Potential anti-inflammatory treatments against cutaneous sulfur mustard injury using the mouse ear vesicant model.

In spite of several decades of research, no effective treatment to skin injuries following exposure to sulfur mustard (HD) has yet been found. In the present study, the mouse ear vesicant model was applied to awake mice in order to evaluate the efficiency of potential anti-inflammatory treatments in preventing HD-induced skin damages. Clinical follow-up and histological evaluation were used to characterize the injuries to the skin and to evaluate the efficiency of the drugs that were applied. Thus, the extent of mouse ear oedema and the histopathological changes following a single application of 0.2 or 1 microL of neat HD for 10 min (representing moderate and severe lesions, respectively), were monitored. Typical HD skin lesions were observed including epithelial and dermal damage. The development of the injury in mouse ears was found to be very similar to that reported in human skin. Screening of post-exposure topical steroids and non-steroidal antiinflammatory drugs (NSAIDs) proved that HD-induced inflammation could be diminished significantly as long as the treatment was applied during the early stages following exposure. A combined application of these drugs approved to be particularly effective in reducing inflammation.

Prophylaxis against organophosphate poisoning by sustained release of scopolamine and physostigmine.

Protection efficacy of continuous prophylactic administration of physostigmine and scopolamine against sarin-induced toxicity was evaluated previously in guinea pigs. The present study in large animals used Beagle dogs, that serve as an animal model with cholinergic sensitivity similar to that of humans. Pretreatment with physostigmine salicylate and scopolamine hydrochloride at dose rates of 2.5 and 1 microg x kg(-1) x h(-1), respectively, was administered via Alzet mini-osmotic pumps. At the time of exposure, the physostigmine salicylate concentration in plasma was 0.7 ng x ml(-1) and the scopolamine hydrochloride concentration was ca. 0.2 ng x ml(-1), both of which are levels known to be well tolerated in humans. Whole-blood cholinesterase inhibition was 15-20%. This regimen conferred full protection against 2.5 x LD50 i.v. of sarin. Albeit the high-dose exposure, cholinergic toxicity symptoms were mild with no convulsions. About 11-14 min following poisoning the treated animals started to walk and 15-20 min following exposure full recovery was observed and the dogs behaved normally. With higher dose rates of physostigmine salicylate and scopolamine hydrochloride, at plasma concentrations of 2.1 and 0.6 ng x ml(-1), respectively, treated dogs regained normal posture 6-10 min after exposure.

Treatment of skin injuries induced by sulfur mustard with calmodulin antagonists, using the pig model.

Sulfur mustard (HD) is a potent cutaneous vesicant that penetrates rapidly through the skin, causing prolonged injuries and leading to severe incapacitation. Although there has been long and intensive efforts to find a treatment for HD skin lesions, no effective treatment is available for HD-induced skin injuries. Recently, ointments containing calmodulin antagonists were found to be effective in preventing skin injuries induced by HD in hairless mice. The present study was designed to investigate the beneficial effects of topical treatments with calmodulin antagonists against HD skin lesions in the pig model. The pig is used as a preferred animal model for human skin in many studies, including vesicants. Neat HD, either in liquid form (0.2-1 microl droplets) or as vapour, was applied to the back skin of female pigs (a cross Large White & Landrace, 10-12 kg) for various exposure durations. Evaluation was based on quantitative analysis of the degree of erythema and area of the lesions, as well as histological evaluation. Calmodulin antagonists (10% pentamide, 1% trifluoperazine, 2% thioridazine) and anaesthetics (20% lidocaine and 3% benoxinate) were dissolved in pluronic F-127 base according to Kim et al. (Eur. J. Pharmacol. 1996; 313: 107-114) or in saline, and were applied either topically as ointments or by intradermal injection, as early as 5 min post-exposure (twice a day for at least 3 days). The results demonstrated that topically applied pluronic base ointments containing lidocaine or pentamide produce beneficial effects when applied immediately after short-term HD exposure to pig skin.

Chronic NMDA receptor stimulation: therapeutic implications of its effect on adenosine A1 receptors.

It is known that stimulation of adenosine A1 receptors has a modulatory effect on the excitability of postsynaptic NMDA receptors. Conversely, acute stimulation of NMDA receptors results in release of adenosine via calcium-independent mechanisms. These findings indicate a close functional relationship between these receptors. It is, therefore, possible that chronic, low level stimulation of the NMDA receptor may have a negative impact on these modulatory processes. To investigate this possibility, we have subjected C57BL mice either to an acute injection of a N6-cyclopentyladenosine (CPA, 0.01 mg/kg) or deoxycoformycin (1 mg/kg) followed by a convulsant dose of N-methyl-D-aspartate (NMDA) (60 mg/kg) or to chronic, low level (20 mg/kg i.p. daily) exposure to NMDA for 8 weeks. One day after the last injection of NMDA, animals were injected either with a convulsant dose of NMDA alone, or with either CPA at 0.001 or 0.01 mg/kg, or with 1 mg/kg deoxycoformycin followed 15 min later by 60 mg/kg NMDA. Neither CPA nor deoxycoformycin were protective when NMDA was given acutely at 60 mg/kg. Chronic treatment with NMDA alone or chronic administration of NMDA followed by 0.001 mg/kg CPA had no significant effect on mortality following a convulsant dose of NMDA. However, when the chronic regimen of NMDA was followed by either 0.01 mg/kg CPA or 1 mg/kg deoxycoformycin, mortality was reduced to 10% (CPA), or eliminated completely (deoxycoformycin). Moreover, combination of chronic NMDA treatment with either CPA (both doses) or deoxycoformycin produced a significant improvement in other measures, i.e., seizure onset, intensity of neurological impairment, and extension of time to death.(ABSTRACT TRUNCATED AT 250 WORDS)

Prophylactic transdermal treatment with physostigmine and scopolamine against soman intoxication in guinea-pigs.

This study was designed to evaluate the prophylactic efficacy of transdermally administered physostigmine (PHY) against soman exposure using guinea-pigs. Transdermal PHY pad (3 cm2 kg-1; 60 micrograms cm-2), containing a vehicle based on propionic acid, was applied onto the dorsal back of the animals, 24 h before exposure to the organophosphate. At the time of exposure, PHY concentrations in brain and plasma were ca. 3.6 ng g-1 and 4.1 ng ml-1, respectively. Brain and whole blood cholinesterase (ChE) activity was inhibited to 70% and 47% of the original activity, respectively. Transdermal PHY by itself protected up to 70% of the animals exposed to 1.5 LD50 of soman (100% mortality was recorded in the control group). Combining transdermal PHY with Scopoderm provided full protection against 1.5 LD50 of soman (protection of 70% against 3 LD50). When the prophylactic treatment was combined with post-exposure therapy (atropine, 10 mg kg-1; toxogonin, 10 mg kg-1) 1 min after 5 LD50 of soman, protection of 90% of the animals was achieved.

Structure-activity relationships of 9-alkyladenine and ribose-modified adenosine derivatives at rat A3 adenosine receptors.

9-Alkyladenine derivatives and ribose-modified N6-benzyladenosine derivatives were synthesized in an effort to identify selective ligands for the rat A3 adenosine receptor and leads for the development of antagonists. The derivatives contained structural features previously determined to be important for A3 selectivity in adenosine derivatives, such as an N6-(3-iodobenzyl) moiety, and were further substituted at the 2-position with halo, amino, or thio groups. Affinity was determined in radioligand binding assays at rat brain A3 receptors stably expressed in Chinese hamster ovary (CHO) cells, using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-(N-methyluronamide)), and at rat brain A1 and A2a receptors using [3H]-N6-PIA ((R)-N6-phenylisopropyladenosine) and [3H]CGS 21680 (2-[[[4-(2-carboxyethyl)-phenyl]ethyl]amino]-5'- (N-ethylcarbamoyl)adenosine), respectively. A series of N6-(3-iodobenzyl) 2-amino derivatives indicated that a small 2-alkylamino group, e.g., methylamino, was favored at A3 receptors. N6-(3-Iodobenzyl)-9-methyl-2-(methylthio)adenine was 61-fold more potent than the corresponding 2-methoxy ether at A3 receptors and of comparable affinity at A1 and A2a receptors, resulting in a 3-6-fold selectivity for A3 receptors. A pair of chiral N6-(3-iodobenzyl) 9-(2,3-dihydroxypropyl) derivatives showed stereoselectivity, with the R-enantiomer favored at A3 receptors by 5.7-fold. 2-Chloro-9-(beta-D-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 microM. 2-Chloro-9-[2-amino-2,3-dideoxy-beta-D-5-(methylcarbamoyl)- arabinofuranosyl]-N6-(3-iodobenzyl)adenine was moderately selective for A1 and A3 vs A2a receptors. A 3'-deoxy analogue of a highly A3-selective adenosine derivative retained selectivity in binding and was a full agonist in the inhibition of adenylyl cyclase mediated via cloned rat A3 receptors expressed in CHO cells. The 3'-OH and 4'-CH2OH groups of adenosine are not required for activation at A3 receptors. A number of 2',3'-dideoxyadenosines and 9-acyclic-substituted adenines appear to inhibit adenylyl cyclase at the allosteric "P" site.

The effects of adenosine A3 receptor stimulation on seizures in mice.

We have previously shown that acute preischemic adenosine A3 receptor stimulation results in an increased postischemic damage, while chronic stimulation of this receptor diminishes it. Since several pathophysiological phenomena are common for both ischemia and seizures, we have explored the effect of acute and chronic administration of the adenosine A3 receptor selective agonist IB-MECA (N6-(3-iodobenzyl) adenosine-5'-N-methylcarboxamide) prior to seizures induced by N-methyl-D-aspartate (NMDA), pentamethylenetetrazole, or electric shock. At 100 micrograms/kg, acutely injected IB-MECA was protective in chemically but not electrically induced seizures. In chronic administration of IB-MECA, significant protection against chemically induced seizures was obtained in all studied measures, i.e., seizure latency, neurological impairment, and survival. Although threshold voltage was unchanged in electrically induced seizures, a chronic regimen of IB-MECA significantly reduced postepileptic mortality. Since the combination of an arteriole-constricting compound 48/80 and hypotension-inducing clonidine injected prior to NMDA results in a significant protection against seizures, and since acute stimulation of adenosine A3 receptor causes both arteriolar constriction and severe hypotension, there is a possibility that the protection obtained by the acutely administered drug may result from inadequate delivery of chemoconvulsants to the brain. It is, however, unknown whether the protective effect of chronically administered IB-MECA is related to the effect of the drug on blood flow, neuronal mechanisms, or both.

Transdermal physostigmine in the treatment of Alzheimer's disease.

Physostigmine has been reported to improve the memory function of some patients with Alzheimer's Disease (AD). However, the drug has a short half-life and a narrow therapeutic window. To overcome these impediments, we developed a continuous transdermal delivery system and tested it for 2 weeks in 12 AD inpatients, using a single-blind design. No major adverse effects were recorded in any of the patients. Physostigmine plasma concentrations were relatively stable (0.56 +/- 0.10 ng/ml) and correlated well with blood acetylcholinesterase inhibition. Six of the 12 patients reported improved vigilance and concentration, and also had higher scores in all four neuropsychological tests employed (Mini Mental State examination, Short Mental Test [SMT], Wechsler's Memory Scale [WMS], and Buschke's Selective Reminding Test). The performance of two additional patients improved in only two tests (SMT and WMS). Transdermal delivery of physostigmine appears to be safe and may be useful for the treatment of a subset of AD patients.

Effect of ultrasound on transdermal drug delivery to rats and guinea pigs.

The effect of therapeutic range ultrasound (1 MHz) on skin permeation of D-mannitol, a highly polar sugar alcohol, inulin, a high molecular weight polysaccharide and physostigmine, a lipophilic anticholinesterase drug was studied in rats and guinea pigs. D-Mannitol and inulin are totally and rapidly excreted, once they have penetrated through the skin into the blood stream, permitting direct in vivo monitoring. For evaluating skin penetration of physostigmine the decrease of whole blood cholinesterase was measured. Ultrasound nearly completely eliminated the lag time usually associated with transdermal delivery of drugs. 3-5 min of ultrasound irradiation (1.5 W/cm2 continuous wave or 3 W/cm2 pulsed wave) increased the transdermal permeation of inulin and mannitol in rats by 5-20-fold within 1-2 h following ultrasound application. Ultrasound treatment also significantly increased (P less than 0.05) the inhibition of cholinesterase during the first hour after application in both physostigmine treated rats and guinea pigs: while in control guinea pigs no significant inhibition of cholinesterase could be detected during the first 2 h after application of physostigmine, the ultrasound treated group showed a 15 +/- 5% (mean +/- SEM) decrease in blood cholinesterase 1 h after ultrasound application. For physostigmine-treated rats the level of cholinesterase inhibition 1 h after ultrasound application was 53 +/- 5% in the ultrasound-treated group and 35 +/- 5% in the controls.