Potential of the rat model of conditioned gaping to detect nausea produced by rolipram, a phosphodiesterase-4 (PDE4) inhibitor.

Rolipram, a phosphodiesterase-4 (PDE4) inhibitor, is of current interest as a cognitive enhancer and as a treatment for inflammatory diseases. Originally developed as an anti-depressant, rolipram's efficacy was limited due to its side effects of nausea and vomiting. The experiments reported here evaluated the potential of rolipram to produce conditioned gaping (a selective measure of nausea in rats) to a flavor in the taste reactivity test (Experiment 1) and to a context (Experiment 2). In Experiment 1, rats were intra-orally infused with 17% sucrose solution prior to being injected with rolipram (Vehicle, 0.03, 0.1 or 0.3 mg/kg). Following 3 conditioning trials, rats conditioned with 0.3 mg/kg rolipram displayed conditioned gaping reactions during the infusion of sucrose. In Experiment 2, rats received 4 conditioning trials in which they were injected with 0.3 mg/kg rolipram and placed into a distinctive chamber. At test, when returned to the chamber rats displayed conditioned gaping. These results demonstrate the ability of the conditioned gaping model to detect the nauseating properties of a rolipram-paired flavor (Experiment 1) and rolipram-paired context (Experiment 2), further validating the potential use of the conditioned gaping model as a pre-clinical screening tool to evaluate the side effect of nausea produced by newly developed drugs.

Correlation between emetic effect of phosphodiesterase 4 inhibitors and their occupation of the high-affinity rolipram binding site in Suncus murinus brain.

We employed an ex vivo [(3)H]rolipram binding experiment to elucidate the mechanism of emetic activity of phosphodiesterase 4 inhibitors. In Suncus murinus (an insectivore used for evaluation of emesis), emetic potential as well as ability to occupy the high-affinity rolipram binding site in brain membrane fraction in vivo were determined for phosphodiesterase 4 inhibitors. In vitro, [(3)H]rolipram bound to the membrane fraction of S. murinus brain with high affinity and its value was comparable to that for rat brain (K(d)=3.6 nM and 3.5 nM, respectively). The test compounds included denbufylline, rolipram, piclamilast, CDP840 and KF19514, each of which possessed similar affinities for the rolipram binding sites in both S. murinus and rat brain. In S. murinus, these compounds induced emesis via intraperitoneal administration. Their ED(50) values were as follows: denbufylline (1.4 mg/kg), rolipram (0.16 mg/kg), piclamilast (1.8 mg/kg), CDP840 (20 mg/kg), and KF19514 (0.030 mg/kg). In addition, these compounds occupied the high-affinity rolipram binding site in vivo as detected by dose-dependent reduction in capacity of ex vivo [(3)H]rolipram binding in brain membrane fractions. A clear correlation was observed between dose required to induce emesis and that to occupy the high-affinity rolipram binding site for individual phosphodiesterase 4 inhibitors. We conclude that the emetic effect of phosphodiesterase 4 inhibitors is caused at least in part via binding to the high-affinity rolipram binding site in brain in vivo.

Mechanisms of rolipram-induced increase in the incidence of mammary adenocarcinoma: histopathological study of a 104-week oral carcinogenicity study in female Sprague-Dawley rats.

The present study was carried out to elucidate the mechanisms behind an increase in the incidence of malignant or multiple mammary tumors as a result of oral administration of rolipram in a 104-week carcinogenicity study. The organs and tissues of Sprague-Dawley (SD) rats of both sexes, which had been subjected to a 104-week oral carcinogenicity study at doses of 0.2, 0.6 and 2.0 mg/kg, were examined. No treatment-related effects were seen in males; however, in females, there was a significant increase in the number of malignant or multiple mammary tumor bearers at a dose of 2.0 mg/kg. No other target organs were identified and the incidence of other tumor types were within the female control range. To clarify the mechanisms behind a rolipram-induced increase in the incidence of mammary adenocarcinoma at time points earlier than 104 weeks, the hormonal changes associated with pituitary adenoma were identified, and estrous cycling in the ovary, uterus, and vagina were examined in female rats treated with rolipram for 52 weeks. The plasma prolactin (PRL) concentration in all female groups exceeded the control value at Week 52, and all these differences were statistically significant. There was also a dose-dependent relationship with PRL-producing pituitary adenomas. Changes in estrous cycling in the uterus and vagina and a decrease in the size and number of corpora lutea in the ovaries of female rats treated with rolipram at 2.0 mg/kg for 52 weeks indicated that an increase in the estrus phase of the cycle corresponded to a marked decrease in the diestrus phase, which might result from the increased plasma estrogen concentration. Together, all of the above mentioned data suggest that rolipram not only stimulates an increase in the number and size of PRL adenomas in the pituitary gland but also in the estrus phase of the estrous cycle. These events might cause progression of the mammary gland tissues from hyperplasia to carcinoma.

Type 4 phosphodiesterase inhibition impairs detection of low odor concentrations in mice.

The cAMP-specific phosphodiesterase PDE4A is abundant in the dendrites, soma and axons of olfactory receptor neurons of the mouse, but it is not present in the cilia, where olfactory transduction initiates. Although the function of PDE4A in mammalian olfaction is unknown, patch clamp studies on deciliated olfactory receptor cells in the newt have shown that adrenaline or cAMP analogs can increase the contrast sensitivity to current injection. We used mice to ask whether increasing the levels of cAMP in sensory neurons by inhibiting PDE4A activity with rolipram could lead to changes in the perception of odorants that correspond to the in vitro cellular responses seen in newts. In an automated olfactometer, rolipram treatment (1mg/kg, i.p.) significantly impaired the detection accuracy of 1-propanol at relatively high dilutions but did not affect detection at lower dilutions. Meanwhile, the ability to discriminate amyl acetate alone from a mixture of amyl acetate+citronellal was not affected by rolipram at any odor dilution. In a different task in which mice were trained to discriminate between cups of scented versus unscented sand, rolipram treatment resulted in poorer discrimination at high and better discrimination at low, odor dilutions. In sum, PDE4 inhibition resulted in a consistent decrement in the ability of mice to detect low concentrations of odorants, but the effects of rolipram on detection of higher concentrations were task-dependent.

Efeito de drogas inibidoras de TNF-a em células mononucleares do sangue periférico de pacientes infectados pelo HTLV-I / Effect of drugs inhibiting TNF-a in peripheral blood mononuclear cells of patients infected by HTLV-I

O Retovírus HTLV-I é o agente etiológico da Mielopatia Associada ao HTLV-I/Paraparesia Espástica Tropical (HAM/TSP), Leucemia de Células T do Adulto (ATL) e outras doenças sistêmicas mediadas pela resposta imune. A infecção pelo HTLV-I induz uma elevada proliferação espontânea das células T do perfil de citocinas com predominância das pró-inflamatórias. Nos indivíduos com HAM/TSP o TNF-a encontra-se elevado e envolvido na lesão tecidual. O surgimento de drogas inibidoras da síntese de TNF-a traz a possibilidade de uma terapêutica, buscando reduzir a inflamação e lesão tecidual. O objetivo deste estudo foi avaliar o poder inibidor destas drogas na produção de TNF-a em PBMC de indivíduos infectados com HTLV-I. PBMC de 37 indivíduos foram avaliados: assintomáticos (n=11), subclínicos (n=7) e com mielopatia (n=19). Foram utilizadas quatro drogas inibidoras da síntese de TNF-a: Pentoxifilina, Forskolin, Rolipram e Talidomida, as quais agem em diferentes etapas da síntese desta citocina. As concentrações espontâneas de TNF-a e IFN-y e com as drogas inibidoras foram avaliados nos sobrenadantes das culturas de PBMC através da técnica de ELISA e os resultados comparados entre os grupos usando o teste Mann-Whitney. A produção espontânea de TNF-a foi mais elevada no grupo com HAM/TSP quando comparado ao assintomático e a diferença estatisticamente significante (p = 0.001). A produção espontânea de IFN-y também foi mais alta no grupo com HAM/TSP quando comparados aos assintomáticos e a diferença estatisticamente significante (p = 0.017). Para avaliação das drogas inibidoras de TNF-a, utilizamos PBMC de indivíduos com de TNF-a e IFN-y espontâneos maiores que 5 pg/ml e os resultados comparados pelo teste estatístico Wilcoxon signed ranks. Pentoxifilina foi utilizada nas doses de 50 e 200 μM. A inibição da produção de TNF-a com 50 μM foi de 71 ± 26% (p = 0.003) e de IFN-y com 50 μM foi de 46 ± 24% (p = 0.001). Forskolin foi utilizado nas doses...

Development of an in vitro model for vascular injury with human endothelial cells.

The aim of the present work was to establish an in vitro screening assay for drug candidates using human endothelial cells as a model for vascular injury after intravenous application. Different endpoints for viability and functionality of endothelial cells were investigated in human umbilical vein endothelial cells (HUVEC) and in immortalised human endothelial cells (IVEC). Cellular viability was determined by measuring ATP content and by the AlamarBlue assay. For comparison, the toxicity of the selected compounds was also tested in a murine fibroblast cell line (3T3 cells). Selected endpoints for endothelial cell-specific function were vascular permeability, determined by measurement of the transendothelial resistance and the diffusion of tracer molecules (FITC-dextran), and the release of prostaglandin and thromboxane as indicators for prothrombotic or vasoconstrictory action. Five compounds (cyclosporin A, mitomycin C, menadione, amrinone and rolipram) were selected due to their known effects on the vasculature. The cytotoxicity of all compounds was similar in endothelial and 3T3 cells. ATP content and AlamarBlue metabolism did not differ significantly except for amrinone. A dose-dependent decrease of transendothelial resistance and an increase in FITC-dextran permeability could be measured in HUVEC cells for the tested compounds, but the sensitivity was not higher than that of the cytotoxicity assays. Increased prostaglandin or thromboxane release was detected for all compounds at cytotoxic concentrations and for rolipram also at non-toxic concentrations. In conclusion, for a first ranking of drug candidates, cytotoxicity assays on any of the three cell types used are appropriate. For a more detailed characterisation of individual compounds, functional assays on HUVEC cells are proposed.