No-observed-adverse-effect-level (NOAEL) assessment as an optimized dose of cholinesterase reactivators for the treatment of exposure to warfare nerve agents in mice.

Despite the international convention on the prohibition of chemical weapons ratified in 1997, the threat of conflicts and terrorist attacks involving such weapons still exists. Among these, organophosphorus-nerve agents (OPs) inhibit cholinesterases (ChE) causing cholinergic syndrome. The reactivation of these enzymes is therefore essential to protect the poisoned people. However, these reactivating molecules, mainly named oximes, have major drawbacks with limited efficacy against some OPs and a non-negligible ChE inhibitor potential if administered at an inadequate dose, an effect that they are precisely supposed to mitigate. As a result, this project focused on assessing therapeutic efficacy, in mice, up to the NOAEL dose, the maximum dose of oxime that does not induce any observable toxic effect. NOAEL doses of HI-6 DMS, a reference oxime, and JDS364. HCl, a candidate reactivator, were assessed using dual-chamber plethysmography, with respiratory ventilation impairment as a toxicity criterion. Time-course modeling parameters and pharmacodynamic profiles, reflecting the interaction between the oxime and circulating ChE, were evaluated for treatments at their NOAEL and higher doses. Finally, the therapeutic potential against OPs poisoning was determined through the assessment of protective indices. For JDS364. HCl, the NOAEL dose corresponds to the smallest dose inducing the most significant therapeutic effect without causing any abnormality in ChE activity. In contrast, for HI-6 DMS, its therapeutic benefit was observed at doses higher than its NOAEL, leading to alterations in respiratory function. These alterations could not be directly correlated with ChE inhibition and had no adverse effects on survival. They are potentially attributed to the stimulation of non-enzymatic cholinergic targets by HI-6 DMS. Thus, the NOAEL appears to be an optimal dose for evaluating the efficacy of oximes, particularly when it can be linked to respiratory alterations effectively resulting from ChE inhibition.

Oxidative stress status assessment of rats' brains injury following subacute exposure to K-oximes.

Oxidative stress status and morphological injuries in the brain of Wistar rats induced by repeated application of selected acetylcholinesterase reactivators - asoxime, obidoxime, K027, K048, K074, and K075 were evaluated. Each oxime in a dose of 0.1 of LD50/kg im was given 2x/week for 4 weeks. Markers of lipid peroxidation (malondialdehyde, MDA), and protein oxidation (advanced oxidation protein products, AOPP), as well as the activity of antioxidant enzymes (catalase, CAT, superoxide dismutase, SOD, glutathione reductase, GR, and glutathione peroxidase, GPx), were estimated in the brain tissue homogenates on day 35 of the study. Brain alterations were carefully quantified by semiquantitative grading scales - brain damage score (BDS). Oxidative stress parameters, MDA and AOPP were significantly highest in the asoxime-, obidoxime- and K075-treated groups (p < 0.001). The activity of SOD and CAT was significantly elevated in the obidoxime-, K048-, and K075-treated groups (p < 0.001). Besides, GR was markedly decreased in the obidoxime- and K074-treated groups (p < 0.01), while treatment with K048, K074 and K075 induced extremely high elevation in GPx levels (p < 0.001). In the same groups of rats, brain alterations associated with polymorphonuclear cell infiltrate were significantly more severe than those observed in animals receiving only asoxime or K027 (p < 0.001). The presented results confirmed that treatment with different oximes significantly improved the oxidative status and attenuated signs of inflammation in rats' brains. Presented results, together with our previously published data can help to predict likely adverse systemic toxic effects, and target organ systems, which are crucial for establishing risk categories, as well as in dose selection of K-oximes as drug candidates.

Theoretical assessment of the performances of commercial oximes on the reactivation of acetylcholinesterase inhibited by the nerve agent A-242 (novichok).

The nerve agents of the A-series are relatively recent chemical weapons with no antidote available yet. Once inside the human body, those chemicals act similarly to the classic nerve agents, by binding to the catalytic residue Serine 203 (Ser203) of human acetylcholinesterase (HssAChE) and thus preventing the proper function of this enzyme. However, there is no experimental evidence yet if the current antidotes for intoxication by nerve agents are also capable of restoring AChE inhibited by the nerve agents of the A-series. In order to launch some light on this issue, we used computational techniques (molecular docking, molecular dynamics and MM-PBSA interaction energy calculations) to assess the performances of the four currently available commercial oximes (2-PAM, HI-6, obidoxime and trimedoxime) when in contact with HssAChE inhibited by the agent A-242. Based on the near-attack conformation (NAC) criterion, our results suggest that the commercial oximes would have limited efficacy to reactivate the enzyme since they are not able to properly approach the adduct Ser203-A-242. Among those oximes, trimedoxime seems to be the most promising, since it showed lower values of energy in the MM-PBSA calculations, a higher stability inside the catalytic anionic center (CAS) of HssAChE, and was able to adopt a position closer to the NAC that could enable the reactivation mechanism.

A preclinical assessment to repurpose drugs to target type 1 diabetes-associated type B coxsackieviruses.

AIM: To screen several antiviral drugs systematically for their efficacy against type B coxsackieviruses. METHODS: Ten drugs with different antiviral mechanisms were analysed for their efficacy against prototype strains of type B coxsackieviruses in A549 cells. Cell viability was quantified in fixed cells using a colorimetric assay. Median effective dose was interpolated from the triplicated experiments and the dose-response curves were generated for each drug-virus combination. Drug cytotoxicity was similarly quantified and selectivity indices calculated. RESULTS: Hizentra, pleconaril, fluoxetine, norfluoxetine, ribavirin, favipiravir, and guanidine hydrochloride were able to abrogate infection by all tested viruses, with the exception of complete inefficacy of pleconaril against coxsackievirus B3 and favipiravir against coxsackievirus B2. The effective doses for Hizentra, enviroxime, ribavirin, favipiravir, and pleconaril were clearly below their therapeutic serum concentrations, while the effective concentrations of fluoxetin, norfluoxetine and itraconazole exceeded their therapeutic serum concentrations. Lovastatin and azithromycin did not efficiently block type B coxsackieviruses. CONCLUSION: Hizentra, enviroxime, pleconaril, ribavirin, and favipiravir are effective against type B coxsackieviruses in vitro in their therapeutic serum concentrations. These antiviral drugs are therefore attractive candidates for type 1 diabetes prevention/treatment trials. They can also be used in other clinical conditions caused by type B coxsackieviruses.

Assessment of ionizable, zwitterionic oximes as reactivating antidotal agents for organophosphate exposure.

Since the development in the 1950's of 2-PAM (Pralidoxime), an antidote that reactivates organophosphate conjugated acetylcholinesterase in target tissues upon pesticide or nerve agent exposure, improvements in antidotal therapy have largely involved congeneric pyridinium aldoximes. Despite seminal advances in detailing the structures of the cholinesterases as the primary target site, progress with small molecule antidotes has yet to define a superior agent. Two major limitations are immediately apparent. The first is the impacted space within the active center gorge, particularly when the active center serine at its base is conjugated with an organophosphate. The reactivating nucleophile will have to negotiate the tortuous gorge terrain to access the phosphorus atom with its most nucleophilic form or ionization state, the oximate anion. A second limitation stems from the antidote crossing the blood-brain barrier sufficiently rapidly, since it is well documented that central acetylcholinesterase inhibition gives rise to cardiovascular and respiratory compromise. The associated hypoxia then leads to a sequelae of events, including poor perfusion of the brain and periphery, along with muscle fasciculation, tremors and eventually seizures. We consider both the barriers confronting and further achievements necessary to enhance efficacy of antidotes.

An easy method for the determination of active concentrations of cholinesterase reactivators in blood samples: Application to the efficacy assessment of non quaternary reactivators compared to HI-6 and pralidoxime in VX-poisoned mice.

Organophosphorus nerve agents, like VX, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). AChE inhibited by VX can be reactivated using powerful nucleophilic molecules, most commonly oximes, which are one major component of the emergency treatment in case of nerve agent intoxication. We present here a comparative in vivo study on Swiss mice of four reactivators: HI-6, pralidoxime and two uncharged derivatives of 3-hydroxy-2-pyridinaldoxime that should more easily cross the blood-brain barrier and display a significant central nervous system activity. The reactivability kinetic profile of the oximes is established following intraperitoneal injection in healthy mice, using an original and fast enzymatic method based on the reactivation potential of oxime-containing plasma samples. HI-6 displays the highest reactivation potential whatever the conditions, followed by pralidoxime and the two non quaternary reactivators at the dose of 50 mg/kg bw. But these three last reactivators display equivalent reactivation potential at the same dose of 100 µmol/kg bw. Maximal reactivation potential closely correlates to surviving test results of VX intoxicated mice.

Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation.

Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.

Quantitative assessment of BRAF V600 mutant circulating cell-free tumor DNA as a tool for therapeutic monitoring in metastatic melanoma patients treated with BRAF/MEK inhibitors.

BACKGROUND: BRAF V600 mutant circulating cell-free tumor DNA (BRAF V600mut ctDNA) could serve as a specific biomarker in patients with BRAF V600 mutant melanoma. We analyzed the value of BRAF V600mut ctDNA from plasma as a monitoring tool for advanced melanoma patients treated with BRAF/MEK inhibitors. METHODS: Allele-specific quantitative PCR analysis for BRAF V600 E/E2/D/K/R/M mutations was performed on DNA extracted from plasma of patients with known BRAF V600 mutant melanoma who were treated with dabrafenib and trametinib. RESULTS: 245 plasma samples from 36 patients were analyzed. In 16 patients the first plasma sample was obtained before the first dosing of dabrafenib/trametinib. At baseline, BRAF V600mut ctDNA was detected in 75 % of patients (n = 12/16). BRAF V600mut ctDNA decreased rapidly upon initiation of targeted therapy (p < 0.001) and became undetectable in 60 % of patients (n = 7/12) after 6 weeks of treatment. During treatment, disease progression (PD) was diagnosed in 27 of 36 patients. An increase of the BRAF V600mut ctDNA copy number and fraction, identified PD with a sensitivity of 70 % (n = 19/27) and a specificity of 100 %. An increase in the BRAF V600mut ctDNA fraction was detected prior to clinical PD in 44 % of cases (n = 12/27) and simultaneously with PD in 26 % of patients (n = 7/27). CONCLUSIONS: Quantitative analysis of BRAF V600mut ctDNA in plasma has unique features as a monitoring tool during treatment with BRAF/MEK inhibitors. Its potential as an early predictor of acquired resistance deserves further evaluation.

Social determinants of health, inequality and social inclusion among people with disabilities / Determinantes sociais da saúde, iniquidades e inclusão social entre pessoas com deficiência / Determinantes sociales de la salud, iniquidades e inclusión social entre personas con deficiencia

OBJECTIVE: to analyze the socio-familial and community inclusion and social participation of people with disabilities, as well as their inclusion in occupations in daily life. METHOD: qualitative study with data collected through open interviews concerning the participants' life histories and systematic observation. The sample was composed of ten individuals with acquired or congenital disabilities living in the region covered by a Family Health Center. The social conception of disability was the theoretical framework used. Data were analyzed according to an interpretative reconstructive approach based on Habermas' Theory of Communicative Action. RESULTS: the results show that the socio-familial and community inclusion of the study participants is conditioned to the social determinants of health and present high levels of social inequality expressed by difficult access to PHC and rehabilitation services, work and income, education, culture, transportation and social participation. CONCLUSION: there is a need to develop community-centered care programs in cooperation with PHC services aiming to cope with poverty and improve social inclusion. .

OBJETIVO: analisar a inclusão sociofamiliar e comunitária e a participação social de pessoas com deficiência, bem como sua inserção em ocupações na vida cotidiana. MÉTODO: estudo qualitativo, com coleta de dados por meio de entrevistas abertas sobre história de vida e observação sistemática. A amostra foi composta por dez pessoas com deficiência, adquirida ou congênita, moradoras de região adstrita a um Núcleo de Saúde da Família. O referencial teórico foi a concepção social da deficiência. Os dados foram analisados segundo abordagem interpretativa reconstrutiva, fundamentada no referencial da Teoria da Ação Comunicativa de Habermas. RESULTADOS: os resultados evidenciaram que a inclusão sociofamiliar e comunitária dos sujeitos do estudo condiciona-se a determinantes sociais da saúde, apresentando índices de iniquidades sociais, expressos pela dificuldade de acesso a serviços de Atenção Primária à Saúde e de reabilitação, trabalho e renda, educação, cultura, transporte e participação social. CONCLUSÃO: conclui-se a necessidade da elaboração de programas de atenção centrados na comunidade, voltados ao enfrentamento da pobreza e à inclusão social, em articulação com serviços de Atenção Primaria à Saúde. .

OBJETIVO: analizar la inclusión social familiar y comunitaria, y la participación social de personas con deficiencia, así como su inserción en ocupaciones en la vida cotidiana. MÉTODO: estudio cualitativo, con recolección de datos por medio de entrevistas abiertas sobre historia de vida y por observación sistemática. La muestra estuvo compuesta por diez personas con deficiencia, adquirida o congénita, habitantes de una región adscrita a un Núcleo de Salud de la Familia. El referencial teórico fue la concepción social de la deficiencia. Los datos fueron analizados según abordaje interpretativo reconstructivo, fundamentado en el referencial de la Teoría de la Acción Comunicativa de Habermas. RESULTADOS: los resultados evidenciaron que la inclusión social familiar y comunitaria de los sujetos del estudio se condiciona a determinantes sociales de la salud, presentando índices de iniquidades sociales, expresados por la dificultad de acceso a servicios de Atención Primaria de la Salud y de rehabilitación, trabajo y renta, educación, cultura, transporte y participación social. CONCLUSIÓN: se concluye que existe la necesidad de elaborar programas de atención centrados en la comunidad, dirigidos al enfrentamiento de la pobreza y a la inclusión social, en articulación con servicios de Atención Primaria a la Salud. .

Co-clinical assessment identifies patterns of BRAF inhibitor resistance in melanoma.

Multiple mechanisms have been described that confer BRAF inhibitor resistance to melanomas, yet the basis of this resistance remains undefined in a sizable portion of patient samples. Here, we characterized samples from a set of patients with melanoma that included individuals at baseline diagnosis, on BRAF inhibitor treatment, and with resistant tumors at both the protein and RNA levels. Using RNA and DNA sequencing, we identified known resistance-conferring mutations in 50% (6 of 12) of the resistant samples. In parallel, targeted proteomic analysis by protein array categorized the resistant samples into 3 stable groups, 2 of which were characterized by reactivation of MAPK signaling to different levels and 1 that was MAPK independent. The molecular relevance of these classifications identified in patients was supported by both mutation data and the similarity of resistance patterns that emerged during a co-clinical trial in a genetically engineered mouse (GEM) model of melanoma that recapitulates the development of BRAF inhibitor resistance. Additionally, we defined candidate biomarkers in pre- and early-treatment patient samples that have potential for predicting clinical responses. On the basis of these observations, we suggest that BRAF inhibitor-resistant melanomas can be actionably classified using protein expression patterns, even without identification of the underlying genetic alteration.

Reversible cholinesterase inhibitors as pre-treatment for exposure to organophosphates: assessment using azinphos-methyl.

Pre-treatment with reversible acetylcholinesterase (AChE) inhibitors before organophosphorous compound (OPC) exposure can reduce OPC-induced mortality. However, pyridostigmine, the only substance employed for such prophylaxis, is merely efficacious against a limited number of OPCs. In search of more efficacious and broad-range alternatives, we have compared in vivo the ability of five reversible AChE inhibitors (pyridostigmine, physostigmine, ranitidine, tacrine and K-27) to reduce mortality induced by the OPC azinphos-methyl. Protection was quantified using Cox analysis by determining the relative risk (RR) of death in rats that were administered these AChE inhibitors in equitoxic dosage (25% of LD01) 30 min before azinphos-methyl exposure. Azinphos-methyl-induced mortality was significantly reduced by all five tested compounds as compared with the reference group that was only exposed to azinphos-methyl without prior pre-treatment (RR = 1). The most efficacious prophylactic agents were K-27 (RR = 0.15) and physostigmine (RR = 0.21), being significantly more efficacious than ranitidine (RR = 0.62) and pyridostigmine (RR = 0.37). Pre-treatment with tacrine (RR = 0.29) was significantly more efficacious than pre-treatment with ranitidine, but the difference between tacrine and pyridostigmine was not significant. Our results indicate that prophylactic administration of the oxime K-27 may be a promising alternative in cases of imminent OPC exposure.

Assessment of the drug interaction potential and single- and repeat-dose pharmacokinetics of the BRAF inhibitor dabrafenib.

The induction of CYP2C9 by dabrafenib using S-warfarin as a probe and the effects of a CYP3A inhibitor (ketoconazole) and a CYP2C8 inhibitor (gemfibrozil) on dabrafenib pharmacokinetics were evaluated in patients with BRAF V600 mutation-positive tumors. Dabrafenib single- and repeat-dose pharmacokinetics were also evaluated. S-warfarin AUC(0- ∞) decreased 37% and Cmax increased 18% with dabrafenib. Dabrafenib AUC(0- τ) and C(max) increased 71% and 33%, respectively, with ketoconazole. Hydroxy- and desmethyl-dabrafenib AUC(0-τ) increased 82% and 68%, respectively, and AUC for carboxy-dabrafenib decreased 16%. Dabrafenib AUC(0-τ) increased 47%, with no change in C(max), after gemfibrozil co-administration. Gemfibrozil did not affect systemic exposure to dabrafenib metabolites. Single- and repeat-dose dabrafenib pharmacokinetics were consistent with previous reports. All cohorts used the commercial capsules. More-frequent monitoring of international normalized ratios is recommended in patients receiving warfarin during initiation or discontinuation of dabrafenib. Substitution of strong inhibitors or strong inducers of CYP3A or CYP2C8 is recommended during treatment with dabrafenib.

Assessment of antidotal efficacy of cholinesterase reactivators against paraoxon: In vitro reactivation kinetics and physicochemical properties.

The search of proficient oximes as reactivators of irreversibly inhibited-AChE by organophosphate poisoning necessitates an appropriate assessment of their physicochemical properties and reactivation kinetics. Therefore, herein acid dissociation constant; pKa, lipophilicity; logP, polar surface area, hydrogen bond donor and acceptor counts of structurally different oximes (two tertiary oximes and thirteen pyridinium aldoxime derivatives) have been evaluated. The experimentally obtained data for pKa has been comparatively analyzed by using non-linear regression. Further the tested oximes were screened through in vitro reactivation kinetics against paraoxon-inhibited AChE. The pKa values of all the examined oximes were within the range of 7.50-9.53. pKa values of uncharged and mono-pyridinium oximes were in good correlation with their reactivation potency. The high negative logP values of pyridinium oxime reactivators indicate their high hydrophilic character; hence oximes with improved lipophilicity should be designed for the development of novel and more potent antidotes. Propane and butane linked oximes were superior reactivators than xylene linked bis-oxime reactivators. It is concluded from the present study that pKa value is not only ruled by the position of oximino functionality in the pyridinium ring, but also by the position of linker. Although, pyridinium oximes are proved to be better reactivators but their lipophilicity has to be improved.

QSAR models for the reactivation of sarin inhibited acetylcholinesterase by quaternary pyridinium oximes based on Monte Carlo method.

Monte Carlo method has been used as a computational tool for building QSAR models for the reactivation of sarin inhibited acetylcholinesterase (AChE) by quaternary pyridinium oximes. Simplified molecular input line entry system (SMILES) together with hydrogen-suppressed graph (HSG) was used to represent molecular structure. Total number of considered oximes was 46 and activity was defined as logarithm of the AChE reactivation percentage by oximes with concentration of 0.001 M. One-variable models have been calculated with CORAL software for one data split into training, calibration and test set. Computational experiments indicated that this approach can satisfactorily predict the desired endpoint. Best QSAR model had the following statistical parameters: for training set r2=0.7096, s=0.177, MAE=0.148; calibration set: r2=0.6759, s=0.330, MAE=0.271 and test set: r2=0.8620, s=0.182, MAE=0.150. Structural indicators (SMILES based molecular fragments) for the increase and the decrease of the stated activity are defined. Using defined structural alerts computer aided design of new oxime derivatives with desired activity is presented.

Good manufacturing practice: manufacturing of a nerve agent antidote nanoparticle suspension.

We have established a current good manufacturing practice (GMP) manufacturing process to produce a nanoparticle suspension of 1,1'-methylenebis-4-[(hydroxyimino)methyl]pyridinium dimethanesulfonate (MMB4 DMS) in cottonseed oil (CSO) as a nerve agent antidote for a Phase 1 clinical trial. Bis-pyridinium oximes such as MMB4 were previously developed for emergency treatment of organophosphate nerve agent intoxication. Many of these compounds offer efficacy superior to monopyridinium oximes, but they have poor thermal stability due to hydrolytic cleavage in aqueous solution. We previously developed a nonaqueous nanoparticle suspension to improve the hydrothermal stability, termed Enhanced Formulation (EF). An example of this formulation technology is a suspension of MMB4 DMS nanoparticles in CSO. Due to the profound effect of particle size distribution on product quality and performance, particle size must be controlled during the manufacturing process. Therefore, a particle size analysis method for MMB4 DMS in CSO was developed and validated to use in support of good laboratory practice/GMP development and production activities. Manufacturing of EF was accomplished by milling MMB4 DMS with CSO and zirconia beads in an agitator bead mill. The resulting bulk material was filled into 5-mL glass vials at a sterile fill facility and terminally sterilized by gamma irradiation. The clinical lot was tested and released, a Certificate of Analysis was issued, and a 3-year International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) stability study started. The drug product was placed in storage for Phase 1 clinical trial distribution. A dose delivery uniformity study was undertaken to ensure that the correct doses were delivered to the patients in the clinic.

Kinetic assessment of N-methyl-2-methoxypyridinium species as phosphonate anion methylating agents.

Organophosphate nerve agents and pesticides are potent inhibitors of acetylcholinesterase (AChE). Although oxime nucleophiles can reactivate the AChE-phosphyl adduct, the adduct undergoes a reaction called aging. No compounds have been described that reactivate the aged-AChE adduct. A family of 2-methoxypyridinium species which reverse aging in a model system is presented. A kinetic study of this system, which includes an SAR analysis, demonstrates that the reaction is highly tunable based on the ring substituents.

Assessment of neuromuscular dysfunction during poisoning by organophosphorus compounds.

Dysfunction of respiratory muscles is a life-threatening complication in poisoning by organophosphorus compounds (OPs). It is both of central and peripheral origin due to impaired cholinergic signalling upon inhibition of acetylcholinesterase (AChE). The dysfunction at neuromuscular synapses is not amenable to anticholinergics and remains a therapeutic challenge. Thus, a clear understanding of the distinct mechanisms occurring at neuromuscular synapses is decisive for the development and improvement of therapeutic strategies, particularly with nerve agent poisoning, where clinical studies are prevented by ethical considerations. Using red blood cell AChE, the kinetics of OP induced inhibition, aging, and spontaneous and oxime-induced reactivation have been elucidated. In a dynamically working in vitro model with real-time determination of membrane-bound AChE, it was shown that the kinetic constants derived from erythrocyte AChE are comparable to muscle AChE in a given species. To assess, whether kinetic considerations of AChE activity are relevant for the neuromuscular function, organotypic spinal cord-skeletal muscle cocultures have been established. In this model neostigmine and VX affected neuromuscular transmission as anticipated from their known actions on AChE. Also oxime-induced restoration of the neuromuscular transmission was observed. These findings were confirmed by functional studies on diaphragm muscles of various species with determination of muscle force generation upon phrenic nerve or indirect electrical field stimulation techniques. Investigations with human intercostal muscles are in progress to assess the conditions in human tissue. The results obtained with paraoxon favourably correlate with data from clinical findings of parathion-poisoned patients where the correlation of neuromuscular transmission with the activity of erythrocyte AChE could be established. In conclusion, a variety of methods are available to follow the microscopic reactions occurring at the synaptic level. Due to the lack of clinical data with different OPs, e.g. nerve agents, well designed animal experiments, reflecting the human situation as close as possible, are indispensable for the development of new drugs against the deleterious OP effects.

Synthesis, toxicological and pharmacological assessment of morpholinooximes.

The synthesis, pharmacology and toxicology of four morpholine derivatives from 1-(2-arylmorpholino)-3-phenyl-3-propanonoxime and the synthesis of two anilides are described. The structures of the synthesized derivatives were proved by IR, 1H NMR and occasionally with 13C NMR. The acute toxicity of the compounds in mice was determined. A comparative pharmacological study of the in vivo effect on the central nervous system was realised by the following screening tests: pentobarbital induced sleeping time, locomotor activity and behaviour despair test for antidepressive activity. The most active compound was 1-(2-phenylmorpholino)-3-phenyl-3-propanonoxime (2b) which showed low toxicity and antidepressive activity at a dose of 1/10 LD50.

Alcohol aversion in the rat: behavioral assessment of noxious drug effects.

Injections of p-chlorophenylalanine or n-butyraldoxime given after rats were first given a 10-minute drinking test with saccharin or ethanol solutions produced a learned aversion to these solutions. These findings suggest that the reduced self-selection of alcohol (preference) resulting from the administration of these drugs, reported by others, is not specifically alcohol-related. The technique described offers a sensitive procedure for the assessment of unpleasant effects of drugs.