Reactivation and aging kinetics of human acetylcholinesterase inhibited by organophosphonylcholines

A great number of structurally different organophosphorus compounds (OPs) was synthesized in the past decades to be used as pesticides or chemical warfare agents. Methyl-fluorophosphonylcholines were found to be highly toxic OPs and the acetylcholinesterase (AChE) reactivator pralidoxime was shown to be unable to reactivate inhibited AChE. In the course of the development of more effective AChE reactivators, we have determined the reactivation rate constants of various oximes with human AChE inhibited by methylfluorophosphonylcholine (MFPCh), methylfluoro--phosphonylcholine (MFPCh) and methylfluorophosphonylhomocholine (MFPhCh). In addition, we investigated the potential influence of aging phenomena on the oxime efficacy. Human AChE inhibited by MFPCh, MFPCh or MFPhCh was extremely resistant towards reactivation by oximes. Nevertheless, the newer compounds, HLö 7 and HI 6, were substantially more potent reactivators than obidoxime and pralidoxime. The low oxime efficacy was not due to rapid aging since no decrease in reactivatability was found over 96 h at 37°C. Within this period a substantial spontaneous reactivation was observed, with MFPCh >MFPCh >MFPhCh, which did not follow pseudo-first-order kinetics. In conclusion, the unexpected results, i.e., high resistance of inhibited AChE towards oxime reactivation and aging, and much lower resistance towards spontaneous reactivation, calls for further experiments at a molecular level for a better understanding of the interactions among AChE, its inhibitors and reactivators.     

In vitro kinetic interactions of pyridostigmine, physostigmine and soman with erythrocyte and muscle acetylcholinesterase from different species

The low effectiveness of atropine and oxime treatment in soman poisoning may be enhanced by carbamates pre-treatment. For ethical reasons medical countermeasures can only be tested in animal models despite the fact of substantial species differences. With this kinetic in vitro study the interactions between pyridostigmine, physostigmine and soman with human, Rhesus monkey, swine and guinea pig erythrocyte AChE were investigated. In addition, the effect of the carbamates on the residual activity and enzyme recovery after soman inhibition was examined with erythrocyte and intercostal muscle AChE from these species with a dynamic in vitro model with real-time determination of AChE activity. Only small to moderate species differences of the inhibition and decarbamylation kinetics were recorded. It was possible to show that with erythrocyte and muscle AChE a similar level of protection by carbamates and reactivation after discontinuation of the carbamates and soman could be observed. Thus, these data indicate that carbamate pre-treatment is expected to protect a critical level of muscle AChE and confirm the presumption that erythrocyte AChE may serve as a surrogate for synaptic AChE. Hence, these and previous data fortify the notion that erythrocyte AChE is a proper tool for in vitro kinetic studies as well as for therapeutic monitoring in experimental and clinical studies.     

Kinetic interactions of a homologous series of bispyridinium monooximes (HGG oximes) with native and phosphonylated human acetylcholinesterase

Inhibition of acetylcholinesterase (AChE) is the main toxic mechanism of organophosphorus compounds (OP) and reactivation of OP-inhibited AChE by oximes is a mainstay of antidotal treatment. The inadequate efficacy of clinically used oximes led to the synthesis of numerous new compounds in the past decades to identify more effective reactivators. Despite of extensive in vitro reactivation studies the structural features for the development of effective oximes are not well understood. In the present study we investigated the kinetic interactions of a homologous series of bispyridinium monoximes bearing C1 to C12 alkylketone groups on the second pyridinium ring with native and cyclosarin-inhibited human AChE. We observed a correlation of the length of the alkyl side chain with an up to 20-fold increased affinity towards native AChE. The effect of the alkyl side chain on the affinity and reactivity towards phosphonylated AChE was moderate, except of a markedly reduced reactivity of C10 and C12 oximes. In comparison to the reference oxime HI-6 all HGG oximes had a lower reactivating potency and these oximes are not considered as promising compounds for the reactivation of cyclosarin-inhibited AChE.     

Reactivating potency of obidoxime, pralidoxime, HI 6 and HLö 7 in human erythrocyte acetylcholinesterase inhibited by highly toxic organophosphorus compounds

The treatment of poisoning by highly toxic organophosphorus compounds (nerve agents) is unsatisfactory. Until now, the efficacy of new potential antidotes has primarily been evaluated in animals. However, the extrapolation of these results to humans is hampered by species differences. Since oximes are believed to act primarily through reactivation of inhibited acetylcholinesterase (AChE) and erythrocyte AChE is regarded to be a good marker for the synaptic enzyme, the reactivating potency can be investigated with human erythro‐cyte AChE in vitro. The present study was undertaken to evaluate the ability of various oximes at concentrations therapeutically relevant in humans to reactivate human erythrocyte AChE inhibited by different nerve agents. Isolated human erythrocyte AChE was inhibited with soman, sarin, cyclosarin, tabun or VX for 30?min and reactivated in the absence of inhibitory activity over 5–60?min by obidoxime, pralidoxime, HI 6 or HLö 7 (10 and 30?μM). The AChE activity was determined photometrically. The reactivation of human AChE by oximes was dependent on the organophosphate used. After soman, sarin, cyclosarin, or VX the reactivating potency decreased in the order HLö 7 > HI 6 > obidoxime > pralidoxime. Obidoxime and pralidoxime were weak reactivators of cyclosarin-inhibited AChE. Only obidoxime and HLö 7 reactivated tabun-inhibited AChE partially (20%), while pralidoxime and HI 6 were almost ineffective (5%). Therefore, HLö 7 may serve as a broad-spectrum reactivator in nerve agent poisoning at doses therapeutically relevant in humans.     

Inhibition, reactivation and aging kinetics of highly toxic organophosphorus compounds: pig versus minipig acetylcholinesterase

Organophosphorus compound-based (OP) chemical warfare agents (nerve agents) represent a continuing threat to military forces and the civilian population. OPs act primarily by inhibiting acetylcholinesterase (AChE), the standard treatment for which includes AChE reactivators (oximes) in combination with antimuscarinic drugs. In the last decades, the efficacy of oximes has been investigated mostly in small animal models. In order to increase the predictive value of animal studies it is desirable to measure numerous physiological and biochemical parameters. This is difficult in small animals. Large animal models fulfil these requirements and swine are increasingly being used in toxicology studies. Swine breeds generally show considerable variability in different characteristics which may be minimised by the use of specially bred minipigs which have a known genetic background and health status. A comparative study was, therefore, initiated to investigate the kinetic properties of the White Landrace pig and Göttingen minipig AChE in respect of inhibition by the pesticide paraoxon; the nerve agents cyclosarin, VX and VR; the reactivation of inhibited AChE by oximes (obidoxime, pralidoxime and HI 6); and the aging and spontaneous reactivation of inhibited AChE. The determination of the respective kinetic constants found similarities between pig and minipig AChE which showed marked differences in comparison with human AChE values. This has to be considered in designing meaningful models for the investigation of oxime efficacy in pig or minipig experiments. The generated data indicate comparable kinetic properties of pig and minipig AChE and may provide a kinetic basis for extrapolation of data from pig studies to humans.     

Kinetic analysis of interactions of paraoxon and oximes with human, Rhesus monkey, swine, rabbit, rat and guinea pig acetylcholinesterase

Previous in vitro studies showed marked species differences in the reactivating efficiency of oximes between human and animal acetylcholinesterase (AChE) inhibited by organophosphorus (OP) nerve agents. These findings provoked the present in vitro study which was designed to determine the inhibition, aging, spontaneous and oxime-induced reactivation kinetics of the pesticide paraoxon, serving as a model compound for diethyl-OP, and the oximes obidoxime, pralidoxime, HI 6 and MMB-4 with human, Rhesus monkey, swine, rabbit, rat and guinea pig erythrocyte AChE. Comparable results were obtained with human and monkey AChE. Differences between human, swine, rabbit, rat and guinea pig AChE were determined for the inhibition and reactivation kinetics. A six-fold difference of the inhibitory potency of paraoxon with human and guinea pig AChE was recorded while only moderate differences of the reactivation constants between human and animal AChE were determined. Obidoxime was by far the most effective reactivator with all tested species. Only minor species differences were found for the aging and spontaneous reactivation kinetics. The results of the present study underline the necessity to determine the inhibition, aging and reactivation kinetics in vitro as a basis for the development of meaningful therapeutic animal models, for the proper assessment of in vivo animal data and for the extrapolation of animal data to humans.     

Metabolism of coumarin and 7-ethoxycoumarin by rat,mouse, guinea pig,Cynomolgus monkey and human precision-cut liver slices

1. The metabolism of 50 μM 7-ethoxycoumarin and 50 μM [3-¹⁴C]coumarin has been studied in precision-cut liver slices from the male Sprague-Dawley rat, female DBA/2 mouse, male Dunkin-Hartley guinea pig, male Cynomolgus monkey and man.

2. In liver slices from all five species 7-ethoxycoumarin was metabolized to 7-hydroxycoumarin (7-HC), which was extensively conjugated with D-glucuronic acid and sulphate. In rat and mouse, 7-HC was preferentially conjugated with sulphate, whereas rates of glucuronidation and sulphation were similar in the other three species.

3. [3-¹⁴C]coumarin was metabolized by liver slices from all five species to various polar products and to metabolite(s) that bound covalently to liver slice proteins. In Cynomolgus monkey and both human subjects studied, 7-HC was the major metabolite that was conjugated with D-glucuronic acid and sulphate, whereas in rat the major metabolites were products of the 3-hydroxylation pathway and unknown metabolites. Major metabolites in mouse liver slices were 7-HC, 3-hydroxylation pathway products and unknown metabolites, and in guinea pig liver slices, 7-HC and unknown metabolites.

4. The metabolism of 7-ethoxycoumarin to free and conjugated 7-HC and [3-¹⁴C] coumarin to total polar products was greater in liver slices from mouse and Cynomolgus monkey than the other three species.

5. With liver slices from all five species there appeared to be little difference in the extent of metabolism of 7-ethoxycoumarin and [3-¹⁴C]coumarin to various products in either a complex tissue culture medium (RPMI 1640 plus foetal calf serum) or a simple balanced salt solution (Earle's balanced salt solution).

6. These results demonstrate that precision-cut liver slices are avaluable in vitro model system for investigating species differences in xenobiotic metabolism. Generally, the observed species differences in coumarin metabolism in vitro agree well with available in vivo data.     

Kinetic analysis of interactions between alkylene-linked bis-pyridiniumaldoximes and human acetylcholinesterases inhibited by various organophosphorus compounds

The therapeutic approach of organophosphorus compound (OP) intoxications is to reactivate the inhibited enzyme acetylcholinesterase (AChE). Numerous studies demonstrated a limited efficacy of standard oxime-based reactivators against different nerve agents such as tabun and cyclosarin. This emphasizes research for more effective oximes. In the present study, reactivation kinetics of tabun-, sarin-, cyclosarin-, VX- or paraoxon-ethyl-inhibited human AChE (hAChE) with a homologous series of bis-ortho-pyridiniumaldoximes, Ortho-4 - Ortho-9, was investigated with a robot-assisted setting, allowing determination of second-order reactivation rate constants as well as model calculations. The reactivation constants of Ortho-4 - Ortho-9 resulted in marked differences of affinity and reactivity depending on the OP structure and the linker length of the oximes. In general, the K_D values decreased with increasing linker length. Reactivity increased from Ortho-4 to Ortho-6 for PXE- and VX-inhibited hAChE and from Ortho-4 to Ortho-7 for GA-inhibited hAChE and decreased again with Ortho-8 and Ortho-9. In contrast, k_r decreased with increasing linker length for sarin- and cyclosarin-inhibited hAChE. In view of the pronounced decrease of K_D from Ortho-4 to Ortho-9, the k_r2 values increased with all tested OP. Hence, the ratios of K_I/K_D and of K_I/k_r2 showed that in almost all cases the affinity of Ortho-N to the native hAChE was higher than to OP-inhibited enzyme. Model calculations indicated that Ortho-6 - Ortho-9 could be superior to obidoxime in reactivating tabun-inhibited hAChE. Finally, these data emphasize the need to develop oximes with a higher selective affinity towards OP-inhibited hAChE in order to minimize possible side effects.     

Detoxification of nerve agents by a substituted β-cyclodextrin: Application of a modified biological assay

Chemical warfare agents (nerve agents) are still available and present a real threat to the population. Numerous in vitro and in vivo studies showed that various nerve agents, e.g. tabun and cyclosarin, are resistant towards standard therapy with atropine and oxime. Based on these facts we applied a modified biological assay for the easy, semi-quantitative testing of the detoxifying properties of the β-cyclodextrin derivative CD-IBA. Cyclosarin, sarin, tabun and VX were incubated with CD-IBA for 1–50 min at 37 °C, then an aliquot was added to erythrocyte acetylcholinesterase (AChE) and the percentage of AChE inhibition was determined. The validity of the assay was confirmed by concomitant quantification of tabun by GC–MS.     

Absorption of the nerve agent VX (O-ethyl-S-[2(di-isopropylamino)ethyl] methyl phosphonothioate) through pig, human and guinea pig skin in vitro

The physico-chemical properties of VX make the skin the most likely route of absorption into the human body. The development of effective medical countermeasures against such percutaneous threat agents relies on the use of appropriate animal models, as the inherent toxicity of nerve agents precludes the use of human volunteers. Previous studies have characterised the mechanism of nerve agent toxicity in rodent models, however, it is generally accepted that one of the most appropriate animal models for human skin absorption is the domestic pig. The purpose of the present study was to measure and compare the skin absorption kinetics of VX in vitro using pig, human and guinea pig skin to highlight any potential species differences in skin permeability. When undiluted VX was applied directly to the skin, the permeability of guinea pig skin was approximately 7-fold greater than human skin. There was no significant difference in the permeability of pig and human skin. When VX diluted with isopropyl alcohol was applied to the skin, the permeability of guinea pig skin was approximately 4-fold greater than human skin. There was no significant difference in the permeability of pig and human skin. From this data it may be inferred that dermatomed, abdominal pig skin is an appropriate model for the human skin absorption of VX.     

Association mechanism of four acetylcholinesterase inhibitors (AChEIs) with human serum albumin: a biochromatographic approach

In this work, the interaction of a series of acetylcholinesterase inhibitors (AChEIs; donepezil, galanthamine, huperzine and neostigmine) with human serum albumin (HSA) immobilized on porous silica particles was studied using a biochromatographic approach. For all the tested AChEI molecules, linear retention plots were observed at all temperatures. An analysis of the thermodynamics (i.e. enthalpy (ΔH°), entropy ((S°*)) of the interaction of the AChEI molecules with the immobilized human serum albumin was also carried out. The (H° and (S°* values for donepezil, galanthamine and neostigmine, were negative due to van der Waals interactions and hydrogen bonding which govern this association with albumin. Whereas the positive values of (H° and (S°* of huperzine binding on HSA indicated a predominance of hydrophobic interactions. The association of AChEIs with HSA was increased linearly with pH. A comparative thermodynamic study with benzodiazepine molecules was also done to determine the potential binding site of these drugs on HSA.     

Potential of two new oximes in reactivate human acetylcholinesterase and butyrylcholinesterase inhibited by organophosphate compounds: An in vitro study

Organophosphate (OP) compounds exert inhibition on cholinesterase (ChE) activity by irreversibly binding to the catalytic site of the enzyme. Oximes are compounds generally used to reverse the ChE inhibition caused by OP agents. In this study, we compared the in vitro reactivation potency of two new oximes (oxime 1: butane-2,3-dionethiosemicarbazone; oxime 2: 3-(phenylhydrazono) butan-2-one) against the inhibition on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities induced by chlorpyrifos, diazinon and malathion. Oximes used clinically (obidoxime and pralidoxime) were used as positive control. For this study, human blood (erythrocytes for AChE determination and plasma for BChE determination) was used and different concentrations of oximes (1–100 μM) were tested. The concentrations of OP used were based on the IC₅₀ for AChE and BChE. Results demonstrated that obidoxime was more effective in reactivate the AChE inhibition induced by OP compounds. However, both newly developed oximes achieved similar reactivations rates that pralidoxime for chlorpyrifos and diazinon-inhibited AChE. For BChE reactivation, none of evaluated oximes achieved positives rates of reactivation, been obidoxime able to reactivate malathion-inhibited BChE only in 24% at the highest concentration. We conclude that both newly developed oximes seem to be promising reactivators of OP-inhibited AChE.     

Centchroman: Binding with plasma proteins of rat,rabbit, guinea pig,and rhesus monkey

Incubation of plasma samples rat, rabbit, guinea pig, and rhesus monkey showed a nonsaturable low affinity binding with ¹⁴C-centchroman. The Scatchard analysis of binding data revealed a low affinity and high capacity binding of centchroman with plasma protein. It was observed that ¹⁴C-centchroman did not compete with ³H-cortisol, ³H-estradiol, non-steroidal estrogenic/antiestrogenic agents, or with the binding sites of other steroidal sex hormones used with plasma samples of different animals. The characterization of centchroman bound complex with polyacrylamide gel electrophoresis showed that centchroman binds with plasma albumin at af of 0.68.     

Species difference in the induction of hepatic CYP1A subfamily enzymes, especially CYP1A2, by 2-methoxy-4-nitroaniline among rats, mice, and guinea pigs

Species difference in the induction of hepatic cytochrome P450 CYP1A subfamily enzymes by 2-methoxy-4-nitroaniline (2-MeO-4-NA) was investigated among male F344 rats, C57BL/6 Cr mice, and Hartley guinea pigs. All species of animals were treated with a single ip injection of 2-MeO-4-NA (0.44 mmol/kg body weight), and changes in levels of the mRNA and protein of hepatic cytochrome P4501A (CYP1A) subfamily enzymes were examined by the methods of RT-PCR and Western blot, respectively. In addition, hepatic microsomal enzyme activities were measured using methoxyresorufin and ethoxyresorufin as substrates of CYP1A2 and CYP1A1, respectively. The overall results of the RT-PCR, Western blot, and measurement of the enzyme activity indicated that 2-MeO-4-NA-mediated induction of hepatic CYP1A subfamily enzymes, especially CYP1A2, occurred only in rats but not any other species of animals examined and that the species difference in the CYP1A induction was not necessarily correlated with that in pharmacokinetics of 2-MeO-4-NA. Furthermore, a luciferase reporter gene assay for screening of the ligands of arylhydrocarbon receptor (AhR) using a rat hepatic cell line suggested that 2-MeO-4-NA is not an AhR ligand. The present findings demonstrate for the first time the species difference in the 2-MeO-4-NA-mediated induction of hepatic CYP1A subfamily enzymes between rats and other rodents, mice and guinea pigs, and further propose an AhR-independent pathway for 2-MeO-4-NA-mediated induction in rats.     

SKF 104353, a high affinity antagonist for human and guinea pig lung leukotriene D4 receptor, blocked phosphatidylinositol metabolism and thromboxane synthesis induced by leukotriene D4

SKF 104353 (2(S)-hydroxyl-3(R)-carboxyethylthio)-3-[2-(8-phenyloctyl) phenyl] propanoic acid) is a synthetic structural analog of leukotrienes D4 and E4 (LTD4, LTE4). This compound binds to guinea pig and human lung LTD4 receptors with affinities (Ki) of 5 +/- 2 and 10 +/- 3 nM, respectively. The Ki values of a reference compound, FPL 55712, were 2200 and 4500 nM, respectively, approximately 400- and 500-fold less effective than SKF 104353. LTD4- and LTE4-induced biosynthesis of thromboxane B2 has been shown to be mediated by LTD4 receptors in guinea pig lung in vitro. SKF 104353 did not induce synthesis of TxB2 in this system at concentrations of 1-20 microM. When SKF 104353 and increasing concentrations of LTD4 were incubated with guinea pig lung, the dose response curve of LTD4-induced TxB2 biosynthesis was shifted to the right with a -log[KB] = 8.4 +/- 0.2. LTD4-induced phosphatidylinositol (PI) hydrolysis in guinea pig lung has been shown to be the major signal transduction mechanism. In this system, SKF 104353 (1-20 microM) did not promote PI hydrolysis. Pretreatment of the [3H]myo-inositol-labeled guinea pig lung with SKF 104353 shifted the LTD4-induced PI hydrolysis dose response curve to the right, indicating that SKF 104353 inhibited LTD4 receptor-mediated intracellular second messenger formation. These results demonstrate that SKF 104353 is a high affinity, specific LTD4 receptor antagonist. It inhibited LTD4-induced PI hydrolysis and TxB2 biosynthesis in guinea pig lung. SKF 104353 may prove to be an important research tool for research on the activities of leukotrienes and of value therapeutically in the treatment of leukotriene-mediated diseases.     

Inhibition of acetylcholinesterase by two genistein derivatives: kinetic analysis,molecular docking and molecular dynamics simulation

In this study two genistein derivatives (G1 and G2) are reported as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and differences in the inhibition of AChE are described. Although they differ in structure by a single methyl group, the inhibitory effect of G1 (IC₅₀=264 nmol/L) on AChE was 80 times stronger than that of G2 (IC₅₀=21,210 nmol/L). Enzyme-kinetic analysis, molecular docking and molecular dynamics (MD) simulations were conducted to better understand the molecular basis for this difference. The results obtained by kinetic analysis demonstrated that G1 can interact with both the catalytic active site and peripheral anionic site of AChE. The predicted binding free energies of two complexes calculated by the molecular mechanics/generalized born surface area (MM/GBSA) method were consistent with the experimental data. The analysis of the individual energy terms suggested that a difference between the net electrostatic contributions (ΔE_ele+ΔG_GB) was responsible for the binding affinities of these two inhibitors. Additionally, analysis of the molecular mechanics and MM/GBSA free energy decomposition revealed that the difference between G1 and G2 originated from interactions with Tyr124, Glu292, Val294 and Phe338 of AChE. In conclusion, the results reveal significant differences at the molecular level in the mechanism of inhibition of AChE by these structurally related compounds.KEY WORDS: Genistein derivatives, Acetylcholinesterase (AChE), Kinetics analysis, Molecular docking, Molecular dynamics simulation, MM/GBSAAbbreviations: ACh, acetylcholine; AChEIs, acetylcholinesterase inhibitors; AChE, acetylcholinesterase; AD, Alzheimer׳s disease; BuChE, butyrylcholinesterase; BuSCh, S-butyrylthiocholine chloride; CAS, catalytic active site; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); GAFF, generalized AMBER force field; G1, 3-(4-methoxyphenyl)-7-(2-(piperidin-1-yl)ethoxy)-4H-chromen-4-one; G2, (S)-3-(4-methoxyphenyl)-7-(2-(2-methylpiperidin-1-yl)ethoxy)-4H-chromen-4-one; iso-OMPA, tetraisopropyl pyrophosphoramide; MD, molecular dynamics; MM/GBSA, molecular mechanics/generalized born surface area; PAS, peripheral anionic site; PDB, protein data bank; PME, particle mesh Ewald; RMSD, root-mean-square deviation; S-ACh, acetylthiocholine iodide; ΔE_ele, electrostatic energy contribution; ΔE_MM, gas-phase interaction energy between receptor and ligand; ΔE_vdw, van der Waals energy contribution; SASA, solvent accessible surface area; ΔG_exp, experimental binding free energy; ΔG_GB, polar desolvation energy term; ΔG_pred, total binding free energy; ΔG_SA, nonpolar desolvation energy term; ΔS, conformational entropy contribution     

异源性Th表位修饰的CTLA4-Ig—msBlys构建及融合蛋白表达

目的构建异源性Th表位修饰的CTLA4-Ig-msBlys的原核表达质粒,并在EcoliBL21中诱导融合蛋白表达,初步检测蛋白特性。方法采用PCR法从质粒pCMV-sBlys扩增sBlys片段,将与卵清蛋白（ovalbumin,OVA）Th表位重组后的msBlys连接CTLA4-Ig片段,克隆入pGEX-KG原核表达载体,用LB固体培养基抗生素筛选、酶切并经琼脂糖凝胶电泳鉴定;将质粒pGEX—KG-CTI。A4-Ig—msBlys转入E．coliBL21菌株中,实现插入基因的融合表达,用SDS-PAGE和Western-blot检测表达产物。结果pGEX—KG—GTLA4-Ig—msBlys构建正确且最终转人E．coliBL21,得到融合蛋白的表达;CTLA4-Ig-msBlys融合蛋白在E．coli中获得表达,表达产物的相对分子质量同预期值一致。结论成功构建原核表达质粒pGEX-KG-CTLA4-Ig-msBlys,并在E．coliBL21表达CTLA4-Ig-msBlys融合蛋白,为下一步探讨Blys在自身免疫性疾病患者的治疗作用奠定了基础。     

Inhibition of rabbit brain 4-aminobutyrate transaminase by some taurine analogues: a kinetic analysis

The use of the antiepileptic drug, 4-aminobutyrate transaminase (GABA-T) inhibitor vigabatrin (VIGA), has been recently cautioned because it is associated to irreversible field defects from damage of the retina. Since novel GABA-T inhibitors might prove useful in epilepsy or other CNS pathologies as VIGA substitutes, the aim of the present investigation was to characterize the biochemical properties of some taurine analogues (TA) previously shown to act as GABA-T inhibitors. These include (+/-)piperidine-3-sulfonic acid (PSA), 2-aminoethylphosphonic acid (AEP), (+/-)2-acetylaminocyclohexane sulfonic acid (ATAHS) and 2-aminobenzenesulfonate (ANSA). Kinetic analysis of the activity of partially purified rabbit brain GABA-T in the presence of VIGA and TA showed that PSA and AEP caused a linear, mixed-type inhibition (Ki values 364 and 1010 microM, respectively), whereas VIGA, ANSA and ATAHS behaved like competitive inhibitors (Ki values 320, 434 and 598 microM, respectively). Among the compounds studied, only VIGA exerted a time-dependent, irreversible inhibition of the enzyme, with Ki and k(inact) values of 773 microM and 0.14 min(-1), respectively. Furthermore, the ability of VIGA and TA to enhance GABA-ergic transmission was assessed in rabbit brain cortical slices by NMR quantitative analysis. The results demonstrate that VIGA as well as all TA promoted a significant increase of GABA content. In conclusion, PSA, ANSA and ATAHS, reversible GABA-T inhibitors with Ki values close to that of VIGA, represent a new class of compounds, susceptible of therapeutic exploitation in many disorders associated with low levels of GABA in brain tissues.     

Efficacy of a combination of acetylcholinesterase reactivators,HI-6 and obidoxime,against tabun and soman poisoning of mice

The bispyridinium oxime HI-6, 1-((((4-amino-carbonyl) pyridinio)methoxy) methyl)-2-(hydroxyimino)-methyl) pyridinium dichloride monohydrate, combined with atropine is an effective treatment for soman (pinacolyl methylphosphonofluoridate) poisoning but is relatively ineffective against tabun (ethyl N-dimethyl phosphoroamidocyanidate) poisoning in mice. This contrasts with those results obtained using the bispyridinium oxime obidoxime [1,1-(oxy bis(methylene)) bis(4-(hydroxyimino)-methyl) pyridinium dibromide]. The purpose of this study was to investigate the efficacy of the combination of HI-6 and obidoxime plus atropine against poisoning by tabun and soman in mice. The combination of ineffective single doses of obidoxime (5 or 10 mg/kg) and HI-6 (25 or 50 mg/kg) improved the treatment of tabun poisoning over either oxime alone. Combinations employing higher concentrations of obidoxime (25 or 50 mg/kg) and HI-6 (100 or 200 mg/kg) resulted in significant toxicity in the absence of organophosphate poisoning. Against soman poisoning the addition of obidoxime to HI-6 did not attenuate the efficacy of HI-6. The half-life of elimination and peak serum concentrations of HI-6 and obidoxime were not altered following administration of the combined injection. Reactivation of tabun-inhibited acetylcholinesterase was found consistently in the diaphragm but not in the brain. Using response surface methods it was possible to estimate the optimal therapy against soman and tabun poisoning (74.5 mg/kg HI-6+31.9 mg obidoxime against 1052 g/kg challenge of tabun and 129 mg/kg HI-6 +0 mg/kg obidoxime against 390 g/kg challenge of soman). It is proposed that reactivation of tabun inhibited acetylcholinesterase at the diaphragm may be responsible for the increased efficacy of the combination of HI-6 and obidoxime against tabun poisoning in mice.     

Blockade of human atrial 5-HT4 receptors by SB 207710, a selective and high affinity 5-HT4 receptor antagonist

The mode of antagonism of 5-hydroxytryptamine-induced positive inotropic effects by the highly selective 5-HT₄ receptor antagonist SB 207710 (1-butyl4-piperidinyl) methyl 8-amino-7-iodo-1,4-benzodioxan-5-carboxylate was investigated on isolated preparations of human right atrial appendage. SB 207 710 caused concentration-dependent (0.1–10 nmol/l) surmountable antagonism of the effects of 5-hydroxytryptamine with a pK_B (mol/l) of 10.1. Due to its high selectivity and affinity, SB 207710 could be a powerful tool for the comparison of human atrial 5-HT₄ receptors with 5-HT₄ receptors of other organs of man and other species.