Methods to induce primary and secondary traumatic damage in organotypic hippocampal slice cultures.

Organotypic brain slice cultures have been used in a variety of studies on neurodegenerative processes [K.M. Abdel-Hamid, M. Tymianski, Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins, J. Neurosci. 17, 1997, pp. 3538-3553; D.W. Newell, A. Barth, V. Papermaster, A.T. Malouf, Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures, J. Neurosci. 15, 1995, pp. 7702-7711; J.L. Perez Velazquez, M.V. Frantseva, P.L. Carlen, In vitro ischemia promotes glutamate mediated free radical generation and intracellular calcium accumulation in pyramidal neurons of cultured hippocampal slices, J. Neurosci. 23, 1997, pp. 9085-9094; L. Stoppini, L.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37, 1991, pp. 173-182; R.C. Tasker, J.T. Coyle, J.J. Vornov, The regional vulnerability to hypoglycemia induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK 801, J. Neurosci. 12, 1992, pp. 4298-4308.]. We describe two methods to induce traumatic cell damage in hippocampal organotypic cultures. Primary trauma injury was achieved by rolling a stainless steel cylinder (0.9 g) on the organotypic slices. Secondary injury was followed after dropping a weight (0.137 g) on a localised area of the organotypic slice, from a height of 2 mm. The time course and extent of cell death were determined by measuring the fluorescence of the viability indicator propidium iodide (PI) at several time points after the injury. The initial localised impact damage spread 24 and 67 h after injury, cell death being 25% and 54%, respectively, when slices were kept at 37 degrees C. To validate these methods as models to assess neuroprotective strategies, similar insults were applied to slices at relatively low temperatures (30 degrees C), which is known to be neuroprotective [F.C. Barone, G.Z. Feuerstein, R.F. White, Brain cooling during transient focal ischaemia provides complete neuroprotection, Neurosci. Biobehav. Rev. 1, 1997, pp. 31-44; V.M. Bruno, M.P. Goldberg, L.L. Dugan, R.G. Giffard, D.W. Choi, Neuroprotective effect of hypothermia in cortical cultures exposed to oxygen glucose deprivation or excitatory aminoacids, J. Neurochem. 4, 1994, pp. 387-392; G.C. Newman, H. Qi, F.E. Hospod, K. Grundhmann, Preservation of hippocampal brain slices with in vivo or in vitro hypothermia, Brain Res. 1, 1992, pp. 159-163; J.Y. Yager, J. Asseline, Effect of mild hypothermia on cerebral energy metabolism during the evolution of hypoxic ischaemic brain damage in the immature rat, Stroke, 5, 1996, pp. 919-925.]. Low temperature incubation significantly reduced cell death, now being 9% at 24 h and 14% at 67 h. Our results show that these models of moderate mechanical trauma using organotypic slice cultures can be used to study neurodegeneration and neuroprotective strategies.

Tyrphostin 4-nitrobenzylidene malononitrile reduces chemotherapy toxicity without impairing efficacy.

In mice, 4-nitrobenzylidene malononitrile (AG1714), which belongs to the tyrphostin family, reduced toxicity induced by doxorubicin and cisplatin without impairing their antitumor efficacy. AG1714 reduced mortality induced by doxorubicin and cisplatin. It prevented, in a dose-dependent manner, cisplatin-induced nephrotoxicity as assessed by measurement of serum creatinine and blood urea nitrogen levels. The protective effect of AG1714 was most pronounced on its administration 2 h before cisplatin. AG1714 also prevented doxorubicin-induced myelosuppression as assessed by the scoring of bone marrow nucleated cells and colony-forming units. Cisplatin-induced small intestinal injury was also protected by AG1714 as assessed by histopathological analysis. In vitro, AG1714 reduced cisplatin-induced apoptosis in a murine fibroblastic cell line (A9) and did not affect doxorubicin-induced apoptosis of B-16 melanoma cells. In contrast to its protective effect against mortality and injury of normal tissues induced by chemotherapy, AG1714 did not impair its antitumor activity and in some tumor models enhanced it. This was evident by using the murine tumors B-16 melanoma, Lewis lung carcinoma, and methylcholanthrene-induced fibrosarcoma and the human tumors SK-28 melanoma and human ovary carcinoma xenografts in nude mice. Experiments in which low and high doses of cisplatin and doxorubicin were administered to tumor-bearing mice demonstrated that AG1714 reduced mortality of high-dose chemotherapy and increased its therapeutic index. AG1714 could provide a novel, useful tool to improve chemotherapy by allowing dose intensification.

Improved oral delivery of desmopressin via a novel vehicle: mucoadhesive submicron emulsion.

PURPOSE: Desmopressin acetate (DDAVP) is used parenterally and intranasally in the control of several diseases. Oral administration of DDAVP, while most desirable, is not practical presently due to low bioavailability. The objective of the present study was to explore the feasibility for employing oil-in-water MucoAdhesive SubMicron Emulsion (MA-SME), a novel mucoadhesive vehicle with polymer-coated droplets, for enhanced oral delivery of DDAVP. METHODS: We used a modified pharmacopeal method, based on measurement of the antidiuretic activity, for the assessment of oral delivery of DDAVP in rats. DDAVP formulated in two MA-SME preparations, in non-mucoadhesive SME (plain-SME), in saline and in other control solutions was administered orally to rats via a stomach tube at a dose of 0.5 units/kg. At various times following DDAVP administration, water was given via a stomach tube. Excretion times for 30% and 60% of the total water load were measured. RESULTS: Excretion times for DDAVP in MA-SME formulations were always longer (up to 2-fold) than those following DDAVP in saline. By contrast, excretion times for DDAVP in plain-SME and in non-SME Carbopol (a Mucoadhesive polymer) solution were virtually identical to those for DDAVP in saline. CONCLUSIONS: Formulations of MA-SME were shown to generate substantial enhancement (up to 12-fold) of the rat oral bioavailability of DDAVP with regard to simple saline solution of the drug. From the results it is also evident that MA-SME, but not plain-SME or non-SME Carbopol solution, is responsible for the enhancement of oral delivery of DDAVP in rats.

Enhanced transdermal delivery of diazepam by submicron emulsion (SME) creams.

Diazepam, a lipophilic drug with CNS activity, serves here as a model to investigate the efficacy of SubMicron Emulsion (SME) as a novel transdermal vehicle. Diazepam was formulated in various topical regular creams and SubMicron Emulsion creams of different compositions. The different formulations were applied topically and protection against Pentamethylenetetrazole induced convulsive effects in mice was monitored. The efficacy of Diazepam applied topically in emulsion creams strongly depends on the oil droplet size and to a lesser degree--on the formulation composition and the oil type. Processing medium-chain-triglyceride (MCT) emulsion with a high-pressure homogenizer causes a drastic reduction in the droplet size, thereby significantly increasing the transdermal activity of Diazepam. In this case both the high-pressure homogenization and the presence of lecithin, an efficient dispersant, contribute to the effective droplet size reduction of below 1 micron, usually between 100-300 nm. The SubMicron Emulsions as vehicles for transdermal delivery of Diazepam generate significant systemic activity of the drug as compared with regular creams or ointments. Transdermal delivery of Diazepam via SME formulations is very effective, and the activity may reach the range of parenteral delivery. A single application of Diazepam in SME cream to mice skin provides pronounced transdermal drug delivery and prolonged protective activity up to 6 hours.

Submicron emulsion vehicle for enhanced transdermal delivery of steroidal and nonsteroidal antiinflammatory drugs.

Significant improvement is demonstrated for transdermal delivery of steroidal and nonsteroidal antiinflammatory drugs (NSAID), including betamethasone valerate and dipropionate, indomethacin, diclofenac, piroxicam, and naproxen, when formulated in a submicron Emulsion (SME) vehicle rather than in standard creams. SMEs comprise oil droplets, with mean size of approximately 100 nm (0.1 micron), dispersed in a continuous water phase. Hydrophobic drugs are incorporated into the oil phase of the SME, resulting in improved penetration and increased efficacy of the incorporated antiinflammatory drug. The performance of medicated topical SME was compared with that of regular topical cream formulations, as measured by the carrageenan-induced paw edema rat model. Indomethacin in SME topical vehicle was 50% more active than in regular cream base, Diclofenac in SME proved to be 40% more active than Voltaren Emulgel. Improvement of steroidal antiinflammatory drugs action in topical SME cream was even more pronounced; that is, up to 3-4-fold. Antiinflammatory drugs in SME also demonstrate noticeable systemic activity, but for regional edema treatment, local delivery is advantageous. The new SME delivery system was tested for primary irritation in humans in a 48-h trial. Low irritancy and excellent human acceptance for SME placebo or diclofenac-loaded SME cream make this novel transdermal/topical DDS attractive for further development.