Neuroprotection for ischaemic stroke: translation from the bench to the bedside.

Neuroprotection seeks to restrict injury to the brain parenchyma following an ischaemic insult by preventing salvageable neurons from dying. The concept of neuroprotection has shown promise in experimental studies, but has failed to translate into clinical success. Many reasons exist for this including the heterogeneity of human stroke and the lack of methodological agreement between preclinical and clinical studies. Even with the proposed Stroke Therapy Academic Industry Roundtable criteria for preclinical development of neuroprotective agents for stroke, we have still seen limited success in the clinic, an example being NXY-059, which fulfilled nearly all the Stroke Therapy Academic Industry Roundtable criteria. There are currently a number of ongoing trials for neuroprotective strategies including hypothermia and albumin, but the outcome of these approaches remains to be seen. Combination therapies with thrombolysis also need to be fully investigated, as restoration of oxygen and glucose will always be the best therapy to protect against cell death from stroke. There are also a number of promising neuroprotectants in preclinical development including haematopoietic growth factors, and inhibitors of the nicotinamide adenine dinucleotide phosphate oxidases, a source of free radical production which is a key step in the pathophysiology of acute ischaemic stroke. For these neuroprotectants to succeed, essential quality standards need to be adhered to; however, these must remain realistic as the evidence that standardization of procedures improves translational success remains absent for stroke.

Neutralization of the haemorrhagic activities of viperine snake venoms and venom metalloproteinases using synthetic peptide inhibitors and chelators.

Envenoming by the West African saw-scaled viper, Echis ocellatus resembles that of most vipers, in that it results in local blistering, necrosis and sometimes life-threatening systemic haemorrhage. While effective against systemic envenoming, current antivenoms have little or no effect against local tissue damage. The major mediators of local venom pathology are the zinc-dependant snake venom metalloproteinases (SVMPs). The high degree of structural and functional homology between SVMPs and their mammalian relatives the matrix metalloproteinases (MMPs) suggests that substrate/inhibitor interactions between these subfamilies are likely to be analogous. In this study, four recently developed MMP inhibitors (MMPIs) (Marimastat, AG-3340, CGS-270 23A and Bay-12 9566) are evaluated in addition to three metal ion chelators (EDTA, TPEN and BAPTA) for their ability to inhibit the haemorrhagic activities of the medically important E. ocellatus venom and one of its haemorrhagic SVMPs, EoVMP2. As expected, the metal ion chelators significantly inhibited the haemorrhagic activities of both whole E. ocellatus venom and EoVMP2, while the synthetic MMPIs show more variation in their efficacies. These variations suggest that individual MMPIs show specificity towards SVMPs and that their application to the neutralization of local haemorrhage may require a synthetic MMPI mixture, ensuring that a close structural component for each SVMP is represented.

Evaluation of potential strontium chelators in an octanol-water system.

90Sr has a physical half-life of sufficient duration to make it a potentially dangerous contaminant from nuclear accidents and radioactive wastes. In the present study, the efficacy of 16 compounds as potential chelators of strontium was tested in vitro. Strontium solubilization from strontium carbonate and its distribution in an octanol-water system (Do/w) was determined in the absence and presence of alpha-ketoglutaric acid, Kryptofix 222, ethylenglycol-bis-(beta-amino-ethylether)-N,N-tetraacetic acid, diethylentriamine pentaacetic acid, Kryptofix 5, disodium chlodronate, disodium ethidronate, oxaloacetic acid, fumaric acid, D-gluconic acid, succinic acid, citric acid, D,L-2,3-diaminopropionic acid, 1,1-cyclohexanediacetic acid, tartaric acid, and trans-1,2-cy-clohexanediol. Kryptofix 222 and Kryptofix 5 significantly increased solubilized strontium, suggesting strontium chelation potential. Since in previous in vivo studies both compounds were also effective in the removal of strontium following internal contamination, it is concluded that the octanol-water system may be useful screening compounds with strontium chelation potential.

In vitro and in vivo evaluations of Chinese traditional (kampo) medicines as anticalculus agents in the rat.

Inhibitory effects of two Chinese traditional (Kampo) medicines, Shigyaku-san and Shikunshi-to, on the in vitro formation of calcium phosphate precipitates and the in vivo deposition of supragingival dental calculus were studied. Both had inhibitory effects on hydroxyapatite transformation and induction; effects of Shigyaku-san and Shikunshi-to were 1/100 and 1/400 of that of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP). When used topically (in drinking water) for 4 weeks, 20-40 mg/ml of Shigyaku-san or Shikunshi-to reduced the formation of dental calculus by 40-60%, while 0.03% (w/v) HEBP decreased it by 70-98%. However, when these drugs were given by gastric intubation for 2 weeks, they were all ineffective, suggesting that the observed anticalculus effects were of topical rather than of systemic origin.

Effects of the Ca2+ chelators EGTA and EDTA on ethanol- or stress-induced gastric mucosal lesions and gastric secretion.

Ca2+ modulates gastric function and dysfunction as well as the release of cysteine proteases and metalloproteinases which have been implicated in the pathogenesis of gastric mucosal lesions. We thus tested the hypothesis that pretreatment with the Ca2+ chelators, ethylene diamine tetraacetic acid (EDTA) and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) might reduce the experimental gastric mucosal damage induced by restraint cold stress or 1.0 ml of 75% ethanol. Other rats were prepared with chronic indwelling gastric cannulas and the effects of EDTA and EGTA on conscious basal gastric acid output were assessed. In addition, rats were pretreated with EGTA or EDTA prior to pylorus ligation and their effects on acid and pepsin output assessed. Both EDTA and EGTA reduced significantly the extent of ethanol-induced gastric mucosal damage as well as the degree of stress-induced gastric lesions. To further characterize the mechanism of Ca2+ chelator protection against ethanol-induced gastric lesions, some rats were vagotomized or adrenalectomized prior to treatment with EGTA or EDTA, followed by ethanol or stress. Both adrenalectomy and vagotomy abolished gastroprotection by EGTA and slightly reduced that induced by EDTA in both models of experimental gastric mucosal injury. Both EDTA and EGTA reduced significantly basal gastric acid output, an effect which persisted for at least 2 h following their administration. Both compounds also decreased significantly acid and pepsin output in pylorus-ligated rats. We conclude that Ca2+ chelators attenuate both acid-dependent and acid-independent gastric lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of effectiveness of several chelators in removing internally deposited strontium from mice following repeated parenteral strontium administration.

Diethylenetriaminepentaacetic acid (DTPA), ethylenglycolbis-(beta-amino-ethylether)-N,N-tetraacetic acid (EGTA), tartaric acid, KRYPTOFIX 222, and KRYPTOFIX 5 were evaluated for their efficacy in mobilization of strontium from the body of mice which had received 20 sc injections of strontium nitrate (95 mg/kg/injection) for 4 w. Twenty-four hours after the last strontium injection, ip administration of 1 of the various chelators or 0.9% saline was initiated and continued daily for 5 d. Mice were housed in metabolic cages, and urine and feces were collected daily for 5 d. After this period, the animals were killed and tissues removed. Tartaric acid, KRYPTOFIX 222, and KRYPTOFIX 5 had no effect on urinary or fecal strontium elimination, whereas DTPA and EGTA significantly decreased the fecal strontium excretion. The concentration of strontium in bone was only lowered in tartaric-treated mice. This study indicates the use of the above chelators is not an effective treatment to enhance the removal of strontium following repeated parenteral strontium administration.

Evaluation of the effects of chelation therapy with time following strontium exposure to mice.

The effect of increasing the time interval between strontium exposure and chelation therapy was studied in male Swiss mice. Diethylenetriaminepentaacetic acid (DTPA), ethyleneglycol-bis-(beta-amino-ethylether)-N,N'-tetraacetic acid (EGTA), 4,7,13,16,21,24-hexaoxa-1,10-diazabycyclo[8.8.8]-hexacosane (Kryptofix 222), tartaric acid, and 1,13-bis(8-chinolyl)-1,4,7,10,13-pentaoxatridecan (Kryptofix 5) were administered intraperitoneally at 0, 6, 12, and 24 h after subcutaneous injection of 1,139 mg/kg of strontium nitrate. Chelating agents were given at doses equal to one-fourth of their respective LD50 values. Daily elimination of strontium into urine and feces was determined for five days after which time the animals were killed, and the concentration of strontium was determined in various tissues. Only Kryptofix 222 was capable of increasing the total urinary elimination of strontium when given immediately after strontium exposure, whereas the amount of total strontium excreted into the feces was significantly enhanced by treatment with EGTA at 0 or 24 h after strontium injection, or with Kryptofix 222 at 6 h after strontium exposure. However, Kryptofix 222, tartaric acid, and Kryptofix 5 reduced the concentration of strontium in bone at 0, 6, or 12 h after strontium injection, whereas at 24 h only Kryptofix 5 significantly lowered the concentration of the metal in bone. The results of this study indicate that the length of time before initiating chelation therapy for strontium removal may influence remarkably the effectiveness of this therapy.

Treatment of reperfusion injury with intracoronary calcium channel antagonists and reduced coronary free calcium concentration in regionally ischemic, reperfused porcine hearts.

The effect of intracoronary diltiazem, EGTA (ethylene-bis-(beta-aminomethylether)-N,N'-tetraacetic acid), nifedipine, verapamil and isotonic saline solution as placebo on reperfusion injury was investigated in regionally ischemic, reperfused porcine hearts. The left anterior descending coronary artery was distally occluded for 45 min and was reperfused for 3 days. Intracoronary infusion was started immediately before reperfusion and continued during 45 min of reperfusion. Infarct size was determined as the ratio of infarcted (tetrazolium stain) to ischemic myocardium (dye technique). Regional systolic shortening was assessed by sonomicrometry. Apart from left ventricular end-diastolic pressure before ischemia and during 45 min of reperfusion, global hemodynamic values in the five treatment groups did not differ; in particular, calculated left ventricular oxygen consumption before and during ischemia was equally low. Intracoronary EGTA decreased coronary venous free calcium concentration to about 70% of baseline value. Infarct size was reduced from 76 +/- 10% (control group, n = 8) to 60 +/- 10% (p less than 0.01) by intracoronary diltiazem (n = 8) and to 55 +/- 15% (p less than 0.01) by intracoronary EGTA (n = 8). Insignificant reductions in infarct size were found after treatment with intracoronary verapamil (63 +/- 18%, n = 8) and intracoronary nifedipine (68 +/- 9%, n = 7). Regional systolic shortening of the risk region, which did not differ among the groups before occlusion and during ischemia, recovered to the greatest extent in the EGTA-treated pigs (p less than 0.01 compared with values in the control group). Treatment with intracoronary calcium antagonists resulted in only marginal improvement of systolic shortening.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipase A2 inhibitors and their possible clinical use in the treatment of acute pancreatitis.

A laboratory method was established for measurement of phospholipase A2 activity in buffer and serum. A series of different phospholipase A2 inhibitors was tested. The most effective inhibitors were Ca2+ chelating compounds like EDTA, DTPA, EGTA, and phytic acid. The calcium salt of EDTA also has some inhibitory effect. Serum phospholipase A2 activity in normal healthy control patients was measured. The activity in 27 patients with acute pancreatitis was tested. The activity was abnormally high in five patients. This activity was in vitro inhibited by EDTA and partly by CaNa2EDTA. The clinical picture of these patients did not differ from that of phospholipase-A2-negative patients. Six patients with acute pancreatitis were treated by intravenous infusion of CaNA2EDTA. Two of them had haemorrhagic pancreatitis and two were suspected of having early haemorrhagic pancreatitis. During the CaNa2EDTA infusion serum amylase and phospholipase A2 activities decreased. All patients recovered. No harmful side effects were noticed.

Methylene blue and other agents as inhibitors of calcium oxalithiasis in vivo.

Growth of calcium oxalate on an established calculus upon a zinc nucleus in the bladder of rats was studied. Animals were fed either a regular solid chow or chow containing a potential crystal inhibitor ad libitum, along with drinking water containing 0.75 per cent ethylene glycol. Chow containing 0.2 per cent methylene blue and 0.5 per cent vitamin C not only decreased the growth rate, but calculi were much softer than those in controls. Safranin O was the only other significant growth inhibitor identified. Ethylenediamonotetraacetic acid and ethylenebis (oxyethylenenitrilo)-tetraacetic acid transformed the additional growth from the mono- to the dihydrate form of calcium oxalate.