MMP-9 inhibitors impair learning in spontaneously hypertensive rats.

Vascular cognitive impairment dementia (VCID) is a major cause of cognitive loss in the elderly. Matrix metalloproteinases (MMPs) are a family of proteases involved in remodeling the extracellular matrix in development, injury and repair. Blood-brain barrier (BBB) disruption due to inflammation mediated by MMPs is a mechanism of white matter injury. Currently there are no treatments besides the control of vascular risk factors. We tested two MMP-9 inhibitors that improved outcome in acute stroke: DP-460 and SB-3CT. We hypothesized that these inhibitors would have a beneficial effect in chronic stroke by reducing edema in white matter and improving behavioral outcomes. Spontaneously hypertensive stroke-prone rats (SHRSPs) with unilateral carotid artery occlusion (UCAO) fed a Japanese Permissive Diet (JPD) were used as a model of VCID. JPD was begun in the 12th week of life. Rats were treated with DP-460 (500 mg/kg) for 4 weeks, or SB-3CT (10 mg/kg) for 8 weeks, beginning at the UCAO/JPD onset. Rats treated with a dextrose or DMSO solution served as vehicle controls. Naïve SHRSPs on a standard diet served as sham control. Magnetic resonance imaging (MRI) analyses of the corpus callosum, external capsule, hippocampus and Morris water maze behavioral tests were conducted. We found an increase in body weight (p = 0.004) and blood pressure (p = 0.007) at 15 weeks with the DP-460 drug. SB-3CT increased body weight at 14 weeks (p = 0.015) and had significant but variable effects on blood pressure. Neither drug affected imaging parameters. Behavioral studies showed an impaired ability to learn with DP-460 (p<0.001) and no effect on learning with SB-3CT. Unchanged MMP-9 levels were detected in DP-460-treated rats via gel zymography. Our findings suggest that MMPs are not major factors in white matter damage in the SHRSP model of VCID and that drugs that are relatively selective for MMP-9 can interfere with learning.

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.

Quantitative analysis of the effect of irrigant solution sequences on dentin erosion.

OBJECTIVE: The purpose of the study was to examine the level of erosion on root canal wall dentin caused by immersion in different irrigant solutions in alternative sequences. METHODS: Dentin specimens from teeth with one root canal were instrumented and randomly divided into five groups. Each group was subjected to 17% EDTA, 17% ethyleneglycoltetraacetic acid (EGTA), or 10% citric acid (CA) and 5.25% sodium hypochlorite (NaOCl) varying the time of irrigant exposure and the order of the irrigants. The specimens were examined by scanning electron microscopy (SEM), and randomized digital images of the dentin surface were taken. The area of tubule openings was measured by a semiautomatic method. RESULTS: No erosion was detected when demineralizing agents were used as a final rinse after NaOCl. However, the erosion of peritubular and intertubular dentin was detected when EDTA, EGTA, or CA were used first followed by 5.25% NaOCl (P < .05), and an increase over 100% in the area of dentin tubule openings was measured (P < .01). CONCLUSIONS: NaOCl used as a final irrigant solution after demineralization agents causes marked erosion of root canal dentin.

[Importance of selective A1 agonists for the protection of brain and heart against injuring factors].

Powerful selective A1 agonist N6-cyclopentyladenosine (CPA) effectively protects the brain (upon decapitation) and the heart (upon intoxication by KCl or ethylen glycol tetra acetate (EGTA)) against the action of injuring factors on experimental animals. CPA weakens or removes damages and/or cell death and probably promotes the regeneration of tissue structures and restoration of their functions. Thus, CPA increases the tolerance of the heart and brain with respect to the introduction of two strong toxicants and even upon decapitation. CPA and 5'-N-ethyl carboxamide adenosine (NECA) induce profound hypothermia, which also takes part in the protection. Selective agonists exhibit two different protective effects against injuring factors.

Calcium loss from root canal dentin following EDTA, EGTA, EDTAC, and tetracycline-HCl treatment with or without subsequent NaOCl irrigation.

The aim of this study was to determine the extent of calcium removal on root canal dentin after 17% EDTA, 17% EGTA, 15% EDTAC, and 1% tetracycline-HCl treatment; with or without subsequent use of 2.5% NaOCl. Extracted single-rooted human teeth were bisected longitudinally and the root halves (n=100) were isolated with nail varnish, leaving the root canal exposed. The samples were immersed in the test solutions for 1 and 5 minutes, after which the amount of calcium ion (Ca(2+)) release into the solutions was determined by flame photometry. Regardless of treatment time, all single (treatment solution only) and combined (treatment solution with subsequent NaOCl application) irrigation regimens removed significantly more Ca(2+) than control treatment (distilled water). Compared with other groups, treatment with 17% EDTA and 17% EDTA + 2.5% NaOCl resulted in the maximum amount of Ca(2+) removal from root canal dentin (p<0.05). All combined-treatment groups except 17% EGTA + 2.5% NaOCl removed significantly more Ca(2+) than their single-treatment versions (p<0.05). Within each test group, extending the treatment time to 5 minutes resulted in significantly more Ca(2+) removal (p<0.05).

Parathyroid hormone 7-84 induces hypocalcemia and inhibits the parathyroid hormone 1-84 secretory response to hypocalcemia in rats with intact parathyroid glands.

Biologic effects of large C-terminal parathyroid hormone (PTH) fragments, opposite to those of N-terminal PTH, have been demonstrated. C-terminal PTH fragments are co-secreted with N-terminal PTH from the parathyroids. The aim of our study was to examine whether C-terminal PTH 7-84 regulates secretion of PTH 1-84 and affects the expression of genes of relevance for parathyroid function, PTH, calcium-sensing receptor (CaR), PTH type 1 receptor (PTHR1), and PTH-related peptide (PTHrP) genes in rat parathyroid glands. PTH 7-84 induced a significant decrease in plasma Ca2+ in rats with intact parathyroid glands. Despite the reduction of plasma Ca2+, no stimulation of PTH 1-84 secretion took place. Furthermore, the PTH 1-84 secretory response to EGTA-induced acute and severe hypocalcemia was significantly inhibited by PTH 7-84. During recovery from hypocalcemia, plasma Ca2+ levels were significantly lower in the PTH 7-84-treated group, as compared with the vehicle group, and at the same time plasma PTH 1-84 levels were significantly suppressed. The expression of PTH, CaR, PTHR1, and PTHrP genes in the rat parathyroid glands was not affected by PTH 7-84. The peripheral metabolism of PTH 1-84 was not affected by PTH 7-84. PTH 7-84 did not cross-react with the rat bioactive PTH 1-84 assay. In normal rats with intact parathyroid glands, PTH 7-84 inhibited the PTH 1-84 secretory response to hypocalcemia and induced a significant decrease in plasma Ca2+. These effects of PTH 7-84 on PTH 1-84 secretion and on plasma Ca2+ levels were not associated with significant changes in PTH, PTHR1, CaR, and PTHrP gene expressions in the rat parathyroid glands. It is hypothesized that PTH 7-84 regulates PTH secretion via an autocrine/paracrine regulatory mechanism.

Clinical pharmacology of DP-b99 in healthy volunteers: first administration to humans.

AIMS: To investigate the safety, tolerability and pharmacokinetics of DP-b99 in healthy volunteers. DP-b99 is a newly developed lipophilic, cell permeable derivative of BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), which selectively modulates the distribution of metal ions in hydrophobic milieu, and is in clinical development as a neuroprotectant for cerebral ischaemic stroke. To our knowledge no BAPTA derivative has ever been administered to man. Here we report the first human administration of DP-b99 in a phase I, two-part, double-blind, randomized placebo controlled study, with single IV doses of 0.003-1.0 mg kg(-1) day(-1) DP-b99 (part 1) or multiple ascending doses of 0.03-1.0 mg kg(-1) day(-1) DP-b99 over 4 days (part 2). METHODS: A double-blind, dose escalating tolerability study of DP-b99 in normal (young - aged between 18 and 40 years and elderly - aged between 65 and 85 years) healthy adult male volunteers was conducted. Part 1 of the study investigated single administration of ascending intravenous doses, and part 2 examined the effects of ascending doses given repeatedly over 4 days. Twenty-four young volunteers in part 1 received single dose administrations and 26 young volunteers in part 2 received repeated ascending dose administrations of either intravenous DP-b99 or placebo. Subsequently, 10 elderly volunteers received repeated intravenous DP-b99 (1 mg kg(-1)) or placebo in part 2 over 4 days. Adverse events were identified by either subject self reporting or based upon laboratory parameters (blood chemistry, complete blood cell count, prothrombin time (PT), activated partial thromboplastin (PTT), physical examination, vital signs (blood pressure, pulse rate, respiratory rate, body temperature) and urinalysis. A comprehensive set of cardiovascular parameters was assessed as well (blood pressure, 12 lead-ECG recordings and continuous bedside cardiac monitoring for 6 h on day 1). RESULTS: The administration of DP-b99 up to the highest dose of 1.0 mg kg(-1) was well tolerated and had an acceptable safety profile up to the highest dose of 1.0 mg kg(-1) tested in both study parts. No serious or severe adverse events were encountered. Eight mild to moderate adverse events were observed in six of the seven young subjects treated with four repeated doses of 1.0 mg kg(-1), with reversible phlebitis being the most frequently reported adverse event. The drug was tolerated better at the injection site by the elderly group compared with the younger subjects. No adverse effects were observed in cardiovascular parameters sensitive to trans-membranous calcium concentrations. The pharmacokinetic parameters were derived by noncompartmental analysis. On day 1 following administration of 1 mg kg(-1) the mean half-life of DP-b99 in young volunteers was 3.47 +/- 0.90 h and in the elderly was 2.11 +/- 0.09 h. On day 4 following the same administration of DP-b99 the mean half-life was 4.36 +/- 1.49 and 2.10 +/- 1.14 h in the young and elderly, respectively. There was higher systemic exposure in the elderly, for example C(max), had a mean 1.6-fold higher exposure on day 1 (95% CI Lower 0.90, Upper 2.74) and 2.5-fold on day 4 (95%CI 1.70, 3.68). This increase is in line with the presumed central role of hepatic blood flow in the elimination of DP-b99. No accumulation was observed after repeated dosing with 1 mg kg(-1) (mean accumulation calculated by AUC(0, 24 h) (day 4) : AUC(0, 24 h) (day 1) and was observed to be between 0.9 and 1.3 (young, elderly). CONCLUSIONS: This study suggests that DP-b99 is well tolerated in healthy young and elderly volunteers within the dose range evaluated. Studies to investigate further the efficacy of the compound are in progress.

Elevated plasma cortisol during induced and spontaneous hypocalcemia in ruminants.

The relationship of cortisol in blood plasma with plasma calcium and phosphorus was studied from 3 days before to 2.5 days after calving in 12 dairy cows (third or more parity). Cows were in three groups: 1) paretic (displayed hypocalcemic and lateral recumbency), 2) nonparetic (plasma calcium at least 8.0 mg/100 ml), and 3) borderline (plasma calcium less than 8.0 mg/100 ml). Cortisol concentrations from 0 to 1.5 days postpartum reflected the state of calcium stress of the groups, paretic more than borderline and borderline more than nonparetic. Phosphorus was lower from 0 to 1 day postpartum in paretic cows. Calcium and phosphorus were negatively correlated (within cow) with cortisol (-.53, -.37). In experiments with goats, cortisol was released in response to hypocalcemia and displayed no activity in initiating an onset of hypocalcemia when given exogenously. Also, the observation that cortisol-treated goats responded less severely with calcium depression and recovered faster from induced hypocalcemia by ethylene glycol-bis (beta-amino-ethyl ester) N,N'-tetraacetic acid infusions suggests cortisol may aid the animal in recovering from hypocalcemia.