Intraspecific variability and pharmacokinetic characteristics of Androctonus mauretanicus mauretanicus scorpion venom.

We evaluated the degree of venom toxicity and protein content of several specimens of Androctonus mauretanicus mauretanicus. The quantity of protein of individual venom obtained after manual extraction from 31 different scorpions varied from a minimum of 0.15 mg to a maximum of 1.53 mg. We determined the venom toxicity, in mice, by estimating the number of LD(50)s of 20 scorpions chosen randomly among the 31 scorpions. It ranged from less than 40 LD(50)s to a maximum of 272 LD(50)s. The correlation between protein content and venom lethality is not systematic. We also determined the pharmacokinetics of the venom and its specific anti-venom in rabbits to compare their distribution and elimination properties. After a subcutaneous injection, high concentrations of venom were measured by ELISA in the vascular space rapidly after the injection (T(max) = 0.5 h). The terminal half-life was 2.8 h, close the one determined after intravenous injection (t(1/2beta) = 3.2 h). The total volume of distribution (Vd(ss) or Vd(beta)) was between 317 and 380 ml/kg. The total body clearance was 82 ml/kg/h. For scorpion anti-venom, the terminal half-life, after intravenous injection, was 20.25 h; the volume of distribution was 83 ml/kg and the total body clearance was 3 ml/kg/h. After intramuscular administration, T(max) was reached at 36 h. The results show that venom lethality varies from specimen to specimen and that pharmacokinetic parameters of venom and anti-venom are totally different. This must be taken under consideration in anti-venom production (anti-venom titre) as well as in therapeutic protocols (dose, injection route) to improve serotherapy.

[Clinical evolution and circulating venom levels in scorpion envenomations in Morocco]. / Evolution clinique et taux circulants du venin dans les envenimations scorpioniques au Maroc.

We conducted a clinical and biological study in Morocco in order to assess the efficacy of antivenom therapy against scorpion stings. Epidemiological and clinical data were collected in 275 patients envenomed by Androctonus mauretanicus mauretanicus and Buthus occitanus scorpions. Patients received antivenom or symptomatic drugs. Blood samples were collected upon hospital admission, at 1 hr and 3 hrs after the treatment. An enzyme linked immunosorbent assay (ELISA) was set up to quantify the venom levels in serum of envenomed patients. Mean serum venom concentrations showed an association between clinical signs and the venom level. The venom concentration at admission, in patients who received 10 ml of antivenom, was significantly reduced after antivenom therapy. The decrease was less important in patients who received only 2 to 5 ml of antivenom. No difference was shown in the venom concentration of patients not treated with antivenom. The clinical signs decreased significantly after antivenom treatment. The absence of antivenom administration increased the risk to develop clinical signs at the end of hospitalisation. This risk was much higher when the delay between scorpion sting and hospital admission increased. The results of our study have demonstrated the efficacy of antivenom in reducing circulating venom and symptoms. Antivenom therapy is more efficient when administered as soon as possible after envenomation and with appropriate quantities of antivenom. This study is favourable to the use of SAS but a prospective study would be useful to confirm these data.

Scorpion envenomation and serotherapy in Morocco.

A clinical and biologic study was conducted in Morocco to assess the efficiency of antivenom therapy for treating victims of scorpion stings. Epidemiologic and clinical data were collected from 275 patients envenomed by Androctonus mauretanicus mauretanicus and Buthus occitanus scorpions. Patients received antivenom or other drugs. Blood samples were collected at the time of hospital admission and 1 hr and 3 hr after treatment. Serum venom levels were quantified by using an ELISA. An association was found between clinical signs of envenoming and the level of venom in serum. Patients classified as grade II (moderate envenoming) had higher serum levels of venom level than patients classified as grade I (mild envenoming). At admission to the hospital, the mean venom concentration was not significantly different between the group not treated with antivenom, the group who received 2-5 ml of antivenom, and the group who received 10 ml of antivenom. A significant decrease in serum venom levels and an improvement in the clinical conditions were observed in patients administered 10 ml of antivenom. The lower decrease in serum venom levels in patients who received 2-5 ml of antivenom was due to lower doses of antivenom. No difference in the venom concentration was observed in patients who were not treated with antivenom. The absence of administration of antivenom increased the risk of developing clinical signs at the end of the hospitalization period. However, this risk was much higher when more than 1 hr elapsed between the time of the scorpion sting and the time of hospital admission. The results demonstrate that antivenom is effective in decreasing circulating venom and morbidity. Serotherapy is more efficient when given as soon as possible after envenomation and with adequate quantities of antivenom.

Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells.

Two membrane glycoproteins acting as energy-dependent efflux pumps, mdr-encoded P-glycoprotein (P-gp) and the more recently described multidrug resistance-associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P-gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood-brain barrier, but localization of MRP has not been well characterized yet. Using RT-PCR and immunoblot analysis, we have compared the expression of P-gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P-gp-encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [3H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P-gp, is not predominantly expressed in the blood-brain barrier endothelial cells and that Mrp1 and the mdr1b P-gp isoform may be present in other cerebral cells.

Modulation of P-glycoprotein activity by glial factors and retinoic acid in an immortalized rat brain microvessel endothelial cell line.

P-glycoprotein (P-gp), a product of the multidrug-resistant (mdr) genes, is expressed in the endothelial cells of the blood-brain barrier (BBB). Effects of glial factors and retinoic acid (RA) on P-gp activity and level were investigated in the immortalized rat brain endothelial cell line RBE4, which expressed immunodetectable P-gp associated with a decrease in accumulation of the P-gp substrates, vinblastine and colchicine. When RBE4 cells were cultured either in the presence of C6-conditioned medium or on C6- or astrocyte-extracellular matrix, intracellular vinblastine and colchicine concentrations were decreased. When the cells were treated with RA, increases in P-gp activities were correlated with increases in P-gp levels. Effects of simultaneous treatments with glial factors and RA were studied in RBE4 cells cultured on astrocyte-extracellular matrix and were shown to be additive on P-gp activity and level. RBE4 cells may serve as a useful in vitro model for basic research on P-gp regulation at the level of the BBB.

Role of P-glycoprotein in colchicine and vinblastine cellular kinetics in an immortalized rat brain microvessel endothelial cell line.

Uptake and efflux of colchicine and vinblastine, whose effects are related to their high-affinity binding to tubulin, were studied in the immortalized rat brain microvessel endothelial cell line RBE4. At 10 nM extracellular drug concentration, uptake equilibrium was approached at 45 hr for colchicine, but at only 3.5 hr for vinblastine. After 1 hr preincubation with 200 nM colchicine or vinblastine, drug efflux fitted biexponential kinetics with an initial fast phase (half-life = 2.2 min and 9.6 min, respectively) and a later slow phase (half-life = 3.6 hr and 1.8 hr, respectively). After 6 hr preincubation with 200 nM colchicine, only the slow phase (half-life = 3.6 hr) could be observed. The colchicine and vinblastine uptake rate was increased by cyclosporin A, an inhibitor of the drug efflux pump P-glycoprotein, which is expressed at the blood-brain barrier. Whereas cyclosporin A decreased vinblastine efflux, its effect on colchicine efflux was apparent after only 13 hr washout and was associated with the re-uptake by cells of colchicine molecules. Differences in uptake kinetics of colchicine and vinblastine could be related to differences in their lipid solubility, and mainly in their binding affinities to tubulin. Differences in efflux kinetics could in addition be explained by the involvement of P-glycoprotein in the efflux of vinblastine, whereas efflux of colchicine was not influenced by this pump. Indeed, binding of colchicine to tubulin would imply that most intracellular colchicine may be inaccessible to P-glycoprotein. In the case of a cytotoxic drug such as colchicine, which is tightly bound to intracellular receptors, the role of P-glycoprotein within the blood-brain barrier would be more to protect the brain against entry of this drug than to detoxify the brain by its extraction.

Synergistic stimulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities by retinoic acid and astroglial factors in immortalized rat brain microvessel endothelial cells.

The immortalized rat brain microvessel endothelial cell line RBE4 was used to investigate the in vitro regulation of two blood-brain barrier specific enzymes, gamma-glutamyl transpeptidase (GTP) and alkaline phosphatase (ALP). The effects of bFGF, astroglial factors, and retinoic acid (a cell differentiation agent) on GTP and ALP activities were separately or simultaneously studied in order to define optimal culture conditions for induction of these two specific enzymes of the blood-brain barrier. In the present study, a phenotypically distinct subpopulation of endothelial cells has been shown to develop from confluent cobblestone monolayers of RBE4 immortalized cerebral endothelial cells. These distinct cells were present within multicellular aggregates and specifically exhibited GTP and ALP activities. Addition of bFGF, astroglial factors, or retinoic acid induced the formation of these three-dimensional structures and in consequence an increase in GTP and ALP activities. For retinoic acid and astroglial factors, this increase could also be explained by the stimulation of either GTP or ALP expression in the phenotypically distinct positive cells associated with aggregates. Simultaneous treatment with retinoic acid and astroglial factors had a synergistic effect on GTP and ALP expression and thus may allow these distinct cells to evolve toward a more differentiated state. Since such results were also obtained with physiological concentrations of retinoic acid, we suggest that addition of this agent might contribute to greater differentiation of cells in in vitro blood-brain barrier models where endothelial cells are cocultured with astrocytes.