A randomised controlled trial of bumetanide in the treatment of autism in children.

Gamma aminobutyric acid (GABA)-mediated synapses and the oscillations they orchestrate are altered in autism. GABA-acting benzodiazepines exert in some patients with autism paradoxical effects, raising the possibility that like in epilepsies, GABA excites neurons because of elevated intracellular concentrations of chloride. Following a successful pilot study,(1) we have now performed a double-blind clinical trial using the diuretic, chloride-importer antagonist bumetanide that reduces intracellular chloride reinforcing GABAergic inhibition. Sixty children with autism or Asperger syndrome (3-11 years old) received for 3 months placebo or bumetanide (1 mg daily), followed by 1-month wash out. Determination of the severity of autism was made with video films at day 0 (D0) and D90 by blind, independent evaluators. Bumetanide reduced significantly the Childhood Autism Rating Scale (CARS) (D90-D0; P<0.004 treated vs placebo), Clinical Global Impressions (P<0.017 treated vs placebo) and Autism Diagnostic Observation Schedule values when the most severe cases (CARS values above the mean ± s.d.; n=9) were removed (Wilcoxon test: P-value=0.031; Student's t-test: P-value=0.017). Side effects were restricted to an occasional mild hypokalaemia (3.0-3.5 mM l(-1) K(+)) that was treated with supplemental potassium. In a companion study, chronic bumetanide treatment significantly improved accuracy in facial emotional labelling, and increased brain activation in areas involved in social and emotional perception (Hadjikhani et al., submitted). Therefore, bumetanide is a promising novel therapeutic agent to treat autism. Larger trials are warranted to better determine the population best suited for this treatment.

Immunoreactivity of a new generation of horse F(ab')2 preparations against European viper venoms and the tetanus toxin.

The immunoreactivity of the current and the new more purified, pasteurized preparations of horse F(ab')2 against the tetanus toxin and Vipera aspis venom was investigated with a biosensor based on technology using the optical phenomenon of surface plasmon resonance. Immunoreactivity data were compared with seroneutralization titres to investigate immunoreactivity-immunoprotection efficacy relationships. The association-dissociation rate and affinity constants of the current and the new tetanus toxin-specific F(ab')2 preparations were similar, at about 10(4) M-1 sec-1, 10(-4) sec-1 and 10(8) M-1, respectively. Similar values were found using a solid immunoradiometric assay. To assess the immunoreactivity of V. aspis venom-specific horse F(ab')2, the mol. wt and percentage of the antigenic fractions of V. aspis venom were determined. Western blotting of electrophoresis gels showed four antigenic fractions of V. aspis venom (mol. wts 17,500, 28,500, 32,000 and 60,000), which represented 6, 3.4, 17.7 and 5% of total venom, respectively. Association and dissociation rate constants were in the same range as those of the tetanus toxin-F(ab')2 interactions for each of the four antigenic fractions. Seroneutralization of both tetanus toxin and V. aspis by the corresponding specific F(ab')2 showed that the LD50 mg-1 protein was 1.76-fold and 1.51-fold higher with the new than with the current preparations, respectively. These improvements in efficacy were in close agreement with the higher immunoreactive fraction ratios, which were 2-fold and 1.8-fold higher with the new preparations. These results demonstrate that the removal of non-IgGT immunoglobulins and the pasteurization treatment have no overall influence on F(ab')2 affinity but improve the specific activity of these new antitoxin horse F(ab')2.

Immunoreactivity and pharmacokinetics of horse anti-scorpion venom F(ab')2-scorpion venom interactions.

The immunoreactivity and pharmacokinetics of a new horse F(ab')2 scorpion antivenom and its effect on Buthus occitanus mardochei venom plasma disposition in the rabbit were studied. The scorpion venom-specific F(ab')2 affinity constant determined by immunoradiometric assay was 1.6 +/- 0.6 10(8) M-1. One group received a F(ab')2 bolus dose of 9.57 mg.kg-1 i.v. bolus or i.m.. The plasma F(ab')2 concentration followed a biexponential decline after i.v. administration with distribution and elimination half-lives of 2.54 +/- 0.36 and 49.52 +/- 3.07 hr, respectively. The total volume of distribution (Vdss or Vd beta) was between 230 and 255 ml.kg-1. Total body clearance was 3.56 +/- 0.34 ml.kg-1.hr-1. After intramuscular administration, Tmax was 48 hr and the absolute bioavailability was 36%. Two other groups of rabbits received i.v.60 micrograms.kg-1 B. occitanus mardochei venom either alone (control group) or followed by 3 mg.kg-1 scorpion venom-specific F(ab')2 administered by intravenous infusion 1.75 hr later. In the rabbits treated with horse F(ab')2 antivenom the venom concentration profile was initially identical to that observed in the control group which received venom alone before F(ab')2 administration. Subsequent infusion of antivenom induced a 1.5-fold elevation of the plasma venom concentration with a Tmax 0.5 hr after F(ab')2 administration. The AUC was 10-fold higher in the F(ab')2-treated group than in the control group in the post-F(ab')2 infusion period. Twelve hours after F(ab')2 administration the venom disposition declined with a terminal half-life equal to that of F(ab')2 (49.49 +/- 7.53 hr). These data show the ability of F(ab')2 to alter venom pharmacokinetics and demonstrate that the scorpion toxins adopt the F(ab')2 elimination properties.

Snake F(ab')2 antivenom from hyperimmunized horse: pharmacokinetics following intravenous and intramuscular administrations in rabbits.

PURPOSE: The pharmacokinetics of a currently available horse F(ab')2 antivenoms to Vipera aspis, V. ammodytes, and V. berus (Ipser Europe) and a new more purified and pasteurized preparation (SAV) was investigated in the rabbit. METHODS: An immunoradiometric assay using an affinity-purified goat IgG horse F(ab')2 specific and the same IgG labelled with iodine 125 as a tracer was developed. The limit of quantification in plasma was 0.032 microgram/ml. Specificity study showed that mouse F(ab')2 and Fab did not cross-react. RESULTS: Pharmacokinetic analysis showed that the plasma F(ab')2 concentration followed a biexponential decline after intravenous bolus administration with distribution and elimination half-lives of 2.66 +/- 0.18 hrs and 49.69 +/- 4.13 hrs, respectively. The total volume of distribution (Vdss or Vd beta) was between 209 and 265 ml.kg-1 and was similar to the volume of the extracellular fluid in the rabbit (300 ml.kg-1). Total body clearance ranged from 3.33 to 3.96 ml.h-1.kg-1. After intramuscular administration which was only investigated with SAV, Tmax was 48 hrs and the absolute bioavailability was 42%. CONCLUSIONS: No difference in pharmacokinetics was observed between the two antivenom preparations following the intravenous administration. In contrast, a reduced rate and extent of absorption was shown following intramuscular administration.

Human hemoglobin conjugated to carboxylate dextran as a potential red blood cell substitute. II--Pharmacotoxicological evaluation.

A solution of human hemoglobin bound to benzene tetracarboxylate substituted dextran, whose physicochemical characteristics are defined in part I, was evaluated in vivo as a potential red blood cell substitute. Further experiments show: - the confirmation of a lack of acute toxicity in mice and guinea pigs after injection of 12.5%, 25% and 50% of the blood mass and the absence of death in rabbits having undergone three successive 25% hemorrhagic shocks in three week intervals. A plasma half-life of 9.5 +/- 0.5 hours in 70-75% hemorrhagic shocks on guinea pigs and the absence of dex-BTC-Hb in thoracic and abdominal cavities. No tissue oedema was noticed. Total hemoglobinuria did not exceed 10% of the injected hemoglobin quantity and only involved free hemoglobin. A lack of death in 70-75% hemorrhagic shocks and survival times ranging from 10 hours to 3 days in total exchange transfusions in guinea pig experiments.

Methemoglobin formation after administration of hemoglobin conjugated to carboxylate dextran in guinea pigs. Attempts to prevent the oxidation of hemoglobin.

In 1990, McGown demonstrated in vitro a limitation of extracellular methemoglobin (metHb) formation by releasing and recycling of ascorbic acid by red blood cells. In order to investigate the autoxidation of free or modified hemoglobin in plasma and the possibility of reproducing McGown's phenomenon in vivo, we performed a 50% blood mass exchange in guinea-pigs with a 70 +/- 5 g/l dex-BTC-Hb solution (metHb < 5%). Methemoglobin was determined according to Evelyn-Malloy's method. We observed a clear but limited oxidation of plasmatic hemoglobin (MetHb approximately 30-40% at t = 12 hrs up to t = 24 hrs). A similar blood mass exchange was performed with the same hemoglobin solution which was previously totally oxidized into metHb. 40% of this methemoglobin was found to be reduced after 12 hrs. These results demonstrated a marked reducing activity by residual blood as shown by others. The addition of potentially protective compounds such as ascorbic acid (non enzymatic intraerythrocytar reduction pathway), methylene blue or riboflavin (enzymatic intraerythrocytar pathway), allowed a significant drop in the methemoglobin level. On the contrary, we didn't observe any reducing effect with reduced glutathione.

Human hemoglobin conjugated to carboxylate dextran as a potential red blood cell substitute. I. Further physico-chemical characterization.

In a previous paper, we described the preparation of a conjugate by reaction of benzene tetracarboxylate-substituted dextran (dex-BTC) with oxyhemoglobin. The first biological experiments carried out on animals showed that a solution of this type of product could be regarded as an oxygen-carrying fluid. The physico-chemical properties of this conjugate have been further characterized in view of its potential application in clinical experiments. Its molecular size distribution and the fraction of intramolecularly cross-linked hemoglobin were determined by elution on various types of chromatographic columns. Moreover the conjugate's oxygen-binding properties were studied in the presence of effectors and of Cl- ions, and the results showed that the allosteric site and the Val 1 alpha residues of conjugated hemoglobin were occupied by dex-BTC. Finally, the fractions obtained after gel filtration exhibited a similar P50 whatever their molecular sizes. So far, all the results obtained are compatible with the use of the conjugate solution as a blood substitute.

Viral validation of the manufacturing process of high purity albumin from placentas.

We report on the viral validation of an industrial purification process which is used to manufacture high purity human albumin from frozen placentas. This process has been used without any modification since 1980 except for a progressive increase in production scale to reach a capacity of 19 tons of placentas per day. The extraction purification process includes five alcohol manufacturing steps, some with strict conditions of alcohol concentration, acid pH, temperature and one including chloroform. These steps are followed by three chromatographies. Albumin is finally submitted to pasteurization both in bulk and in the final container. Selected steps of this process have been tested for their ability to remove or inactivate viruses. Viruses used were HBV, HIV-1, HIV-2 and model viruses poliovirus, avian reovirus, MuLV, Sindbis, SV40 and Aujeszky's. In vitro infectivity titration assays were used for all viruses except for HBV where DNA and antigen titrations were performed. Reduction factors obtained were from 10 to 29 log10 depending on the viral marker. Moreover, testing done on regular production batches demonstrated the absence of HBV, HIV-1 and HCV genomic sequence in the final lots. Viral risk calculation for HBV, HIV-1 and HCV was made considering the maximal theoretically possible contamination of the starting material. This calculation showed the very large safety margin the manufacturing process with respect to virus transmission for these viruses or possibly other unknown ones.

Fixation of aldehydic dextrans onto human deoxyhemoglobin.

A procedure commonly used to transform native adult human hemoglobin (Hb) into a physiological oxygen carrier consists of a pyridoxylation of the protein to lower its oxygen affinity, followed by its polymerization in the presence of glutaraldehyde, with or without further reduction, to increase its circulating half-life. This series of reactions yields derivatives presenting a great molecular heterogeneity that have to be fractionated for use in vivo. Hemoglobin derivatives with low oxygen affinity and a narrow distribution of molecular weights were obtained by linking a dextran polyaldehydic derivative to deoxyhemoglobin at pH 8. From oxygen-binding measurements carried out in the presence of inositolhexaphosphate, a strong effector of hemoglobin, it appeared that the allosteric site of hemoglobin was blocked, probably by crosslinking bonds, which stabilizes its deoxy structure. On the other hand, when the reaction was performed in the presence of inositolhexaphosphate, the resulting conjugates exhibited an oxygen affinity identical to that of unmodified hemoglobin. After treatment with NaBH4, the polymer-hemoglobin derivatives were stable and possessed a reversible oxygen-carrying capacity similar to that of blood. The conjugates prepared from oxyhemoglobin all possessed a lower P50 than native hemoglobin whatever the reaction conditions.

Analytical control procedures of immunoreactivity for IgG and Fab fragments specific to haptens.

This study investigates immunoreactivity control procedures, i.e., specificity, affinity constant (Ka), and specific active binding sites (SABS), for polyclonal anticolchicine, monoclonal antidigitoxin IgG and Fab fragments, and antidigoxin Fab fragments (Digidot). Preliminary control procedures for IgG and Fab fragment purity indicated that all reagents were immunologically pure. All IgG and Fab fragments exhibited similar cross-reactivity and Ka. No decrease in percentage of Fab fragment SABS was observed after papain cleavage of anticolchicine and antidigitoxin IgG. Nevertheless, only 4.3 +/- 1.2% of nonimmunopurified anticolchicine polyclonal Fab fragments and 76.2 +/- 2.3 to 88.7 +/- 2.5% of different batches of immunopurified anti-digoxin Fab (Digidot) were active, the latter percentage being in the range of the 85% specified by the manufacturer. Only 58 +/- 3% of digitoxin-specific monoclonal IgG was active and 67 +/- 7% of its Fab fragments. Results show the importance of determining the ratio of SABS to presumed total specific binding sites for pharmaceutical monoclonal and polyclonal antibody preparations against haptens.

Colchicine-specific Fab fragments alter colchicine disposition in rabbits.

High-affinity goat antibodies and Fab fragments (Ka = 1.1 x 10(10) M(-1) specific to colchicine were prepared to study their effect on colchicine pharmacokinetics in rabbits. First, colchicine disposition kinetics were investigated in four control rabbits after administration of 0.1 mg/kg i.v. Total and free plasma and urine colchicine were assayed by specific radioimmunoassay. The mean elimination half-life of total plasma colchicine was 16 +/- 2.9 h. Colchicine has a large volume of distribution (8.8 +/- 1.8 l/kg) and a low systemic clearance (114.6 +/- 3.4 ml.h-1.kg-1). Renal clearance represented 30.7 +/- 1.9% of total body clearance. The free plasma colchicine fraction was 70% after equilibrium dialysis. Second, 1.5 h after injection of 0.1 mg/kg colchicine, four rabbits were infused over 0.25 h with colchicine-specific Fab fragments at a half-stoichiometrically equivalent dose compared to the colchicine dose. Within 15 min after Fab infusion, total colchicine concentrations increased 10- to 16-fold. Mean area under the plasma concentration-time curves increased 20-fold compared to controls. The free plasma fraction decreased to an undetectable level over a period of 2 h. The Fab fragment administration also produced, respectively, a 24- and 17-fold decrease in the volume of distribution and systemic clearance. Colchicine recovered in urine was significantly higher than in the control group: 44.7 +/- 2.3 and 30.9 +/- 2% of the dose, respectively (P less than .05). These data suggest that high-affinity colchicine-specific Fab fragments can sequestrate and extract colchicine from tissues to the vascular compartment with subsequent colchicine excretion by the renal route.

A potential blood substitute from carboxylic dextran and oxyhemoglobin. I. Preparation, purification and characterization.

Human adult hemoglobin (Hb) from peripheral venous blood was covalently linked, under its oxygenated form, to a dextran polymeric effector to yield conjugates with low oxygen affinity. The polymeric effector was synthesized by directly coupling benzene tetracarboxylic anhydride to dextran (MW approximately 10,000) under mild conditions. The reaction parameters were chosen so that the resulting polymeric derivative was only very little cross-linked and possessed benzene tricarboxylate site concentrations lesser than 1 site for 10 glucose units, as it was found that for higher concentrations the polymer exhibited anticlotting properties. The reaction between oxyHb and the polymer (dex-BTC) was carried out in water in the presence of a water-soluble carbodiimide (EDCI). By optimization of the reaction conditions, a dextran-Hb conjugate with a P50 of 21 torr (37 degrees C, 50mM Bis-tris buffer pH 7.4, 0.14M NaCl, 40 mM glucose) and a potential O2 release of 0.30 ml/g Hb between oxygen partial pressures of 100 and 40 torr, was obtained. The viscosity and oncotic pressure of a solution containing about 6% of conjugated Hb were respectively 1.8 cSt (37 degrees C) and 28 torr (25 degrees C). This solution could be stored frozen at -20 degrees C for a long time, without modification of its properties.

Hemoglobin linked to polyanionic polymers as potential red blood cell substitutes.

A variety of chemical modifications of hemoglobin (Hb) have been proposed in order to transform it into cell-free oxygen carriers. These modifications are intended to increase the plasmatic half-life of the protein and to lower its affinity for oxygen. We have designed and prepared derivatives of dextran and polyoxyethylene functionalized so that, after their covalent fixation onto Hb, they can act as permanent effectors and thus lower the oxygen affinity of the protein while decreasing its renal excretion. The main characteristic of these functionalized polymers is that they possess a relatively high density of anionic groups. Benzene hexacarboxylate (BHC) and tetracarboxylate (BTC) linked to water-soluble polymers such as polyoxyethylene or dextran, yielded polymeric derivatives which, even after reaction with oxyHb, gave rise to conjugates with a lower oxygen affinity than the native protein. We showed on a conjugate obtained by the fixation of a BHC-monosubstituted polyoxyethylene onto oxyHb, that the low oxygen affinity was due to the preferential binding of the polymer-linked BHC to the beta-terminal valine residues. In the reaction of dextran-linked benzene tetracarboxylate (dex-BTC) with oxyHb, a lot of parameters had to be optimized in order to obtain conjugates well fitted to the purpose of blood substitute. Thus the polymer content in BTC and the amounts of reagents used for the reaction determined the oxygen-binding properties, the molecular weights and the biochemical characteristics of the conjugates, as well as the viscosity and oncotic pressure of the solutions. This optimization resulted in products which are now studied in-vivo.

A potential blood substitute from carboxylic dextran and oxyhemoglobin. II. Physicochemical and physiological assessments. Preliminary results on guinea pig.

The hemoglobin solution covalently linked to dextran benzene tetracarboxylate (dex-BTC) described in Part I, offered the following characteristics: [Hb] = 70 g/l, non modified Hb less than 15%, metHb less than 5%, P50 close to blood value, viscosity and oncotic pressure near to physiological values (37 degrees C). It was biocompatible, stable in plasma, non hypotensive in rat model, non pyrogenic and did not present abnormal toxicity (on mice, according to the french Pharmacopea). Nevertheless, on account of the presence of dextran in the conjugate and due to the literature, we observed an anaphylactoïd reaction in rats consecutive to the injections: absence of hypotension but presence of a massive oedema resulting from an important increase in the capillary permeability with harmful consequences to the vascular retention and survival time. As guinea pigs are insensitive to dextran, we are beginning to assess this conjugate solution in this animal. The first results are very promising: a lack of mortality after large injection, an almost complete vascular retention, a weak urinary loss (only non conjugated Hb), and a plasmatic half-disappearance time close to 7h. Results from haemorrhagic shocks to a final hematocrit less than 0.10 l/l and total and isovolemic exchange transfusions seem to prove a real oxygen-carrying capacity of this new Hb solution.

A potential blood substitute from carboxylic dextran and oxyhemoglobin. III. Evaluations by perfusion of normal and ischemic guinea-pig heart.

Hemodynamic parameters of six groups of guinea-pig hearts were studied by the working heart technic of Neely. Three groups were respectively perfused with: Krebs-Henseleit, purified native hemoglobin 1gm/dl and hemoglobin conjugated to dextran benzene tetracarboxylate 1 gm/dl. Three other groups were perfused under the same conditions except that after 30 mn of perfusion, 10 mn of total ischemia were produced followed by 30 mn of reperfusion with the previous solutions. All solutions contained 10 g/l of BSA. Hearts perfused with Hb solutions without ischemia or after ischemia show better parameters than with Krebs-Henseleit. These observations suggest that contrary to previously published results, purified Hb and more, dextran-BTC-Hb appear to be perfusable and are less deleterious for heart than saline without hemoglobin.

Possible importance of chromatographic purification position in a blood substitute elaboration process.

Purification of hemoglobin (Hb) solutions suggested as oxygen carriers is an imperative necessity. All processes currently used clear out the solutions of almost the totality of impurities that are able to negatively influence the transfusional efficiency of Hb. We have studied the stability (metHb measurement) of Hb purified by DEAE dextran chromatography (Spherodex, 10 mM phosphate buffer, pH 7.20) which eliminates lipopolysaccharides, enzymes and non heminic-proteins ... without denaturing Hb. In spite of the improvements due to this purification method on the transfusional efficiency of non modified Hb solutions, the lack of enzymes involved in the protection of Hb against autooxidation (superoxide dismutase, peroxidase, catalase ...) makes it much more vulnerable to the reagents used during the following chemical processes: pyridoxylation, polymerization or macromolecule binding, thereby leading to an important oxidation to metHb. These observations led us to question the optimal position of purification in a process of modified Hb preparation. We have shown the importance of this chromatographic position in working on pyridoxylated Hb bound to monomethoxypolyoxyethylene. Spherodex chromatography appears to be a very satisfactory purification method, provided it occurs only at the last step of the process. Many authors have not been attentive to this phenomenon which could be found with other Hb modifications. This therefore imposes to study the best place to incorporate a particular purification step into a hemoglobin preparatory procedure.

Importance of purification on transfusional efficacy of hemoglobin solutions.

It has only been realized quite recently how important is the purification of hemoglobin solution for use in transfusion and several techniques have been published. We used ion exchange chromatography with which the main "contaminants" (glycoproteins, enzymes, phospholipids) are absorbed by the gel, whereas hemoglobin is not retained. The solutions studied here are non-modified hemoglobin and its homologue pyridoxylated hemoglobin (PLP-Hb). Physico-chemical analyses, usually undertaken to characterize hemoglobin solutions, show no difference before and after purification, except that the enzymatic activity almost disappears. In order to appreciate the benefits of purification, total exchange transfusions were carried out on rats. Without reperfusion, purification of the hemoglobin solution allowed a significantly longer survival time which was even more significant with PLP-Hb solution. Urinary loss did not seem to be affected by purification. With reperfusion in order to compensate these renal losses, PLP-Hb solutions gave survival times up to three days. However, the inevitable death of the animals poses the problem of instability of these purified solutions following enzyme loss.

Reversal of murine colchicine toxicity by colchicine-specific Fab fragments.

High-affinity Fab fragments (2 x 10(10) M-1) specific to colchicine were produced to evaluate their potency in reversing murine colchicine intoxication. Intraperitoneal injection of a 4.46 mg/kg colchicine dose was lethal for 100% of mice. 1.5 h after colchicine administration, a group of 10 mice was treated with colchicine-specific Fab fragments at a half-stoichiometrical dose compared to the colchicine dose by intravenous and intraperitoneal routes. 70% of the Fab-infused mice survived (P less than 0.01). This high efficiency of colchicine-specific Fab fragments in reversing acute murine colchicine toxicity suggests that Fab fragments would be an efficient antidote for the treatment of human colchicine poisoning.

Preparation of unaltered hemoglobin from human placentas for possible use in blood substitutes.

Hemoglobin extracted from human placentas could be used as the basis of blood substitutes provided it could be prepared on a large scale with appropriate oxygen-binding properties. Unfortunately, the industrial conditions under which it is extracted, produce hemoglobin with high oxygen affinity and which is no longer influenced by the classical effectors. These characteristics were shown to be caused by a degradation of the alpha-chain brought about by an arginine carboxypeptidase present in the placental tissues and leading to the disappearance of the C-terminal arginine residue. This carboxypeptidase which is released from the tissues during the process of crushing the frozen placentas, degrades the protein during the chromatographic purification procedure. The addition of an inhibitor of this carboxypeptidase (for example, arginine) as soon as the placentas are thawed and during the chromatographic process, makes it possible to obtain placental hemoglobin with oxygen-binding properties quite similar to those of HbA prepared from peripheral venous blood.