BAPTA, a calcium chelator, neuroprotects injured neurons in vitro and promotes motor recovery after spinal cord transection in vivo.

AIM: Despite animal evidence of a role of calcium in the pathogenesis of spinal cord injury, several studies conducted in the past found calcium blockade ineffective. However, those studies involved oral or parenteral administration of Ca++ antagonists. We hypothesized that Ca++ blockade might be effective with local/immediate application (LIA) at the time of neural injury. METHODS: In this study, we assessed the effects of LIA of BAPTA (1,2-bis (o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid), a cell-permeable highly selective Ca++ chelator, after spinal cord transection (SCT) in mice over 4 weeks. Effects of BAPTA were assessed behaviorally and with immunohistochemistry. Concurrently, BAPTA was submitted for the first time to multimodality assessment in an in vitro model of neural damage as a possible spinal neuroprotectant. RESULTS: We demonstrate that BAPTA alleviates neuronal apoptosis caused by physical damage by inhibition of neuronal apoptosis and reactive oxygen species (ROS) generation. This translates to enhanced preservation of electrophysiological function and superior behavioral recovery. CONCLUSION: This study shows for the first time that local/immediate application of Ca++ chelator BAPTA is strongly neuroprotective after severe spinal cord injury.

Results of Membrane-Activated Chelator Stroke Intervention randomized trial of DP-b99 in acute ischemic stroke.

BACKGROUND AND PURPOSE: DP-b99, a lipophilic moderate-affinity chelator of zinc, was postulated to improve recovery after acute ischemic stroke. We evaluated the safety and therapeutic effects of DP-b99 in patients with acute hemispheric ischemic stroke. METHODS: The Membrane-Activated Chelator Stroke Intervention trial was a randomized, double-blind, placebo-controlled, multicenter, parallel-group trial of intravenous DP-b99 administered for 4 consecutive days (NCT00893867). Acute ischemic stroke patients within 9 hours of onset, but untreated by alteplase, with a baseline National Institutes of Health Stroke Scale score of 10 to 16, and evidence of language dysfunction, visual field defect, and neglect were eligible. The primary efficacy analysis compared distributions of functional status measured by modified Rankin score in the intent-to-treat population of patients with any post-treatment outcome, adjusted for initial severity. Functional and neurological recovery were secondary measures. Home time was an exploratory end point. RESULTS: Enrollment terminated at n=446 after the planned interim analysis determined futility; follow-up continued. Final modified Rankin score distributions were equal between DP-b99 and placebo-treated groups (P=0.10; P(adj) adjusted for baseline age and National Institutes of Health Stroke Scale=0.21). Fewer patients recovered to modified Rankin score ≤1 in the DP-b99-treated group (45/218; 20.6%) than after placebo (63/219; 28.8%) (P=0.05; P(adj)=0.10). Similarly, fewer patients attained National Institutes of Health Stroke Scale ≤1 after DP-b99 (42/218; 19.3%) than placebo (56/219; 25.6%; P=0.10; P(adj)=0.26). Mortality was similar between DP-b99 and placebo intent-to-treat groups (36/218; 16.5% vs 33/219; 15.1%; P=0.68). Home time was unchanged by treatment (median 36 vs 36.5 days; P=0.25). CONCLUSIONS: Despite encouraging preclinical and phase II trial data, DP-b99 shows no evidence of efficacy in treating human ischemic stroke.

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.

The Membrane-Activated Chelator Stroke Intervention (MACSI) Trial of DP-b99 in acute ischemic stroke: a randomized, double-blind, placebo-controlled, multinational pivotal phase III study.

RATIONALE: Zinc is both a direct neurotoxin and a signaling mediator in multiple early and late detrimental processes following ischemia. DP-b99, a lipophilic moderate-affinity chelator of zinc, is a first-in-class multitargeted neuroprotective agent for ischemic stroke. DP-b99 has completed several Phase I studies and two double-blind placebo-controlled Phase II trials, which supported the safety of DP-b99 and were consistent with a beneficial effect on poststroke recuperation. AIM: Membrane-Activated Chelator Stroke Intervention is a Phase III study. The primary objective is to evaluate the safety and therapeutic effects of intravenous 1.0 mg/kg/day DP-b99, initiated within nine-hours of stroke onset in patients with moderately severe hemispheric acute ischemic stroke, through the analysis across the whole distribution of scores of the primary efficacy endpoint of the modified Rankin Scale, 90 days after the stroke. METHODS: The Membrane-Activated Chelator Stroke Intervention study is a randomized, double-blind, placebo-controlled, multicenter, multinational, parallel-arm trial comparing a placebo group to a group treated with intravenous DP-b99 for four consecutive days. Non-rtPA-treated acute ischemic stroke patients--with a baseline NIHSS score of 10-16 and a clinical syndrome that includes language dysfunction, visual field defect and/or neglect--will be stratified on a 1:1 basis to one of the two treatments. Half will be randomized within 0-4.5 h of stroke onset. Follow-up after the four treatment days will occur on days 12, 30 and 90. An interim futility analysis will be performed after primary endpoint data have been collected for 50% of 770 subjects planned to be enrolled. A data and safety monitoring board will assess safety data and will oversee the interim analysis. CONCLUSION: This Phase III Membrane-Activated Chelator Stroke Intervention trial is based on promising data derived from previous Phase I and II DP-b99 trials and capitalizes on lessons learned from failures of past stroke studies in relation to neuroprotection, patient selection and data analysis.

The role of metals in modulating metalloprotease activity in the AD brain.

Biometals such as copper and zinc have an important role in Alzheimer's disease (AD). Accumulating evidence indicates that copper homeostasis is altered in AD brain with elevated extracellular and low intracellular copper levels. Studies in animals and cell cultures have suggested that increasing intracellular copper can ameliorate AD-like pathology including amyloid deposition and tau phosphorylation. Modulating copper homeostasis can also improve cognitive function in animal models of AD. Treatments are now being developed that may result in redistribution of copper within the brain. Metal ligands such as clioquinol (CQ), DP-109 or pyrrolidine dithiocarbamate (PDTC) have shown promising results in animal models of AD, however, the actual mode of action in vivo has not been fully determined. We previously reported that CQ-metal complexes were able to increase intracellular copper levels in vitro. This resulted in stimulation of phosphoinositol-3-kinase activity and mitogen activated protein kinases (MAPK). Increased kinase activity resulted in up-regulated matrix metalloprotease (MMP2 and MMP3) activity resulting in enhanced degradation of secreted A beta. These findings are consistent with previous studies reporting metal-mediated activation of MAPKs and MMPs. How this activation occurs is unknown but evidence suggests that copper may be able to activate membrane receptors such as the epidermal growth factor receptor (EGFR) and result in downstream activation of MAPK pathways. This has been supported by studies showing metal-mediated activation of EGFR through ligand-independent processes in a number of cell-types. Our initial studies reveal that copper complexes can in fact activate EGFR. However, further studies are necessary to determine if metal complexes such as CQ-copper induce up-regulation of A beta-degrading MMP activity through this mechanism. Elucidation of this pathway may have important implications for the development of metal ligand based therapeutics for treatment of AD and other neurodegenerative disorders.

The lipophilic metal chelators DP-109 and DP-460 are neuroprotective in a transgenic mouse model of amyotrophic lateral sclerosis.

One of the hypotheses for the development of familial amyotrophic lateral sclerosis (ALS) is that mutations in the superoxide dismutase 1 enzyme lead to aberrant properties of the copper within the active site of the enzyme which then causes increased oxidative damage. The lipophilic metal chelators DP-109 and DP-460 which chelate calcium, copper, and zinc were tested in the G93A-transgenic ALS mouse model. Both compounds significantly extended survival, DP-109 (5 mg/kg/day) by 10%, DP-460 (10 mg/kg/day) by 9%. While the effect on survival was relatively small, chelator treatment also improved motor performance, dramatically reduced cell loss in the lumbar spinal cord and decreased reactive astrocytosis and microgliosis. Markers of oxidative damage, tumor necrosis factor (TNF)-alpha and alpha-synuclein were reduced in the lumbar spinal cord of G93A mice treated with DP-109 or DP-460 as compared with vehicle-treated animals. Furthermore, the treatment induced protein expression of the transcription factor hypoxia inducible factor-1alpha and mRNA levels of vascular endothelial growth factor as a corresponding target gene. In line with previous studies using metal chelators in the G93A animal model, our results suggest that these compounds have neuroprotective capacities in ALS.

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.

Cell-permeant calcium buffer induced neuroprotection after cortical devascularization.

An excitotoxic cascade resulting in a significant intracellular calcium load is thought to be a primary mechanism leading to neuronal death after ischemia. One way to protect neurons from injury is through the use of cell-permeant calcium buffers. These molecules have been reported to be neuroprotective via their ability to increase the cell's overall Ca(2+) buffering load as well as by attenuating neurotransmitter release. However, their efficacy when given after injury has yet to be determined. We used diffusion-weighted magnetic resonance imaging (DWI), histological, and immunohistochemical methods to determine the neuroprotective efficacy of 2-aminophenol-N, N, O-triacetic acid acetoxymethyl ester (APTRA-AM) after focal cerebral ischemia. Injured animals were given two injections of APTRA-AM at 1 and 12 h after injury. Animals were imaged prior to injury and then at 12, 24, 48 h and 3 and 7 days after injury. After 7 days the animals were euthanized for correlative cresyl violet histology and immunohistochemistry. Injury resulted in a decrease in the apparent diffusion coefficient (ADC) of the injured area within the first 12 h of injury, which returned to normal by 7 days. In contrast, animals injected with APTRA-AM showed no significant change in the ADC at any time point studied. Tissue analysis showed that APTRA-AM significantly reduced the infarct size by 85% and extent of inflammatory cell infiltration by 94%. The results clearly demonstrate significant neuroprotection by APTRA-AM when given after injury.

Effectiveness of ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA) against cerium toxicity.

Therapeutic efficacy of EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid) against cerium intoxicated mice was studied. Administration of cerium showed significant decrease in haemoglobin percentage, RBC counts and blood glucose level with an increase in the activity of serum transaminases and WBC counts. Decrease in the activity of alkaline phosphatase and glycogen content was noted in liver and kidney after cerium exposure. Light and electron microscopical investigations showed that these changes were recouped considerably with the administration of EGTA suggesting its therapeutic efficacy against cerium toxicity.

Effects of an intracellular Ca(2+) chelator on insulin resistance and hypertension in high-fat-fed rats and spontaneously hypertensive rats.

We explored the possibility that a sustained elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) may be a cellular abnormality common to both insulin resistance and hypertension. In high-fat diet (HFD) fed rats, the steady-state glucose infusion rate (GIR) during the euglycemic hyperinsulinemic clamp was reduced by 40% (P <.05) and mean arterial pressure (MAP) was elevated by 20 mm Hg (P <.01) in comparison to the normal chow-fed rats. Intravenous injection of 5,5'-dimethyl derivative of bis(o-aminophenoxy)ethane-N,N,N',N' tetraacetic acetoxymethyl ester (dimethyl-BAPTA/AM), an effective intracellular Ca(2+) chelator, 90 minutes before the clamp not only restored about 50% of the reduced GIR, but also normalized MAP in the HFD rats. The chelator injection also significantly increased GIR by 25% (P <.01) and reduced MAP about 30 mm Hg (P <.01) in the spontaneously hypertensive rats (SHR). In addition, we have recently shown in the HFD rats that an injection of dimethyl-BAPTA/AM normalizes elevated [Ca(2+)](i) in adipocytes. These results together demonstrate that lowering [Ca(2+)](i) simultaneously ameliorates both insulin resistance and hypertension and provide presumptive evidence that sustained high levels of [Ca(2+)](i) may play a common pathophysiologic role in these 2 diseases.

DP-b99 (D-Pharm).

DP-b99 is a derivative of the calcium chelator BAPTA that is under development as a neuroprotectant for the potential treatment of stroke, head trauma and neurological damage associated with coronary artery bypass graft. By March 2003, phase II clinical trials in acute stroke and traumatic brain injury were ongoing.

Effects of correction of hypocalcemia on cardiac function in the newborn lamb.

The effect of correction of hypocalcemia on cardiac function in the neonate is unclear. Therefore, we evaluated changes in hemodynamic variables and cardiac contractility in a hypocalcemic neonatal lamb model. Baseline serum ionized hypocalcemia was induced via chelation with intravenous EGTA. Cardiac contractility, measured as end-systolic elastance, did not change with correction of hypocalcemia in this model. In contrast, the variable dP/dt(max), a load-dependent index of contractility, showed an increase with calcium administration while the end-diastolic volume decreased, suggesting that diastolic function or early systolic function had changed. Systemic vascular resistance, cardiac output, and heart rate did not change significantly with correction of hypocalcemia. The results of this study suggest that in our newborn lamb model, cardiac contractility is insensitive to ionized calcium and may reflect cardiac immaturity, but that there may be an effect of calcium administration on diastolic or early systolic function.

Neuroprotection in vitro and in vivo by cell membrane-permeant Ca2+ chelators.

1. A method of attenuating excitotoxic and hypoxic/ischaemic neurodegeneration in vitro and in vivo using cell-permeant Ca(2+)-chelating agents is described. 2. The mechanism of neuroprotection may depend on both pre- and post-synaptic effects on intracellular Ca2+ dynamics. 3. This method is unique, because it targets Ca2+ ions, the presumed triggers of neurodegeneration, rather than a specific cellular receptor.

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.