular changes (degeneration, necrosis, and regeneration). Even low-severity microscopic observations were evidenced by significant gene expression changes. Furthermore, KIM-1 showed significant up-regulation even in the absence of morphological changes. In contrast, traditional and specific markers demonstrated low sensitivity or specificity. In conclusion, this study suggested KIM-1 as a sensitive molecular marker of different levels of tubular injury, and it is likely to represent a potential tool for early screening of nephrotoxicants.

The calcium channel blocker lacidipine reduces the development of atherosclerotic lesions in the apoE-deficient mouse.

Background Lacidipine is a widely used calcium-channel blocker, which has both long-lasting antihypertensive activity and also antioxidant properties. Previous studies have demonstrated the ability of lacidipine to reduce the development of atherosclerotic lesions in several animal models. Objective The present study investigated the anti-atherosclerotic potential of lacidipine in the apoE-deficient mouse, an experimental model of atherosclerosis showing progressively complex and widespread lesions which closely resemble the inflammatory-fibrous plaques seen in humans. Methods Lacidipine was administered daily by gavage for 10 weeks at dose levels of 0 (control), 0.3, 1.0 and 3.0 mg/kg. Results Lacidipine administration reduces the extension of atherosclerotic lesions in the aorta of the apoE-deficient mouse without affecting plasma lipid levels. We also show that apoE-deficient mice have four-fold higher values of the proatherogenic peptide, endothelin, compared with the wild-type C57BL/6 mouse and that lacidipine administration reduced, in a dose-dependent manner, the concentrations of plasma endothelin. Conclusion Lacidipine has anti-atherogenic effects in the apoE-deficient mouse, and reduces plasma endothelin concentrations.

A robust experimental protocol for pharmacological fMRI in rats and mice.

Pharmacological Magnetic Resonance Imaging (phMRI) methods have significantly expanded the stimulation repertoire available to preclinical fMRI research, by allowing to selectively probe the activity of specific brain circuitries and neurotransmitter systems. However, the application of phMRI to animal models is constrained by a number of experimental factors. Firstly, in order to prevent motion artefacts and reduce restraint-induced stress, phMRI studies are typically performed under anaesthesia. Moreover, several psychoactive drugs produce blood pressure changes and alterations in respiratory frequency that may perturb central haemodynamic readouts of brain function. Hence, the quality and outcome of phMRI studies is critically dependent on the ability to monitor and control peripheral physiological parameters (i.e. blood pressure, arterial blood gases) that could alter phMRI readouts. Here we provide a thorough methodological description of a robust protocol to measure drug-induced cerebral blood volume changes in anaesthetised rats and mice. We show that the protocol ensures stable physiological parameters and robust phMRI response to the psychostimulant drug d-amphetamine in three different rat strains. We also document the successful application of the protocol to map the central effects produced by d-amphetamine in C57Bl/6J mice, a strain commonly used as background for the generation of transgenic lines, thus paving the way to the implementation of phMRI in genetically engineered animals.

Successful xenografting of cryopreserved primary pancreatic cancers.

Abstract. In order to assess the suitability of cryopreserved neoplastic tissues for xenografting into nude (nu/nu) mice, we compared the take rate in 28 samples of pancreatic ductal carcinoma. Eleven fresh samples were implanted in nu/nu mice, and 17 were frozen in cryopreserving solution and implanted at a later time. All samples were examined for the presence of neoplastic tissue in cryostat sections. A total of 15 tumors grew in the animals; five from the freshly implanted samples and ten from those cryopreserved. Ten xenografted tumors were characterized for alterations in p53, K-ras, and p16 genes, which were found in six, eight, and nine cases, respectively. Our results demonstrate that the take rate for xenografting is comparable between cryopreserved and fresh tissue samples. The procedure allows for the exchange of tumor material between institutions and permits the establishment of centralized facilities for the storage of an array of different primary tumor samples suitable for the production of in vivo models of cancers.

Glutamine synthetase activity in rat urine as sensitive marker to detect S3 segment-specific injury of proximal tubule induced by xenobiotics.

Abstract The possibility of detecting segment-specific injury of the proximal tubule by means of urinary enzymes was investigated in rats. Urinary glutamine synthetase, an enzyme exclusively localized in the S3 segment, and N-acetyl-β-d-glucosaminidase, prevalently a S1-S2, but S3 enzyme also, were determined after single treatment with 100 mg/kg body wt. of hexachloro-1:3-butadiene (HCBD; i.p.), toxic for the S3 segment, or 25 mg/kg body wt. of potassium dichromate (s.c.), toxic for the S1-S2 segments. Excretion of total urinary proteins was also measured. In addition, a dose-response relationship was determined between three doses (50, 100 and 200 mg/kg body wt.) of HCBD and glutamine synthetase activity in urine. Glutamine synthetase activity, measured according to a new assay for urine based on modification of methods developed for organs, increased in the urine only when the S3 segment of the proximal tubule was damaged, as demonstrated by histological findings of the kidneys. HCBD caused early excretion of the enzyme related to the necrosis of the S3 segment, whereas potassium dichromate caused a slight increase only when the resulting lesion to this segment (vacuolization) began to develop. On the contrary, N-acetyl-β-d-glucosaminidase activity showed the peak of excretion 24 and 34 h after treatment with HCBD or potassium dichromate, respectively, according to the histological findings of necrosis of the S3 segment (the former) and vacuolization of the S1-S2 segments (the latter). Excretion of total urinary proteins reached the peak 24 h (HCBD) and 48 h (potassium dichromate) after treatment. HCBD at 200 mg/kg body wt. caused a peak of glutamine synthetase activity in urine 10 h after injection, whereas the peak caused by doses of 50 and 100 mg/kg body wt. occurred 24 h following treatment. The peak of enzyme activity in urine significantly increased with the dose. The results suggest that the measurement of urinary activity of S3 segment-specific enzyme as glutamine synthetase allows us to detect early S3 segment-specific injury of the proximal tubule. In addition, the method for urinary enzyme activity appears sensitive, simple and fast.

Synthesis of a palladium(II)-dithiocarbamate complex: biological assay and nephrotoxicity in rats

Abstract. A new palladium(II)-dithiocarbamate complex, [Pd(ESDT)Cl]n, was synthesised and its chemical characteristics are discussed. This complex was examined for its cytotoxic properties in human tumour cell lines; for comparison, the cytotoxicity of cisplatin was evaluated under the same experimental conditions. In particular, Pd(II)-complex cytotoxicity on ovarian carcinoma C13 cells, resistant to cisplatin, showed that there seemed to be no cross-resistance between [Pd(ESDT)Cl]n and cisplatin. The effects on the kidney were also studied. Biochemical investigation on urinary parameters showed that the effects after a single injection are similar to those of cisplatin, with an increase of urinary proteins and enzyme excretion in urine, and a significant decrease of glutamine synthetase activity in the renal tissue. In addition, the Pd(II)-complex caused a significant decrease of p-aminohippuric acid uptake in renal cortical slices relative to cisplatin. On the other hand, histopathological findings showed that the effects of the Pd(II)-complex are more severe and diffuse than the damage caused by cisplatin. Biochemical and histopathological findings show that the Pd(II)-complex affects the pars recta and pars convoluta, in contrast to cisplatin, which only affects the pars recta.

Neuroimaging Evidence of Altered Fronto-Cortical and Striatal Function after Prolonged Cocaine Self-Administration in the Rat

Cocaine addiction is often modeled in experimental paradigms where rodents learn to self-administer (SA) the drug. However, the extent to which these models replicate the functional alterations observed in clinical neuroimaging studies of cocaine addiction remains unknown. We used magnetic resonance imaging (MRI) to assess basal and evoked brain function in rats subjected to a prolonged, extended-access cocaine SA scheme. Specifically, we measured basal cerebral blood volume (bCBV), an established correlate of basal metabolism, and assessed the reactivity of the dopaminergic system by mapping the pharmacological MRI (phMRI) response evoked by the dopamine-releaser amphetamine. Cocaine-exposed subjects exhibited reduced bCBV in fronto-cortical areas, nucleus accumbens, ventral hippocampus, and thalamus. The cocaine group also showed an attenuated functional response to amphetamine in ventrostriatal areas, an effect that was significantly correlated with total cocaine intake. An inverse relationship between bCBV in the reticular thalamus and the frontal response elicited by amphetamine was found in control subjects but not in the cocaine group, suggesting that the inhibitory interplay within this attentional circuit may be compromised by the drug. Importantly, histopathological analysis did not reveal significant alterations of the microvascular bed in the brain of cocaine-exposed subjects, suggesting that the imaging findings cannot be merely ascribed to cocaine-induced vascular damage. These results document that chronic, extended-access cocaine SA in the rat produces focal fronto-cortical and striatal alterations that serve as plausible neurobiological substrate for the behavioral expression of compulsive drug intake in laboratory animals.