Flex center method versus center method for endothelial corneal evaluation in eye banking: a comparative analysis.

Specular microscopy can provide a non-invasive morphological analysis of the cornea endothelial cell layer. A variety of analysis programs are available to determine corneal endothelial quality. The flex-center endothelial analysis method (Konan Inc) is a newer technique including the outermost digitized cells and thus increases the number of cells for analysis. The aim of this study is to analyze whether the new flex-center method, increases the possibilities of corneal endothelium evaluation before implants. For this purpose 67 corneas were studied by both methods at the Eye Bank of the Tissue Establishment of Córdoba. Although we have found differences in the resulting of number of cells in the area analysed, no significant differences were found with respect to the endothelial cell count, coefficient of variation cell area, and the percentage of hexagonal cells recorded. Based on this data, both methods can be used satisfactorily in eye banking.

In vitro susceptibility of high virulence microorganisms isolated in heart valve banking.

Storage preparation of human heart valves for implants generally includes incubation in an antimicrobial disinfection solution and cryopreservation. Changes in patterns of microorganisms susceptibility to antibiotics is a variable process of that promote its inefficiency. The aim of this study has been an evaluation of in vitro susceptibility of high virulence microorganisms isolated in our tissue bank for 14 years in order to evaluate the efficiency, and to promote changes for further antibiotics mixtures as well. Data presented in this study show that microorganisms isolates in valve banking display susceptibility patterns similar to those shown in other clinical circumstances, and the most commonly used antibiotics regimes are useful to date. An antibiotic cocktail containing aminoglicoside in addition to ciprofloxacin and vancomycin is an efficient mixture to be used in valve banking. Further studies will be necessary for monitoring patterns changes of in vitro susceptibility of microbiological isolates in tissue banking.

Ultra-low exposure to α-7 nicotinic acetylcholine receptor partial agonists elicits an improvement in cognition that corresponds with an increase in α-7 receptor expression in rodents: implications for low dose clinical efficacy.

Αlpha-7 neuronal nicotinic receptors (NNRs) are considered targets for cognitive enhancement in schizophrenia and Alzheimer's disease. AZD0328 is an alpha-7 NNR partial agonist that enhances cognition in rodents and nonhuman primates at sub-microgram to microgram doses. We hypothesized that increased expression of the alpha-7 receptor contributes to this beneficial activity at low doses and tested this by examining the effect of AZD0328 using in vivo and ex vivo binding, RT-PCR and cognitive function in rodents. AZD0328 (0.00178 mg/kg) was subcutaneously administered to mice 4, 24, 48 and 72 hours prior to testing in novel object recognition and produced a significant increase in cognition at 4, 24 and 48 h post-dosing. In vivo binding was examined in rat brain using [(3)H]AZ11637326 and there was a dose-dependent reduction in receptor binding at higher doses of AZD0328 (0.001-3 mg/kg), and a second alpha-7 partial agonist, SSR180711 (0.01-30 mg/kg). Lower doses of both compounds (0.0001 mg/kg) produced a significant increase in binding of [(3)H]AZ11637326. Ex vivo binding using [(125)I]-α-bungarotoxin, showed a significant increase in receptor number (B(max.)) in the frontal cortex or hippocampus with no significant effect on receptor affinity (K(d)) 2 h post administration of AZD0328. [(3)H]AZ11637326 administered 1.5 h following AZD0328 produced a significant increase in specific binding in rat brain regions. We found that the effect on receptor number was long-lasting, with [(125)I]-α-bungarotoxin binding increased in rats given AZD0328 for 2-48 h, but this was not accompanied by increased mRNA synthesis. SSR180711 produced a similar increase in B(max.) and specific binding with no effect on K(d). Therefore, trace dose of alpha-7 partial agonists has rapid onset and produces a profound, sustained effect on novel object recognition in mice that corresponds by dose to an increase in receptor number in rat brain. These findings provide an explanation for the acute and sustained benefit of alpha-7 receptor activation in working memory in nonhuman primates and guidance for drug development initiatives and treatment regimens for nicotinic partial agonists.

Implementation of a Quality Plan (ISO 9002) In a Regional Tissue Bank.

Quality control and standardized preservation methods are essential in the field of transplantation. The International Organization for Standardization (ISO) has established a common set of manufacturing, trade and communications standards that are applicable worldwide and that provide the basis of a quality plan for Tissuebreak Banks.The Sectorial Tissue Banking (STB) of the Regional Blood Transfusion Center (RBTC) of Córdoba (Spain) is a non-profit-making tissue bank, established in 1992 to provide tissues for surgical procedures to the hospitals in a regional area. In 1998, the STB as a part of the RBTC embarked upon the path of becoming ISO-certified: after two years of the implementation of the project, STB attained ISO 9002 certification, thus becoming one of the first tissue banks in Europe to achieve this qualification. In this paper we describe the process of becoming ISO-certified, to demonstrate the positive impact that it has had on our entire organization.The assistance of an outside consultant who provided the necessary information for implementing an ISO quality management system was required. The initial improvement was: a well-defined quality manual to address all elements of the ISO 9002 standard, an improved document control system, detailed standard operating procedures (SOPs) and improved employees training processes. A quality committee team and developed quality indicators were created. The internal quality auditing program was established by the selection of employees from a cross-section of the organization, who were trained in internal auditing processes. A formal corrective action system was developed and implemented to facilitate process improvement. The consultant conducted a pre-certification audit, and one month later the certification audit was performed.In conclusion, the implementation of an ISO quality program in the STB has helped our center to establish a control process in the manufacturing of products and services to meet the expectations of our customers, by providing components and services that comply with the national regulatory standards and requirements.

Efficacy of AR-R15896AR in the rat monofilament model of transient middle cerebral artery occlusion.

The monofilament technique of transient middle cerebral artery occlusion (MCAO) was used in 3 separate studies to evaluate the efficacy of the low-affinity, use-dependent N-methyl-d-aspartate receptor antagonist, AR-R15896AR. First, a dose-response curve was attempted. Wister Kyoto rats received 2 hours of MCAO. Five minutes later, a 30-minute intravenous infusion of AR-R15896AR was given, followed by subcutaneous implantation of Alzet minipumps that were calibrated to maintain specified plasma levels (approximately 682, 1885, or 2682 ng/mL) of AR-R15896 (free base) for 1 week. The highest plasma level attained significantly decreased the percentage of damage to the subcortex, cortex, and total brain. Second, the high-dose, 1-week treatment regimen was repeated to determine if neuroprotection would extend to 8 weeks after MCAO. Indeed, in separate groups of animals, significant reduction in the percentage of damage, which was generally confined to the cortex and subcortex, was observed at 1, 2, 4, and 8 weeks. Third, verification was achieved in another laboratory. Lister Hooded rats received 60 minutes of transient MCAO. At 70 minutes, an acute dose of AR-R15896AR (20.3 mg/kg) was injected intraperitoneally and the rats were killed 23 hours later. This treatment group also exhibited significant reduction in the volume of infarction in the subcortex, cortex, and total brain. The outcome of these investigations supports the ongoing Phase II clinical trials in patients with acute stroke.

-(S)-Alpha-phenyl-2-pyridine-ethanamine Dihydrochloride-, a low affinity uncompetitive N-methyl-D-aspartic acid antagonist, is effective in rodent models of global and focal ischemia.

[(S)-Alpha-phenyl-2-pyridine-ethanamine dihydrochloride] (ARL 15896AR) is a low affinity uncompetitive N-methyl-D-aspartic acid receptor antagonist that was tested in animal models of anoxia and ischemia. Pretreatment of rodents with ARL 15896AR extended survival time during exposure to hypoxia. With the rat four-vessel occlusion model of global ischemia (20 min), oral dosing commencing at reflow, resulted in significant protection of the CA1 hippocampal neurons. ARL 15896AR was, however, ineffective in the rat two-vessel occlusion model and in the gerbil models of forebrain ischemia, the latter due to an inability to attain suitable plasma levels. In the spontaneously hypertensive rat model of middle cerebral artery occlusion (MCAO) (2 hr plus 22 hr reflow), acute dosing with ARL 15896AR (i.p.) beginning from 30 min before or up to 1 hr post-MCAO significantly reduced cortical infarct volume. The ability of ARL 15896AR to influence infarct size, as well as functional correlates was examined in SHR after 90 min of MCAO. T2 weighted magnetic resonance images taken at 2 and 6 days post-MCAO revealed significantly smaller lesion sizes in the group receiving injections with ARL 15896AR beginning 30 min after occlusion. Spontaneously hypertensive rats were subsequently tested (30-42 days post-MCAO) and found to be deficient in skilled use of the forepaws (staircase test). The contralateral forepaw was most severely impaired, however, ARL 15896AR treatment prevented motor impairment in only the ipsilateral forepaw. Histopathological examination of cortical infarct size was unremarkable between treated and control rats. The findings indicate that ARL 15896AR exhibits neuroprotection in global and focal models of ischemia

Brain uptake and biotransformation of remacemide hydrochloride, a novel anticonvulsant.

The brain uptake and biotransformation of remacemide hydrochloride [(+/-)-2-amino-N-(1-methyl-1,2-diphenylethyl)acetamide monohydrochloride; FPL 12924AA] were studied in the rat. The brain uptake indices (BUI) for remacemide and its pharmacologically active metabolite FPL12495 [(+/-)-1-methyl-1,2-diphenylethylamine monohydrochloride] were 51 and 130%, respectively. The BUI of [14C] remacemide and [14C]FPL12495 were not affected by increasing amounts of unlabeled remacemide or FPL12495, respectively. Likewise, the BUI of remacemide was not affected by dl-amphetamine or beta-phenethylamine. A mixture of [3H]remacemide hydrochloride (3H label in the glycine moiety) and [14C]remacemide hydrochloride (14C label in 1,2-diphenyl-2-aminopropane moiety) was administered by intracarotid injection. The ratio of 14C/3H in the brain was equal to that in the injection mixture, indicating that remacemide enters the brain intact. HPLC analysis of brain extracts of rats given [14C] remacemide hydrochloride by intracarotid injection revealed that 97.8 +/- 0.2% (mean +/- SD) of the radioactivity was present as remacemide, whereas 1.9 +/- 0.2% of the radioactivity was present as FPL12495. Finally, in vitro studies revealed that remacemide is hydrolyzed by whole-brain homogenates to the pharmacologically active metabolite FPL12495. Data indicate that remacemide enters the brain by passive diffusion and undergoes deglycination at the blood-brain barrier or within the brain to give FPL12495.

Determination of papaverine and cocaine by use of a precipitation system coupled on-line to an atomic absorption spectrometer.

A continuous-precipitation flame-atomization atomic absorption spectrometric method for the determination of papaverine and cocaine hydrochlorides is proposed. The method is based on the precipitation of reineckates by injection of Reinecke's salt into a carrier containing the alkaloids and their subsequent retention on a stainless steel filter. In this way, papaverine and cocaine hydrochlorides can be determine over the ranges 5-85 and 50-850 micrograms ml-1 with a relative standard deviation of 1.3 and 3.2%, respectively, and a sampling frequency of 150 h-1. The proposed method is more sensitive and selective for papaverine than it is for cocaine and can be applied to the determination of papaverine HCl in pharmaceutical preparations.

Automatic determination of amylocaine and bromhexine by atomic absorption spectrometry.

An automatic method for the determination of amylocaine and bromhexine hydrochloride by atomic absorption spectrometry (AAS) is proposed. The drugs were determined indirectly by formation of reineckates, extraction into 1,2-dichloroethane and measurement of chromium in the organic phase. The chemical conditions and experimental variables influencing the performance of the flow system of the liquid-liquid extractor were established. The proposed method allows the determination of amylocaine and bromhexine at concentrations between 3 and 120 micrograms ml-1 with a relative standard deviation of 1 and 3%, respectively, even in the presence of other synthetic drugs.

Quantitation of the novel anticonvulsant remacemide in rat and dog plasma and urine: application of the plasma methodology to measure the plasma protein binding of remacemide.

Sensitive and selective methods have been developed for quantitation of the novel anticonvulsant remacemide in rat and dog plasma and urine. The methods employed liquid-liquid extraction (urine) or ion-exchange solid-phase extraction (plasma), with an internal standard, followed by high-performance liquid chromatography with ultraviolet detection. The detection limit for both methods was 10 ng/ml. Overall accuracy was 0.00% for plasma and -1.4% for urine with a precision of 6.04 and 3.87% for plasma and urine, respectively. The standard curves were linear for both plasma and urine over a wide concentration range (9.96-2490 ng/ml). The plasma method was also applied to measurement of in vitro plasma protein binding of remacemide in rat, dog and human plasma.

Biological profile of the metabolites and potential metabolites of the anticonvulsant remacemide.

Remacemide hydrochloride ((+/-)-2-amino-N-(1-methyl-1,2-diphenylethyl)- acetamide hydrochloride or FPL 1292AA) is a novel compound undergoing clinical trials for patients with generalized tonic/clonic and complex partial epilepsy. Remacemide exhibits efficacy against maximal electroconvulsive shock (MES) in rodents and seizures elicited by N-methyl-D,L-aspartate (NMDLA) in mice. Using rat synaptic membrane fractions, remacemide was shown to possess relatively weak noncompetitive binding to the ionic channel site of the NMDA (N-methyl-D-aspartic acid) receptor complex. With the hypothesis that activity against NMDLA-elicited seizures might be reflected by transformation to a more active metabolic species, the aim of the present study was to evaluate potential pharmacological effects of the 9 identified metabolites of remacemide which were all found in human and dog urine. Moreover, specific entities were recognized in plasma (including the rat's), as well as dog and rat cerebrospinal fluid. Five putative metabolites were also examined. A major route of metabolic transformation of remacemide in rats yields the formation of a pharmacologically active more potent desglycine derivative, namely FPL 12495 (+/-). Potency over the parent compound is revealed in the MES test in mice and rats, the NMDA-induced convulsions/mortality test in mice, and especially involving in vitro displacement of MK801 binding to the channel subsite of the NMDA receptor. The S isomer (FPL 12859) of this desglycinate is even more potent, while the R isomer is less potent than the corresponding racemate. Unlike the non-competitive NMDA antagonist, MK801, these desglycinates did not prevent kindled seizures. Three other identified metabolites show efficacy in the mouse and rat in vivo tests, namely the N-hydroxy-desglycinate (FPL 15053) and the p-hydroxy-desglycinates (FPL 14331 and FPL 14465). FPL 15053 exhibited modest activity in all tests. The only in vivo activity exhibited by the 2 p-hydroxy-desglycinates was evidenced in the MES test following i.p. and i.v. dosing. However, FPL 14331 was active in the MK801 binding assay. An oxoacetate metabolite, PFL 15455, failed to demonstrate any biological activity. Of potential metabolites tested 2 beta-hydroxy-desglycinates (FPL 14991 and FPL 14981) displayed modest activity in the MES test, however, only FPL 14981 prevented NMDLA-induced convulsions/mortality in mice and was 2-fold more active regarding MK801 binding. The hydroxy-methyl derivative of remacemide (FPL 13592) and its desglycinate (FPL 15112) prevented MES-induced convulsions only after i.v. administration; only the desglycine derivative displaced MK801 binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Preliminary methodology for controlled cost-benefit study of drug impact: the effect of cimetidine on days of work lost in a short-term trial in duodenal ulcer.

The phase II/III U.S. clinical drug trials for cimetidine (Tagamet) in duodenal ulcer were examined for their potential application to cost-benefit analysis. Data on "time lost from work" were obtained from a special protocol added to these short-term, double-blind trials of cimetidine. Sixty-four outpatients from the clinical trials remained from an original pool of 217 after exclusion of those subjects who were either retired, unemployed, or of uncertain employment status. Cimetidine was significantly more effective than placebo in reducing "time lost from work" during ulcer disease. The patients' "time lost from work" occurred as a strikingly all-or-nothing phenomenon. We conclude that a prospective clinical trial is appropriate for gathering economic data.

Renal vascular response to heat stress in baboons--role of renin-angiotensin.

To determine if hyperthermia in the baboon caused a reduction of renal blood flow (RBF) similar to that reported in man, we repeatedly exposed six unanesthetized male baboons (Papio cynocephalus) to ambient temperatures of 42.5-49.0 degrees C for 55-175 min. Internal temperatures rose 1.0-2.0 degrees C. On the average, RBF fell 23.7% per degrees C, renal vascular resistance (RVR) rose 34.0% per degrees C, and mean arterial pressure (MAP) fell by only 2.9 Torr. Plasma renin activity (PRA), measured in four baboons, rose 97.5% per degrees C. To investigate the role of the renin-angiotensin system in this renal response, we infused propranolol or saralasin (1-sar-8-ala-angiotensin II), an angiotensin II antagonist, systemically in 14 experiments on three baboons. Both propranolol and saralasin infusions prevented most of the reduction in RBF during hyperthermia. Propranolol prevented the increase in PRA. We conclude that renal vasoconstriction accompanies moderate hyperthermia in the awake baboon, and much of this response is mediated by a beta-adrenergic release of renin.