In vitro Research Concerning Effect of Clopidogrel Alone and on Combination with Aspirin and Dypiridamoleon Sedimentation of Erythrocytes.

Based on extended theory of Derjaguin, Landau and Overbeeck (xDLVO) concerning aggregation of colloids and biological cells it was hypothesized that platelet antiaggregant agents have to reduce the aggregation of erythrocytes also. Applying Einstein-Stokes theory of sedimentation of spheres in viscous media it was concluded that sedimentation of erythrocytes is in fact sedimentation of aggregates of the approximately same size. Consequently, an expected outcome was that addition of antiaggregants in vitro to blood samples from patients with rheumatic or cardiovascular diseases will be the decrease of erythrocytes sedimentation. Starting from usual practice of dual antiaggregant therapy (aspirin and clopidogrel) effects of clopidogrel were compared with effects of clopidogrel plus small concentrations of aspirine and dipyridamole (smaller that their concentrations in plasma after in vivo administration) in order to put in evidence a possible synergic effect at platelet membrane level. Whole blood (0.8ml) was collected on 0.1ml 1% EDTA and then was added 25 or 50µl normal saline solution of clopidogrel or of the combination acetylosalycilic acid, clopidogrel and dypyridamole. The final concentrations were 1, 2 and µg/ml, of the same order as cumulated concentration of clopidogrel and its metabolites in clinical pharmacokinetics. Experiments were performed on a number of 40 human blood samples obtained from 2 groups of 20 patients. Sedimentation of erythrocytes was recorded using a camera and captured data were stored on a computer. Global analysis evidenced that in presence of antiaggregants the clusters of the sedimentation curves shifted down and into right, indicating a decrease and delay of sedimentation. Initial slopes and extent of sedimentation decreased linearly on clopidogrel concentration within the 1-3µg/ml range. For comparison of mean curves corresponding to different clopidogrel concentrations it was applied a metric from biopharmacy: areas under plasma concentrations curves (AUC) of drugs. The areas under average sedimentation curves decreased linearly at clopidogrel concentration within the 1-3µg/ml range. The same experiments were performed and similar results were obtained with the triple antiaggregant combination (clopidogrel, acetylosalycilic acid and dipyridamole). Apparently, a synergism between the tested antiaggregants appeared at studied concentration but the number of data was not sufficient to prove the statistical significance of the difference between clopidogrel alone and in triple combination.

In vitro Research of the Concentration Dependence of Effect of Adrenaline on Platelets Aggregation.

PURPOSE: Paper intended to present experimental evidences that adrenaline has a direct effect of inducing platelets aggregation in the concentration range 1-8µM. MATERIAL/METHODS: Platelet rich plasma from patients of Colentina Clinical Hospital, following an informed consent. The platelet rich plasma (PRP) was prepared by centrifuging the anticoagulated sample at 200 G for 10 minutes. Aggregation was evaluated by optical aggregometry, classical method of Born, using Helena PACKS-4 Aggregometer. RESULTS: The curves transmission light-time followed the structure: a lag-time, a first phase aggregation, more or less linear, defined by a "Slope 1", a second wave of aggregation defined by "slope 2" and a "saturation" phase. Slope 1 increases with the concentration of adrenaline. The second slopes of the aggregation curves, maximum aggregation and areas under curves depended linear on adrenaline concentration. CONCLUSIONS: Adrenaline, in concentrations in the 1-8µM, induce aggregation of human platelets from platelet rich plasma. Linear regression models for slope and area were practically identical suggesting a rather unique than biphasic mechanism of action of adrenaline during the time course of aggregation.

Application of Biopharmaceutical and Clinical Metrics in Analyzing Modification of Platelets Aggregation Curves by Calcium Ion.

In context of Evidence Based Medicine concept, Good Clinical Practice rules specify that "data generated should be reliable and robust".Reliability and robustness are further translated in requirements concerning statistical and clinical significance of results. Paper presents main aspectsconnected with comparison of evolutions of endpoints as function of different parameters like time, administered dose, proportion of active components etc., leading to problems of comparison of curves, with direct application to comparison of platelets aggregation curves in presence of different concentrations of ionic calcium. Theoretical part presents comparison of curves in biopharmacyusing f2 metric and area under curve metric, and comparison of survival curves in clinical studies.Platelet aggregation test was performed using Bornturbidimetric light transmission method using Helena PACKS-4 Aggregometer. Blood samples were collected from patients in internal medicine ward of Colentina Clinical Hospital. Platelet rich plasma (PRP) was obtained by centrifugation at 200G. Washed platelets where extracted by centrifugation of PRP at 2700G. The supernatant was replaced with sodium chloride 0.9%. Platelets aggregation was induced by adding different concentrations of calcium gluconate into cuvettes which contained washed platelets. After digitalization, curves were compared using similarity factor f2and areas under curves. Paper puts in evidence that both type of comparison, after mathematical and statistical evaluation, have to define a clinical threshold for clinical significance. In case of f2, in dissolution studies the threshold is 10%, in case of bioequivalence based on area under curves threshold is 20%. Establishment of the threshold for significant clinical difference in comparison of aggregation curves is not only a problem of statistics.Graphical representation of data suggested significant differences between curves obtained with different concentrations of calcium ion. Application of both f2 method and log-rank test let to conclusion that differences were statistical significant. Representation of aria under curves as function of calcium concentration put in evidence an approximate linear dependence. In spite of apparently objective character of mathematical approach, the problem of comparison of aggregation curves remains practically unsolved since we do not know the threshold between clinical significant and non-significant results.

Topicalities in the Sudden Unexpected Death Syndrome in Infants.

Sudden Infant Death Syndrome (SIDS) represents the third cause in postnatal mortality. The pathogenesis is multifactorial. SIDS victims can present sign of preexisting chronic asphyxia, persistent increase in dendritic spine and delayed maturation of synapses in medullary respiratory centers, if a decreased reactivity of 5 hydroxytryptamine 1A (5-HT1A) and 5-HT2A in the dorsal nucleus of the vagus, solitary nucleus and ventrolateral medulla. SIDS is an exclusion diagnosis, so that inexplicable SIDS is the accepted term. The objective of this report is to present current data about the pathogenesis oh this syndrome and the medico-legal measures applied in preventive and curative aim. The "face-up" sleeping position has cut in half the SIDS frequency. In conclusion, the infant sleep studies represent an important line for the future research to provide sufficient explanation of the sudden death in these infants.

Development of a new HPLC method for simultaneous determination of clopidogrel and its major metabolite using a chemometric approach.

This paper presents the development and validation of a new HPLC-UV method for simultaneous quantitative determination of clopidogrel and its hydrolysis product clopidogrel carboxylic acid (CCA) from bulk material and dosage formulations. Development of the chromatographic method is based on a design of experiments (DOE) approach. A Box-Behnken experimental design was used to build the mathematical models and to choose the significant parameters for the optimization by simultaneously taking resolution, capacity factor and peak symmetry as responses. Derringer's desirability function was used for the selection of the optimum experimental conditions in terms of mobile phase composition, column temperature and flow rate. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision, robustness and system suitability. The method was further employed for the study of clopidogrel bisulfate hydrolysis kinetics under different pH conditions, with special emphasis on the acidic hydrolysis studies, since different clopidogrel salts are suffering pre-systemic metabolism trough hydrolysis under the acidic pH of the stomach. This effect is generally difficult to quantify, since CCA is also the main circulating metabolite, and no differentiation between the pre-systemic and systemic CCA can be made. In the acidic environment created by a 0.1N HCl solution CLO degradation was slow at room or body temperature (25 and 37°C respectively), less than 5% of the initial CLO amount being hydrolyzed after 48h. Under forced conditions (85ºC) however, 17.8% of CLO was transformed into CCA within 48 hours.

Sodium Ion Effect on Separation Of Butyrylcholinesterase from Plasma by Ion-Exchange Chromatography.

Purpose of the paper was the optimization of mobile phase in natrium chloride gradient in the chromatographic separation of butyrylcholinesterase from human plasma. MATERIALS/METHODS: Butyrylcholinesterase (BuChE) was isolated from human plasma using a diethylamminoethyl-cellulose column, by elution with 0.02M acetate buffer pH=4.0, gradually increasing NaCl percentage from 10% to 80%. The procedure lasted approximately 28 days. RESULTS: Absorbance of the successive collected fractions at 280 nm presented a maximum at 60 % NaCl concentration. Activity of obtained BuChE was maximum at the same concentration. Another observed effect of NaCl was the decrease of resistance of the column to flow of the elution fluid. In the absence of NaCl the flow rate was 7 mL/h. Increasing of NaCl concentration induced a continuous increase of the flow to a value of 21 mL/h at 60% NaCl solution. After this concentration the flow remained practically constant. The effect on the ionic exchange is essentially an effect on chromatographic partition coefficient, leading, as a rule, to a peak having a Gaussian form. Fitting separately of ascending and descending parts of the apparent peak, led to practically the same exponential coefficient. CONCLUSIONS: Separation of proteins and particularly of BuChE on a chromatographic DEAE Cellulose column can be considered as a method for separation and purification of BuChE from human plasma. Optimum concentration of NaCl is 60 %. Exponential fittings in the neighbourhood of maximum indicated a prevalence of effects of NaCl on the chromatographic partition face to effects on gel-sol equilibrium of stationary phase.

Brief history of syphilis.

Before the discovery of Treponema pallidum as the etiologic agent, the origins of syphilis have been the subject of several debates. Diverse therapeutic agents were employed in an attempt to cure the disease. Examining the milestones in the history of syphilis, the present article reviews the existing theories that tried to explain the origins of the disease, the approach in art, the cultural and the evolution of the treatments from the empiric means to the discovery of penicillin.

Obstetrical trauma--therapeutic dilemmas?

UNLABELLED: Obstetrical trauma is frequent among newborns alive and could represent a cause of perinatal death. The aim of this paper consists of presenting the authors experience regarding the diagnosis and treatment of skeletal and thoracic-abdominal organs related to traumatic birth. MATERIAL AND METHODS: Between 2000 and 2010, 33 patients with trauma at birth were included in the study. The type of the lesion and the therapeutical approach and results were analyzed. RESULTS: Two categories of results were clearly differentiated: excellent for obstetric trauma involving limbs and soft parts and negative, disappointing results with 10 deaths in 12 cases treated for obstetric trauma of abdominal organs. CONCLUSIONS: Bone and soft tissue birth trauma are relatively easy to identify and treat, but the medical approach of thoraco-abdomnial organs birth trauma need clinical experience and technical possibilities.

Pharmacokinetic profile evaluation of di-µ-hydroxo-bis(quercetinatooxovanadium(IV)) complex.

The present study was designed to evaluate the pharmacokinetic (PK) properties of di-µ-hydroxo-bis(quercetinatooxovanadium(IV) (HOBQOV) complex, with hypoglycemic and hypolipidemic properties, in rats after a single i.p. dose of 100 mg/kg b.w. The HOBQOV complex pharmacokinetics is described by a bicompartmental model and the plasma concentration decrease is described by two phases that might be correlated to the distribution and elimination processes. The obtained results suggest a PK profile of the complex suitable for a profound distribution in the animal organism with possibility of interaction with the cytosolic enzymes.

Quality of life of patients with esophageal replacement for congenital and acquired esophageal anomalies.

AIM: To assess the long-term quality of life (QoL) and symptoms experienced by patients who underwent esophageal replacement for congenital and acquired children esophageal pathology. MATERIAL AND METHODS: The study group comprised 71 patients divided into 2 groups, group I: 34 patients who underwent gastric tube esophagoplasty, and group II: 37 patients who underwent colic tube esophagoplasty. QoL score was assessed using a 24-item original questionnaire covering aspects of eating, disease-specific symptoms, physical and psychological development, and social integration. RESULTS: QoL scores based on patient and parental responses were very close. Long-term postoperative functional outcomes for the two groups were comparable. CONCLUSIONS: The QoL scores and general life status were not influenced by the used technique and this pathology should be approached according to the particularity of each case and surgeon's personal experience and preference.

The immediate insertion, loading and provisional prosthetic restoration of dental implants.

The immediate insertion and loading of dental implants although used in the past as an alternative method for the surgical phase of the dental implant treatment is today becoming more and more popular due to its immediate and spectacular results. With information on bone density and a careful patient selection, this method has increased chances of success. Its main advantage is the high degree of patient comfort with a great aesthetic effect. This article presents the theoretical and practical technique used on two clinical cases of dental implantation and their outcome.