Inhibitor Report for: Trichlorfon~Metrifonate

Organophosphate (OP) insecticides have been used indiscriminately, based on their high dissipation rates and low residual levels in the environment. Despite the toxicity of OPs to beneficial insects is principally devoted to the acetylcholinesterase (AChE) inhibition, the physiological mechanisms underlying this activity remain poorly understood. Here we showed the pharmacological pathways that might be involved in severe alterations in the insect locomotion and grooming behaviors following sublethal administration of the OP Trichlorfon (Tn) (0.25, 0.5 and 1microM) in Phoetalia pallida. Tn inhibited the acetylcholinesterase activity (46+/-6, 38+/-3 and 24+/-6nmol NADPH/min/mg protein, n=3, p<0.05), respectively. Tn (1microM) also increased the walking maintenance of animals (46+/-5s; n=27; p<0.05). Tn caused a high increase in the time spent for this behavior (344+/-18s/30min, 388+/-18s/30min and 228+/-12s/30min, n=29-30, p<0.05, respectively). The previous treatment of the animals with different cholinergic modulators showed that pirenzepine>atropine>oxotremorine>d-tubocurarine>tropicamide>methoctramine induced a decrease on Tn (0.5microM)-induced grooming increase, respectively in order of potency. Metoclopramide (0.4microM), a DA-D2 selective inhibitor decreased the Tn-induced grooming activity (158+/-12s/30min; n=29; p<0.05). Nevertheless, the effect of the selective DA-D1 receptor blocker SCH 23390 (1.85microM) on the Tn (0.5microM)-induced grooming increase was significative and more intense than that of metoclopramide (54+/-6s/30min; n=30; p<0.05). Taken together the results suggest that a cross-talking between cholinergic M1/M3 and dopaminergic D1 receptors at the insect nervous system may play a role in the OP-mediated behavioral alterations.

The present experiment was designed to elucidate whether chronic dietary treatment with nimodipine (3 months, 1000 ppm) enhances water maze spatial navigation, passive avoidance behavior and locomotor activity, and whether such a treatment with nimodipine would interact with the therapeutic effect of acute metrifonate treatment. In young medial septum-lesioned rats, nimodipine had no effect by its own on cognitive or motor behavior, and did not enhance the water maze and passive avoidance behavior improving action of metrifonate (3 and 10 mg/kg. p.o.). Nimodipine treatment of aged rats did not markedly affect the deficit in motor performance. Single and combined nimodipine and metrifonate (3 and 10 mg/kg, p.o.) treatment of aged rats resulted in shorter escape distance values to the hidden water maze escape platform compared to those of control aged rats. The passive avoidance performance of aged rats was more effectively facilitated by a combined nimodipine and metrifonate treatment than by either of the drugs on their own. Following a washout period of 2.5 months the rats that were treated previously with nimodipine no longer performed better than aged controls in the water maze test. Furthermore, after the washout period metrifonate 10 mg/kg was no longer effective in improving the water maze behavior of the now 26-month-old rats irrespective of their chronic pretreatment. Taken together, these findings indicate that chronic nimodipine and acute metrifonate treatment may more effectively stimulate cognitive functioning than either of the treatments on their own.

We reviewed 130 admissions of organophosphate poisoning and analyzed the causes of death. Fenitrothion, malathion, dichlorvos, trichlorfon and fenitrothion/malathion were the most frequent chemicals involved. Mortality was 25% (32/130); delay in discovery and transport (18 cases), insufficient respiratory management (8 cases), and severe underlying or co-existing diseases (6 cases) were noted in the lethalities. Insufficient respiratory management consisted of delay in endotracheal intubation (5 cases) and failure in weaning (3 cases). About 3/4 of the severely serum cholinesterase-depressed cases needed ventilators. This suggests that better respiratory management would improve the outcome of organophosphate poisonings. Close observation of the clinical symptoms is essential, and detection of changes in serum cholinesterase may be helpful.

Organophosphate (OP) insecticides have been used indiscriminately, based on their high dissipation rates and low residual levels in the environment. Despite the toxicity of OPs to beneficial insects is principally devoted to the acetylcholinesterase (AChE) inhibition, the physiological mechanisms underlying this activity remain poorly understood. Here we showed the pharmacological pathways that might be involved in severe alterations in the insect locomotion and grooming behaviors following sublethal administration of the OP Trichlorfon (Tn) (0.25, 0.5 and 1microM) in Phoetalia pallida. Tn inhibited the acetylcholinesterase activity (46+/-6, 38+/-3 and 24+/-6nmol NADPH/min/mg protein, n=3, p<0.05), respectively. Tn (1microM) also increased the walking maintenance of animals (46+/-5s; n=27; p<0.05). Tn caused a high increase in the time spent for this behavior (344+/-18s/30min, 388+/-18s/30min and 228+/-12s/30min, n=29-30, p<0.05, respectively). The previous treatment of the animals with different cholinergic modulators showed that pirenzepine>atropine>oxotremorine>d-tubocurarine>tropicamide>methoctramine induced a decrease on Tn (0.5microM)-induced grooming increase, respectively in order of potency. Metoclopramide (0.4microM), a DA-D2 selective inhibitor decreased the Tn-induced grooming activity (158+/-12s/30min; n=29; p<0.05). Nevertheless, the effect of the selective DA-D1 receptor blocker SCH 23390 (1.85microM) on the Tn (0.5microM)-induced grooming increase was significative and more intense than that of metoclopramide (54+/-6s/30min; n=30; p<0.05). Taken together the results suggest that a cross-talking between cholinergic M1/M3 and dopaminergic D1 receptors at the insect nervous system may play a role in the OP-mediated behavioral alterations.

Acylpeptide hydrolase (APH) unblocks N-acetyl peptides. It is a major serine hydrolase in rat blood, brain, and liver detected by derivatization with (3)H-diisopropyl fluorophosphate (DFP) or a biotinylated fluorophosphonate. Although APH does not appear to be a primary target of acute poisoning by organophosphorus (OP) compounds, the inhibitor specificity of this secondary target is largely unknown. This study fills the gap and emphasizes blood APH as a potential marker of OP exposure. The most potent in vitro inhibitors for human erythrocyte and mouse brain APH are DFP (IC(50) 11-17 nM), chlorpyrifos oxon (IC(50) 21-71 nM), dichlorvos (IC(50) 230-560 nM), naled (IC(50) 370-870 nM), and their analogs with modified alkyl substituents. (3)H-diisopropyl fluorophosphate is a potent inhibitor of mouse blood and brain APH in vivo (ED(50) 0.09-0.2 mg/kg and 0.02-0.03 mg/l for ip and vapor exposure, respectively). Mouse blood and brain APH and blood butyrylcholinesterase (BChE) are of similar sensitivity to DFP in vitro and in vivo (ip and vapor exposure), but APH inhibition is much more persistent in vivo (still >80% inhibition after 4 days). The inhibitory potency of OP pesticides in vivo in mice varies from APH selective (dichlorvos, naled, and trichlorfon), to APH and BChE selective (profenofos and tribufos), to ChE selective or nonselective (many commercial insecticides). Sarin administered ip at a lethal dose to guinea pigs inhibits blood acetylcholinesterase and BChE completely but erythrocyte APH only partially. Blood APH activity is therefore a sensitive marker for exposure to some but not all OP pesticides and chemical warfare agents.

Alzheimer's disease (AD) is characterized in the brain by the deposition of amyloid protein outside the neuron, resulting in the formation of plaques, and inside the neuron with neurofibrillary tangles. It is not yet known what causes these pathologic changes, although age and genetics are major risk factors. The cholinesterase inhibitors tacrine and donepezil block acetylcholinesterase and therefore preserve the neurotransmitter acetylcholine. Three other investigational cholinesterase inhibitors are rivastigmine, metrifonate, and galanthamine. Existing therapies being studied for use in AD include vitamin E, estrogen preparations, and anti-inflammatory agents. The physician's role is to care for both the AD patient and the family, which are profoundly affected by this disease.

Forty-seven patients with probable Alzheimer disease (AD) completed a 6-month double-blind study to compare metrifonate with placebo. The Alzheimer Disease Assessment Scale cognitive subscale score of the metrifonate group treated to a 50-70% inhibition of red blood cell acetylcholinesterase activity differed significantly from the placebo group score by 1.8 points (p < 0.03) due to a deterioration in cognitive performance in the placebo group (p < 0.01). Statistically significant deterioration also occurred in the Mini-Mental State Examination scores (p < 0.01) in the placebo-treated group. Adverse effects were uncommon and did not require adjustment of the dose of metrifonate or discontinuation of treatment. These findings extend our previous report of a favorable effect of metrifonate on cognitive symptoms in AD by showing clinical, not only statistical, significance.

Metrifonate is a cholinesterase inhibitor with a long-lasting inhibition that raises brain acetylcholine levels. It is well-absorbed and has limited binding to serum proteins. In preliminary studies of its utility in the treatment of Alzheimer disease's (AD), it led to improvements of cognition or reduced the rate of decline of cognition compared with placebo. It also benefited the global function of these patients. Side effects include nausea, cramping, and diarrhea. Metrifonate has promise as a well-tolerated treatment of the symptoms of AD.

The efficacy and safety of metrifonate, an acetylcholinesterase inhibitor, was evaluated clinically in patients diagnosed with mild to moderate Alzheimer's disease (AD). This was a prospective, 30-week, multicenter, double-blind, randomized, parallel group, dose-finding study, which included a 2-week screening period, a 12-week treatment period, and follow-up visits at 8 and 16 weeks post-treatment. Patients received placebo or metrifonate once daily. Metrifonate-treated patients received a loading dose of 0.5 mg/kg (25 to 45 mg), 0.9 mg/kg (45 to 80 mg), or 2.0 mg/kg (100 to 180 mg) for 2 weeks, followed by a maintenance dose of 0.2 mg/kg (10 to 20 mg), 0.3 mg/kg (15 to 25 mg), or 0.65 mg/kg (30 to 60 mg) for 10 weeks. Four hundred eighty patients were enrolled. Percentages of patients completing double-blind treatment were 96% in the placebo group and 89 to 94% in the metrifonate group. Metrifonate significantly improved cognitive ability, as assessed by the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog), and enhanced global function, as assessed the Clinicians's Interview-Based Impression of Change with Caregiver Input (CIBIC-Plus). At 3 months, in the intent-to-treat patients, the treatment difference for the change in ADAS-Cog score in favor of metrifonate was 2.94 points (95% CI, 1.61 to 4.27; p = 0.0001). These patients also exhibited a 0.35-point improvement on the CIBIC-Plus relative to the placebo patients (95% CI, 0.15 to 0.54; p = 0.0007). Patients receiving lower drug doses had scores intermediate to those of the placebo and the 0.65 mg/kg metrifonate groups on both performance scales. The drug was well tolerated; side effects were predominantly gastrointestinal in nature, and no hepatic toxicity was observed. Therefore, in this study, metrifonate safely improved the cognitive deficits and benefited the global function of AD patients.

Metrifonate, a pro-drug that is transformed non-enzymatically into a potent inhibitor of acetylcholinesterase (AChE), has been used in the tropics for over 30 years for the treatment of schistosomiasis. A pilot study, and Phase I and Phase II studies of metrifonate in Alzheimer's disease (AD) patients conducted prior to the current study showed benign, dose-dependent adverse event profiles consisting primarily of gastrointestinal events, optimal daily dosing with a loading phase (in the absence of a loading dose phase, 6-8 weeks were required to attain steady-state AChE inhibition levels), and an improvement in Alzheimer's Disease Assessment Scale (ADAS) scores. The current open-label study was designed to evaluate the safety and tolerability of relatively high loading doses, followed by lower maintenance doses of metrifonate in the same patient population, and to determine the maximum tolerated dose (MTD) of metrifonate. Accordingly, the first cohort of 8 probable AD patients (per National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association [NINCDS-ADRDA] criteria) were administered once-daily loading doses of 2.5 mg/kg (125-225 mg) for 14 days, followed by 4.0 mg/kg (200-360 mg) for an additional 3 days. These patients were maintained on once-daily doses of 2.0 mg/kg (100-180 mg) for 14 days. AChE inhibition for this cohort ranged from 88% to 94%. On Day 28 of 31, this cohort was discontinued due to moderate to severe side effects in 6 patients; consequently, the second cohort of 8 probable AD patients received a once-daily loading dose of 2.5 mg/kg (125-225 mg) for 14 days followed by a once-daily maintenance dose of 1.5 mg/kg (75-135 mg) for 35 days. This maintenance dose yielded an AChE inhibition level ranging from 89% to 91%. In spite of an AChE inhibition level comparable to that achieved with the higher dose, the reduced dose was associated with a more favorable adverse event profile which was mainly gastrointestinal and musculoskeletal in nature. The maximum tolerated dose was established at 1.5 mg/kg/day (75-135 mg/day) for maintenance dosing in AD patients.

Carboxylesterases (CbxE) can be inhibited by organophosphorus esters (OPs) without causing clinical evidence of toxicity. CbxE are thought to protect the critical enzyme acetylcholinesterase (AChE) from OP inhibition in animals. CbxE and AChE are both present in neuroblastoma cells, but, even though these cells have potential to be an in vitro model of OP toxicity, the effect of OPs on CbxE and the relationship of CbxE inhibition and AChE inhibition have not yet been examined in these cells. Therefore, this study examined concentration-related OP-induced inhibition of CbxE in human SH-SY5Y and mouse NB41A3 neuroblastoma cells with 11 active esterase inhibitors: paraoxon, malaoxon, chlorpyrifos-oxon, tolyl saligenin phosphate (TSP), phenyl saligenin phosphate (PSP), diisopropyl phosphorofluoridate (DFP), mipafox, dichlorvos, trichlorfon, dibutyryl dichlorovinyl phosphate (DBVP), and dioctyl dichlorovinyl phosphate (DOVP). All could inhibit CbxE, although the enzyme was less likely to be inhibited than AChE following exposure to 9 of the test compounds in the human cell line and to all 11 of the test compounds in the murine cell line. Species differences in concentration-related inhibitions of CbxE were evident. When cells were exposed first to an OP with a low IC50 toward CbxE (PSP), followed by an OP with high affinity for AChE (paraoxon or malaoxon), inhibitions of CbxE and AChE were additive. This indicated that CbxE did not protect AChE from OP-induced inhibition in this cell culture model.

This review starts with an historical background of the pharmacological development of tacrine almost fifty years ago (1949). Tacrine is the first drug to be tested, clinically, on a large scale and to be registered (1993) for treatment of Alzheimer's disease. For the first time, clinical results of four second generation cholinesterase inhibitors (ChEI) (donepezil, ENA 713, eptastigmine and metrifonate) are reviewed and compared with other ChEI such as tacrine, physostigmine and galanthamine. Data based on more than 6000 patients show that second generation drugs are well tolerated and show evidence of clinical efficacy. Differences are mainly due to frequency of side effects, number of drop outs and percentage of improved patients. These results also demonstrate the presence of clinical efficacy for all ChEI tested so far. Clinical mechanism of action, levels of efficacy and differences among various ChEI are discussed. Future potential indications are suggested. The present data indicate that optimization of effects prolongation and maintenance of clinical gains will depend on further knowledge of the compounds pharmacodynamic properties.

The effects of 21-day treatment with the acetylcholinesterase inhibitors metrifonate (80 mg kg(-1) per os (p.o.)) and tacrine (3 mg kg(-1) p.o.), twice daily, on cortical and hippocampal cholinergic systems were investigated in aged rats (24-26 months). Extracellular acetylcholine levels were measured by transversal microdialysis in vivo; choline acetyltransferase and acetylcholinesterase activities were measured ex vivo by means of radiometric methods. Basal cortical and hippocampal extracellular acetylcholine levels, measured 18 h after the last metrifonate treatment, were about 15 and two folds higher, respectively, than in control and tacrine-treated rats. A challenge with metrifonate further increased cortical and hippocampal acetylcholine levels by about three and four times, respectively. Basal extracellular acetylcholine levels, measured 18 h after the last treatment with tacrine were not statistically different from those of the control rats. A challenge with tacrine increased cortical and hippocampal extracellular acetylcholine levels by about four and two times. A 75% inhibition of cholinesterase activity was found 18 h after the last metrifonate administration, while only a 15% inhibition was detectable 18 h after the last tacrine administration. The challenge with metrifonate or tacrine resulted in 90 and 80% cholinesterase inhibition, respectively. These results demonstrate that in aging rats a subchronic treatment with metrifonate results in a long-lasting, cholinesterase inhibition, and a persistent increase in acetylcholine extracellular levels which compensate for the age-associated cholinergic hypofunction. Metrifonate is therefore a potentially useful agent for the cholinergic deficit accompanying Alzheimer's disease.

The conditions (sensitive period and doses) for producing hypoplasia in guinea-pig offspring after treatment of pregnant guinea-pigs with the organophosphorous agent trichlorfon (metrifonate, 0,0-dimethyl 2,2,2-trichloro-1-hydroxyethylphosphonate) were examined. The results showed that the minimal dose required was 100 mg/kg on three consecutive days. For the cerebellum the most sensitive period was 42-44 day of gestation, for cortex the most vulnerable period was gestational days 48-50. The doses could be given either per os or subcutaneously. Almost all regions of the brain were reduced in weight. Cerebellum was the most vulnerable region, but also the medulla and hypothalamus were greatly reduced in weight. The mechanism behind the teratogenic effect is not known, but alkylation of DNA or an effect on its repair mechanism are possible effects.

Alzheimer's disease is a chronic neurodegenerative disorder that is characterized by memory impairment, cognitive dysfunction, behavioral disturbances, and deficits in activities of daily living. A consistent observation in these patients is that cholinergic neurons are affected and deteriorate over time, leading to decreased levels of acetylcholine (ACh). Acetylcholinesterase (AChE) inhibitors, which attempt to prevent the breakdown of ACh, may be classified as short acting, intermediate acting, and long acting based on AChE regeneration time. Metrifonate is converted by a nonenzymatic process to the long-acting cholinesterase inhibitor 2,2-dichlorovinyl dimethyl phosphate (DDVP). Acetylcholinesterase inhibition produced by metrifonate occurs rapidly, is dose dependent, can be detected by inhibition measured in red blood cells, and can be reversed by oxime administration. Metrifonate and DDVP improved performance in young rats; cognitive improvement in aged rats also was observed. Both agents were well tolerated and did not have significant effects on various preclinical pharmacologic safety tests.

Preliminary studies suggest that non-cognitive behavioural and personality alterations in Alzheimer's disease may benefit from agents which inhibit central acetylcholinesterase (AChE). A double-blind, placebo-controlled, 26-week study of the AChE inhibitor metrifonate using the NeuroPsychiatric Inventory (NPI) to assess the effects of treatment on neuropsychiatric symptoms observed statistically significant mean change differences favouring treatment in the total NPI score and in symptoms of depression, apathy and hallucinations, as well as a nearly significant difference in aberrant motor behaviours. These data are consistent with previous studies and are believed to represent the first large prospective, controlled study demonstrating a beneficial effect of AChE inhibitor therapy on neuropsychiatric symptoms in Alzheimer's disease. The nature of non-cognitive symptom responses to AChE inhibitor therapy and their potential impact on caregivers is discussed.

The promising results of early trials in Alzheimer's disease with the acetylcholinesterase inhibitor metrifonate prompted initiation of the Metrifonate in ALzheimer's Trial (MALT). MALT is an international, randomized, double-blind, placebo-controlled, parallel-group study which was designed to determine, over a 26-week period, the efficacy, tolerability and safety of two doses of metrifonate in patients with probable Alzheimer's disease. A total of 605 patients were randomized to receive either a daily dose of oral metrifonate 40/50 mg (n = 200, by body weight <65 kg/> or =65 kg) or metrifonate 60/80 mg (n = 197, by body weight <65 kg/> or = 65 kg). Patients were assessed in the key symptom domains of Alzheimer's disease, i.e. cognition, behavioural and psychiatric disturbances, activities of daily living and global function. In summary, administration of metrifonate for 26 weeks to the intent-to-treat population significantly benefited cognitive performance, global function and certain aspects of behaviour and functional ability compared with placebo. These efficacy results were associated with high levels of acetylcholinesterase inhibition and a good safety and tolerability profile. The protocol of MALT is discussed here. Full results of the study will be available in a separate publication.

OBJECTIVE: To evaluate the efficacy and safety of metrifonate, an acetylcholinesterase inhibitor, in patients clinically diagnosed with probable Alzheimer's disease (AD) of mild to moderate severity. METHODS: A prospective, 36-week, multicenter, double-blind, randomized, parallel group study of metrifonate in probable AD patients, including a 2-week screening period, a 26-week double-blind treatment period, and a follow-up visit at 8 weeks post-treatment. A total of 24 ambulatory clinics in the United States in a variety of settings, including contract research organizations, public health facilities, and universities. Patients met diagnostic criteria for probable AD as defined by the work group of the National Institute for Neurological and Communicative Diseases and Stroke and the Alzheimer's Disease and Related Disorders Association. Patients had Mini-Mental State Examination (MMSE) scores of 10 to 26 and Ischemic Scores (Rosen Modification) of <4. A total of 408 patients were enrolled. Percentages of patients completing double-blind treatment were 88% and 79% in the placebo and metrifonate groups, respectively. Rates of discontinuation due to adverse events were 4% in the placebo group and 12% in the metrifonate group. Placebo or metrifonate was administered once daily. Metrifonate-treated patients received a loading dose of 100 to 180 mg based on weight (2.0 mg/kg) for 2 weeks, followed by a maintenance dose of 30 to 60 mg based on weight (0.65 mg/kg) for 24 weeks. Primary efficacy variables were the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) and the Clinician's Interview-Based Impression of Change with Caregiver Input (CIBIC-plus). Secondary efficacy variables included the Neuropsychiatric Inventory (NPI), the Disability Assessment in Dementia, the Global Deterioration Scale (GDS), the ADAS-Noncognitive subscale (ADAS-Noncog), the MMSE, and the Clinician's Interview-Based Impression of Severity with Caregiver Input (CIBIS-plus). Outcome measures reflected changes from baseline at week 26 for all variables. Safety was assessed with incidences of premature termination, treatment-emergent events and mortality, and routine safety evaluations. RESULTS: After 26 weeks of metrifonate therapy, a 2.86-point treatment difference (p = 0.0001) was observed in the ADAS-Cog scores of the intent-to-treat AD patients. The treatment difference in the mean CIBIC-plus score at this time was 0.28 points (p = 0.0071). At week 26, treatment differences also were observed in the mean NPI total score (p = 0.0161). Analysis of the remaining secondary efficacy variables showed treatment differences that favored metrifonate but did not reach statistical significance. Metrifonate adverse events were predominantly mild in intensity. No hepatotoxicity was observed. CONCLUSIONS: Metrifonate was safe and well-tolerated. It enhanced not only the cognitive and global function, but also the behavioral function of patients diagnosed with mild to moderate AD. Therefore, metrifonate appears to be useful in the symptomatic treatment of AD.

Metrifonate is converted nonenzymatically to 2.2, dimethyl dichlorovinyl phosphate (DDVP), an inhibitor of acetylcholinesterase (AChE). This 21-day, randomized, double-blind, placebo-controlled trial of metrifonate in patients with Alzheimer's disease (n = 27) evaluated four doses, each administered orally once daily. All patients received a loading dose (LD) for 6 days followed by a maintenance dose (MD) for 15 days. The treatment groups were: panel 1, LD = 1.5 mg/kg (75-135 mg), MD = 0.25 mg/kg (12.5-25 mg); panel 2, LD = 2.5 mg/kg (125-225 mg), MD = 0.40 mg/kg (20-35 mg); panel 3, LD = 4.0 mg/kg (200-335 mg), MD = 0.65 mg/kg (30-60 mg); and panel 4, LD = 4.0 mg/kg (200-335 mg), MD = 1.0 mg/kg (50-90 mg). All metrifonate doses were well tolerated. Most adverse events were mild to moderate in intensity, gastrointestinal in nature, and transient. Mean area under the concentration-time curve (AUC) and maximum concentration (Cmax) for both metrifonate and DDVP increased in relation to dose. Metrifonate and DDVP had similar, largely dose-independent mean values for time to Cmax (tmax) and half-life (t1/2). There was little or no accumulation of either metrifonate or DDVP with long-term administration. After 21 days of treatment, mean percent erythrocyte AChE inhibition was 14%, 35%, 66%, 77%, and 82% for placebo and panels 1 through 4, respectively. Cognitive improvement was observed with the two highest metrifonate doses. These results reflect favorable safety and pharmacokinetic profiles for the use of metrifonate in the treatment of Alzheimer's disease.

Metrifonate, through its pharmacologically active metabolite 2,2-dichlorovinyl dimethylphosphate (DDVP), is a long-acting cholinesterase inhibitor for the symptomatic treatment of mild-to-moderate Alzheimer's disease. Clinical studies with Alzheimer patients have demonstrated the favourable safety and tolerability profile of this drug. Metrifonate, at therapeutic doses for Alzheimer's disease, achieves high levels of cholinesterase inhibition, i.e. > or =70%, without the need for dose escalation. This is a consequence of the low rate of fluctuation of enzyme activity during therapy with metrifonate. This, in turn, is due to the protracted hydrolytic transformation of metrifonate into DDVP, the resulting smooth onset of cholinesterase inhibition, and the subsequent long duration of action which far outlasts the presence of the active drug in the body. Both metrifonate and DDVP are rapidly metabolized and eliminated from the body. Further, their metabolism does not involve the cytochrome P450 system and both compounds show low plasma protein binding. These pharmacokinetic features account, at least in part, for the favourable safety and tolerability profile of metrifonate as they suggest a minimal risk of drug-drug interactions with other likely co-medications in the long-term therapy of Alzheimer patients.

Metrifonate, administered orally to patients with probable Alzheimer's disease in a once-daily dose, readily enters the brain and inhibits brain acetylcholinesterase (AChE) activity in a dose-dependent fashion. Metrifonate is a prodrug, converted non-enzymatically to 2,2-dichlorovinyl dimethyl phosphate, a long-acting inhibitor of AChE that produces stable enzyme inhibition over time. In combination, these pharmacologic characteristics lead to a reduced side effect profile in comparison with several other cholinesterase inhibitors. Both preliminary and confirmatory pivotal studies have shown that significant cognitive improvement is achieved with this medication in comparison with placebo in patients with probable Alzheimer's disease. Moreover, these studies also have demonstrated that metrifonate benefits the global function--a measure comprising domains of cognition, function, activities of daily living, and behavior--of patients with Alzheimer's disease. The medication is generally well tolerated, and no significant laboratory abnormalities occur. Therefore, metrifonate is a useful treatment for the symptoms of Alzheimer's disease.

We compared the tissue concentration of dipterex and the inhibition of the neuropathy target esterase (NTE) activity among groups of hens (n = 8 each) which were intravenously (i.v.), subcutaneously (s.c.) or orally (p.o.) administered the insecticide dipterex. The tissue concentrations of dipterex in the s.c. group were higher than those in the i.v. and p.o. groups. When dosed subcutaneously, the tissue concentration of dipterex was high in the brain, spinal cord and muscle at 3 hr after dosing and then concentrated in the spinal cord and muscle for the subsequent 3 hr. When dosed intravenously or orally, dipterex was evenly dispersed in various tissues. All hens treated with dipterex showed acute neurotoxic signs within 15 min after dosing. The hens dosed intravenously recovered from this acute poisoning within 3 hr, and the hens dosed orally recovered within 6 hr, while the hens dosed subcutaneously recovered within 24 hr after dosing. One hen in the s.c. group exhibited acute neurological sequelae following the acute poisoning. In addition, the loss of body weight was the largest in the s.c. group (157 +/- 49 g), moderate in the i.v. group (133 +/- 91 g), small in the p.o. group (96 +/- 54 g) and the smallest in the PMSF (phenylmethanesulfonyl fluoride, which was dosed to promote delayed neuropathy) group (80 +/- 49 g). In the untreated hens, the activity of NTE in both the cerebrum and cerebellum was higher than that in the midbrain (p < 0.01). There was no difference in NTE activity between the cerebrum and cerebellum. In both the cerebrum and midbrain, the inhibition of NTE activity in the p.o. group was less than that in the i.v. and s.c. groups, and no difference was found between the i.v. and s.c. groups. In the cerebellum, the inhibition of NTE activity in the s.c. group was larger than that in the i.v. and p.o. groups. These results indicate that the s.c. dosing of dipterex results in a stronger neurotoxicity compared to i.v. and p.o. dosing. However, it was difficult to induce the clinical signs of delayed neuropathy with any administration of dipterex in hens, even when the promotion of delayed neurotoxicity of dipterex was attempted with PMSF or double doses of dipterex itself.

The incidence of schistosomiasis, a parasitic infestation caused by trematodes, or flukes, has decreased significantly in Egypt and other Middle Eastern countries. The education of people in the countryside is a primary reason. The water supply in these countries is now good, and, in most areas, women no longer use the stagnant river tributaries for washing clothes. Medications, which can be used to prevent the infection or kill the parasite include praziquante and metrifonate.

Large scale preventive treatments against bovine hypodermosis have been performed in the Canton of Grisons and in different Jura areas. Either Metrifonate (Neguvon 10% spot-on, Bayer AG) or Ivermectin "microdoses" (i.e. 0.1 ml Ivomec ad. inj-MSD AGVET-per animal) have been applied. The treatment of dairy cows with Neguvon at the then recommended dose (24 ml/animal) showed a low efficacy of 80%, without any correlation to the date of treatment. An increase of the dose to 36 ml/animal revealed an efficacy of 93%, while a weight-dependent dose of 6 ml/100 kg body weight reduced the warble infestation by 97.3% without any drug-related side-effects. The few observed side-effects-salivation and mild meteorism--are likely to be due to the destruction of Hypoderma lineatum larvae in the gullet of treated cows. No case of abortion was reported. The Ivomec--"microdose" applied to young animals or dry cows showed an efficacy > 99.8%. The available drugs would thus allow a quick and efficacious control of bovine hypodermosis, provided they would be used systematically and at an appropriate dose in endemic areas.

The efficacy of metrifonate, a well-tolerated cholinesterase (ChE) inhibitor, in attenuating the normal aging- and corticosterone-induced impairments of radial maze performance of rats was compared. Middle-aged Fischer 344 rats were screened for their spatial orientation performance in the Morris water escape task. Good and bad performers were selected: good performers (N= 22) were treated with subcutaneous sustained-release corticosterone pellets, resulting in hippocampal cell damage and impaired spatial orientation in the radial maze; age-induced bad performers (N = 20) were tested without additional pharmacological intervention. Metrifonate (MFT), administered daily during radial maze testing, 30 min before training, at a dose of 15 mg/kg p.o., facilitated the acquisition of the task in age-impaired rats, but not in corticosterone-impaired rats.

Radioligand assay showed that the cholinesterase (ChE) reactivators dipiroxime and benzyxime, but not carboxime, modulate selective absorption of some cholinolytics (tributam, pediphen, aprophen) in rat brain. Significant suppression of the specific binding of muscarine antagonists was recorded after chlorophos (2.LD50) intoxication. Under such conditions, the ChE reactivators induce increase in the number of binding sites and in the parameters of the constant of cholinolytic absorption on the brain membranes. It was also established by equilibrium dialysis that the binding of cholinolytics in blood plasma under the effect of ChE reactivators is reduced, leading to redistribution of their free and bound fractures, which is most favorable for tissue sorption.

Metrifonate is an organophosphorous compound that has been used in the treatment of schistosomiasis. In this study, we investigated the effects of metrifonate on the impairment of learning and on central cholinergic dysfunction in scopolamine-treated and basal forebrain-lesioned rats. Oral administration of metrifonate (5.0-15.0 mg/kg) ameliorated the scopolamine- and basal forebrain. lesion-induced learning impairment in the water maze and passive avoidance tasks. Metrifonate (50 and 100 mg/kg) also significantly increased extracellular acetylcholine levels but decreased choline levels in the cerebral cortex of the basal forebrain-lesioned rats. The basal forebrain lesion decreased the cholinesterase activity in the cerebral cortex, and metrifonate (100 mg/kg) further reduced the cholinesterase activity. However, cholinesterase inhibition was not observed at the dose that ameliorated learning impairments. These results indicated that metrifonate ameliorated the impairment of learning in both scopolamine-treated and basal forebrain-lesioned rats by not only increasing extracellular acetylcholine levels by inhibiting cholinesterase, but also by undefined other mechanism(s). This finding suggests the usefulness of metrifonate for the therapy of Alzheimer's disease.

Several studies have indicated the possibility of using cholinesterase (ChE) inhibitors as therapeutic drugs for Alzheimer's disease. Metrifonate (MTF) is an organophosphorus compound that has been used in the treatment of schistosomiasis. In this study, we investigated the effects of MTF on the impairment of learning and memory, decreased ChE activity and extracellular acetylcholine (ACh) levels in basal forebrain (BF)-lesioned rats. The oral administration of MTF improved the BF-lesion-induced impairment of performance on passive avoidance task. Further, MTF reduced ChE activity in the cerebral cortex. In vivo brain microdialysis studies showed that MTF significantly increased the release of ACh, but decreased that of choline (Ch) in the cerebral cortex of BF-lesioned rats. These results indicated that MTF ameliorates the impairment of performance on passive avoidance task in BF-lesioned rats by increasing the extracellular ACh levels by inhibiting ChE. This suggested that MTF may be useful as a therapeutic drug for Alzheimer's disease.

The cholinergic system is known to show deterioration during aging and Alzheimer's disease. In response, a therapeutic approach to Alzheimer's disease has been to attempt to compensate for the decrease in central cholinergic function by potentiating the activity of the remaining intact cholinergic cells with cholinesterase inhibitors. In this study treatment with the long-lasting cholinesterase inhibitor metrifonate enhanced acquisition of eyeblink conditioning in aging rabbits without producing interfering side effects. The effects of metrifonate on central and peripheral cholinesterase activity were evaluated, as was the involvement of plasma atropine esterase activity on the central and peripheral response to metrifonate. Results demonstrate that metrifonate can produce predictable, dose-dependent ChE inhibition. Associative learning in the aging rabbit was improved by metrifonate-induced steady state ChE inhibition within a range of 30-80%. Metrifonate was behaviorally effective in the absence of the severe side effects which typically plague cholinesterase inhibitors, suggesting that metrifonate is a possible treatment for the cognitive deficits resulting from normal aging and Alzheimer's disease.

The cholinergic system is known to show deterioration during aging and Alzheimer's disease (AD). In response, a therapeutic approach to AD has been to attempt to compensate for the decrease in central cholinergic function by potentiating the activity of the remaining intact cholinergic cells with cholinesterase (ChE) inhibitors. In this study treatment with the long-lasting ChE inhibitor metrifonate facilitated acquisition and retention of eyeblink conditioning in aging rabbits. Metrifonate treatment resulted in steady-state, dose-dependent acetylcholinesterase (AChE) inhibition in red blood cells. Maximal behavioral efficacy was achieved with AChE inhibition of approximately 40%, with no further improvements resulting from increased levels of inhibition. Metrifonate was behaviorally effective in the absence of the severe side effects that can plague ChE inhibitors, supporting metrifonate as a possible treatment for the cognitive deficits resulting from normal aging and AD.

One of the main characteristics of Alzheimer's disease (AD) is the cerebrovascular deposition of the amyloid beta-peptide (A beta), which is derived from a larger beta-amyloid precursor protein (beta APP). The majority of beta APP is processed by either a secretory of lysosomal/endosomal pathway. Carboxyl-truncated soluble derivatives of beta APP (sAPP) are generated by the proteolytic processing of full-length beta APP by either alpha- or beta-secretase enzyme. Our objective is to determine whether the processing of beta APP can be regulated by cholinesterase inhibitors, some of which were shown to produce a moderate improvement in memory and cognitive functions in patients with Alzheimer's disease. Here we have analyzed the levels of sAPP derivatives in cultured cells treated with different drugs by immunoblotting samples of conditioned media. The immunoreactive protein bands were developed by probing with the monoclonal antibody 22C11. Treating neuroblastoma, pheochromocytoma and fibroblast cells with high dose of either 3,4-diaminopyridine, metrifonate, or physostigmine did not inhibit the secretion of sAPP. Treating glioblastoma with either 3,4-diaminopyridine or metrifonate showed an increase in secretion of sAPP. However, treatment of cells with tacrine reduced release of sAPP in conditioned media of cell lines studied. The difference in action of metrifonate, physostigmine, and tacrine on beta APP is independent of their anticholinesterase activities. Our results suggests that noncatalytic functions of cholinesterase inhibitors can be utilized to alter the metabolism of beta APP, which might in turn affect the process of deposition of A beta, a key component of the cerebrovascular amyloid detected in AD.

The present experiment was designed to elucidate whether chronic dietary treatment with nimodipine (3 months, 1000 ppm) enhances water maze spatial navigation, passive avoidance behavior and locomotor activity, and whether such a treatment with nimodipine would interact with the therapeutic effect of acute metrifonate treatment. In young medial septum-lesioned rats, nimodipine had no effect by its own on cognitive or motor behavior, and did not enhance the water maze and passive avoidance behavior improving action of metrifonate (3 and 10 mg/kg. p.o.). Nimodipine treatment of aged rats did not markedly affect the deficit in motor performance. Single and combined nimodipine and metrifonate (3 and 10 mg/kg, p.o.) treatment of aged rats resulted in shorter escape distance values to the hidden water maze escape platform compared to those of control aged rats. The passive avoidance performance of aged rats was more effectively facilitated by a combined nimodipine and metrifonate treatment than by either of the drugs on their own. Following a washout period of 2.5 months the rats that were treated previously with nimodipine no longer performed better than aged controls in the water maze test. Furthermore, after the washout period metrifonate 10 mg/kg was no longer effective in improving the water maze behavior of the now 26-month-old rats irrespective of their chronic pretreatment. Taken together, these findings indicate that chronic nimodipine and acute metrifonate treatment may more effectively stimulate cognitive functioning than either of the treatments on their own.

We investigated the effects of acute oral pretraining treatment with an indirect acetylcholinesterase inhibitor, metrifonate, on water maze spatial navigation in medial septum-lesioned rats. We observed that metrifonate (30 mg/kg, orally) (1) does not alter the pattern of exploration of lesioned rats at the water maze pool or retrieval of spatial memory, (2) effectively reverses the acquisition defect, (3) enhances reversal learning, and (4) improves acquisition of water maze navigation by facilitating the encoding of the spatial representation of a specific environment. These results indicate that metrifonate does not improve escape performance to the hidden platform by modulating exploration strategy, but that metrifonate enhances the speed and accuracy of development and durability of spatial memory engrams, and facilitates learning capacity that depends on activity of the septo-hippocampal projection.

We studied the effect of nimodipine (1000 ppm mixed in food), an L-type calcium-channel antagonist, administered for 4 months, on the cortical EEG activity in young and aged rats. Nimodipine treatment decreased cortical high voltage spindles (HVSs) in aged rats, but did not prevent the diminution of spontaneous locomotor activity. The threshold dose of metrifonate, a cholinesterase inhibitor, for suppression of HVSs was lower in nimodipine compared to placebo treated aged rats (30 mg/kg versus 60 mg/kg; p.o.). In young rats, nimodipine did not decrease HVSs, protect from scopolamine (0.1 or 0.8 mg/kg, i.p.) induced EEG slowing or augment the effect of metrifonate to suppress slow waves induced by scopolamine. The present results suggest that a chronic nimodipine treatment modulates thalamocortical arousal and thereby adds to the therapeutic effects of metrifonate to restore normal cortical electrical arousal in aged rats.

The study compared the efficacy of acute versus chronic metrifonate treatment to improve initial and reversal learning of the water maze spatial navigation task in medial septal-lesioned rats. Acute oral administration of 30 mg/kg metrifonate at 30 min, but not at 150 or 360 min, before training improved the initial acquisition of the water maze task. In contrast, improvement of initial learning performance of medial septal-lesioned rats pretreated for 21 days with metrifonate was observed irrespective of the timing of metrifonate treatment relative to behavioral testing. Reversal learning was assessed after a four-day wash-out period. No drug treatment was administered during this part of the study. All the medial septal-lesioned rats that had received only acute treatment with metrifonate during the initial learning stage were now as impaired as vehicle treated medial septal-lesioned rats. However, the group subchronically pretreated with metrifonate performed better than the vehicle-treated medial septal-lesioned controls. These results indicate that both acute and subchronic treatment with metrifonate can facilitate spatial learning in medial septal-lesioned rats and the transient nature of this beneficial effect after single acute administration is transformed into a long-lasting improvement by subchronic treatment.

The effects of metrifonate were investigated in 4-6- and 22-24-month-old rats. Extracellular acetylcholine levels were measured by transversal microdialysis in vivo. Baseline extracellular acetylcholine levels in the cerebral cortex and hippocampus were 42% and 60% lower, respectively, in old than in young rats. Old rats did not discriminate between familiar and novel objects. In old rats, metrifonate (80 mg/kg p.o.) brought about 85% inhibition of cholinesterase activity in the cortex and hippocampus, a 4-fold increase in extracellular acetylcholine levels in the cortex only, and restored object recognition. In young rats, metrifonate caused 75% cholinesterase inhibition in the cerebral cortex and hippocampus, a 2-fold increase in cortical and hippocampal extracellular acetylcholine levels, and no effect on object recognition. The slight cholinesterase inhibition following metrifonate (30 mg/kg) in aged rats had no effect on cortical acetylcholine levels and object recognition. In conclusion, metrifonate may improve the age-associated cholinergic hypofunction and cognitive impairment.

Sulprofos, disulfoton, azinphos-methyl, methamidophos, trichlorfon, and tebupirimphos were screened for neurotoxic potential, in accordance with U.S. EPA (FIFRA) requirements. Each organophosphate was administered through the diet for 13 weeks to separate groups of Fischer 344 rats at four dose levels, including a vehicle control. For each study, 12 rats/sex/dietary level were tested using a functional observational battery (FOB), automated measures of activity (figure-8 maze), and detailed clinical observations, with half of the animals perfused at term for microscopic examination of neural and muscle tissues. Separate groups of satellite animals (6/sex/dietary level) were used to measure the effect of each treatment on plasma, erythrocyte (RBC), and brain cholinesterase (ChE) activity. The results show that measures of ChE activity were consistently the most sensitive indices of exposure and assisted in the interpretation of findings. All treatment-related neurobehavioral findings were ascribed to cholinergic toxicity, occurring only at dietary levels that produced more than 20% inhibition of plasma, RBC, and brain ChE activity. Neurobehavioral tests provided no evidence of additional cumulative toxicity after 8 weeks of treatment. The FOB and motor activity findings did not alter the conclusions and generally did not reduce the neurobehavioral no-observed-effect level (NOEL) for any of the six compounds, relative to the results from detailed clinical observations as conducted in these studies. The one exception occurred with tebupirimphos, where the NOEL for motor activity was one dose level lower than the NOEL for the FOB and clinical observations. These results support the value of detailed clinical observations to screen for the neurotoxic potential of organophosphates and a general standard of more than 20% inhibition of brain ChE activity for cholinergic neurotoxicity.

The Hungarian Congenital Abnormality Registry (HCAR) was established in 1962 due to the growing public health importance of congenital abnormalities (CAs). The HCAR is a population-based registry; it collects and keeps permanent records of medical and personal information about malformed newborns and infants (notification of CAs Is mandatory for physicians) in order to develop baseline data for different types of CAs, to search for increases in the incidence of specific CAs, to provide rates, and identify geographic areas of concern for cluster investigation. In the last 25 years numerous studies have been carried out. In the 1970s two significant increases were noticed: i) in the rate of terminal transverse type of limb reduction defects and ii) in the rate of hypospadias. As a result of our epidemiological investigations and case-control studies we were able to identify the possible factors which caused these increases. In the first case, for example, the Ministry of Health changed the policy of using high oestrogens dosage (which increased the risk of this defect 6.1 times) for artificial abortions in 1978 and after this time the increase was stopped. In 1989-1990 a geographical cluster was found. An extremely high rate of cases with Down's syndrome (27%) was identified in a small village and the case-control study and environmental investigations pointed suspicion on the excessive use of a chemical, trichlorfon at local fish farms. All mothers of babies with Down's syndrome had consumed contaminated fish during critical period. The use of this chemical was prohibited and no CAs were recorded after this time at this place. A special case-control surveillance system has been operating since 1980 in Hungary based on the HCAR. The objective of this system is to obtain information on pregnancy exposures and other risk factors for the study and identification of causes of CAs. The teratogenicity of several drugs taken during pregnancy has been examined so far, most of them ended up with negative results. Our system is suitable for surveillance, research and intervention.

Treatment of Alzheimer's disease has in the past been limited to empirical trials of psychotropics for relief of behavioral complications. At present, tacrine and doneprezil are the only FDA-approved antidementia agents available. In the very near future, however, other cholinesterases inhibitors (e.g., ENA 713, metrifonate, long-acting physostigmine) are expected to be approved for clinical use. The evidence at this point suggests that they have modest but meaningful clinical effects and possible long-term benefits. Clinical use of the newer agents is likely to be influenced by their side-effect profiles, which consist largely of cholinergic effects, although without the hepatotoxic effects associated with tacrine. To what extent these agents are accepted by patients and physicians remains to be seen. On the one hand, benefits are modest; on the other, these medications are increasingly safe. Continuing research is clarifying the role of cholinergic therapy in relieving behavioral symptoms, as well as the possible side effects on rates of illness progression, institutionalizaton, and even mortality. In the not-too-distant future, physicians can expect to see a variety of medications, now in early stages of development, that are intended to affect cholinergic systems in other ways. Further down the road, a host of mechanism-based therapeutic strategies, which hope to deal with the first cause of this devastating illness, will have been assessed in clinical trials.

We reviewed 130 admissions of organophosphate poisoning and analyzed the causes of death. Fenitrothion, malathion, dichlorvos, trichlorfon and fenitrothion/malathion were the most frequent chemicals involved. Mortality was 25% (32/130); delay in discovery and transport (18 cases), insufficient respiratory management (8 cases), and severe underlying or co-existing diseases (6 cases) were noted in the lethalities. Insufficient respiratory management consisted of delay in endotracheal intubation (5 cases) and failure in weaning (3 cases). About 3/4 of the severely serum cholinesterase-depressed cases needed ventilators. This suggests that better respiratory management would improve the outcome of organophosphate poisonings. Close observation of the clinical symptoms is essential, and detection of changes in serum cholinesterase may be helpful.

The effects of the centrally acting anti-cholinesterase metrifonate (MFT) and its metabolite dichlorvos (2,2-dichlorovinyl dimethyl phosphate; DDVP) on local cerebral glucose utilization (LCGU) have been studied in 3- and 27-month-old rats, using the autoradiographic [14C]deoxyglucose technique. In 3-month-old rats, MFT (80 mg/kg i.p.) increased LCGU significantly in 17 of the 54 regions studied, including insular, cingulate, and temporal cortices, ventral hippocampus, thalamus, lateral habenula, substantia nigra, and superior colliculus. In these regions, the average MFT-induced increase in LCGU was 23% above control. The average hemispheric LCGU increased by 10% (p < 0.01). DDVP (5 mg/kg) increased LCGU in 19 regions (average increase 26%). The average hemispheric LCGU increased by 9% (p < 0.01). Regional distributions of MFT- and DDVP-induced increases in LCGU were similar and overlapped the distribution of the acetylcholinesterase activity. In 27-month-old rats, MFT was active in 18 regions (average increase 25%). The whole-brain mean LCGU increased by 10% (p < 0.01). MFT compensated for the age-related hypometabolism in some brain areas including insular, temporal, and retrosplenial cortices, substantia nigra, and superior colliculus. The effects of MFT on LCGU were preserved in old rats, at variance with other anticholinesterases (tacrine, physostigmine). Which are less active in the aged rat brain.

We investigated the ability of a cholinesterase inhibitor, metrifonate, to desynchronize cortical EEG activity. Metrifonate suppressed immobility-related high voltage spindling activity in young and aged rats at doses of 30 and 60 mg kg-1, p.o., and 10, 30 and 60 mg kg-1, p.o., respectively. The increase in EEG 1-20 Hz amplitude induced by scopolamine (0.2 mg kg-1, i.p.) was fully alleviated by metrifonate (30 and 100 mg kg-1, p.o.) and partially alleviated by a reference cholinesterase inhibitor, THA (3 and 6 mg kg-1, i.p.). Nucleus basalis (NB) lesions induced by quisqualic acid decreased frontal cortical choline acetyltransferase activity by 80% and increased cortical EEG slow waves. Metrifonate and THA did not reverse NB lesion-induced EEG abnormality. We conclude that metrifonate enhances cholinergic desynchronization of cortical EEG waves and that a severe defect of presynaptic NB cholinergic fibres limits the therapeutic effects of metrifonate.

The potential of the pesticide trichlorfon to induce mitotic aneuploidy has been investigated in genetically engineered human lymphoblastoid cell lines. Trichlorfon failed to induce micronuclei in the AHH-1 and MCL-5 cell lines when treated in media at normal cell culture pH (pH 7.3). Under a treatment pH of 5.5, trichlorfon exposures resulted in the induction of both chromosome loss and chromosome non-disjunction as measured by fluorescence in situ hybridisation (FISH) using a pan-centromeric probe for all human centromeres and centromere probes specific for chromosomes 2, 7 and 18. At treatment concentrations greater than 20 micrograms/ml trichlorfon also induced structural chromosome damage resulting in the production of centromere negative micronuclei.

The present study investigated if short-term treatment with an L-type Ca2+-channel inhibitor, nimodipine, can stimulate cognitive functioning and cortical electroencephalograph (EEG) arousal, and potentiate the effect of a cholinesterase inhibitor, metrifonate. Pretraining administration of nimodipine (3, 10 and 30 mg/kg, p.o.) had no effect on water maze and passive avoidance behavior of young neurologically intact controls, or water maze and passive avoidance performance failure induced by scopolamine pretreatment (i.p.; 0.4 mg/kg during the water maze and 2.0 mg/kg during the passive avoidance study), medial septal lesioning, or aging. Furthermore, nimodipine (3, 10 and 30 mg/kg, p.o.) had no effect on the improvement by metrifonate (10 mg/kg, p.o.) of the water maze and passive avoidance failure induced by scopolamine pretreatment or medial septal lesioning, nor did it affect the potential of metrifonate (30 mg/kg. p.o.) to improve the water maze or passive avoidance behavior of aged rats. Finally, nimodipine (3, 10 and 30 mg/kg, p.o.) had no effect on spontaneously occurring thalamically generated neocortical high-voltage spindles or spectral EEG activity of young controls, nor did it alleviate the spectral EEG abnormality induced by scopolamine (0.2 mg/kg, i.p.) administration. Also, the combination of nimodipine 3 or 10 mg/kg and a subthreshold dose of metrifonate 10 mg/kg could not suppress high-voltage spindles or scopolamine treatment-induced spectral EEG activity abnormalities. According to the present results, short-term treatment with nimodipine does not stimulate cognitive functions or increase cortical EEG arousal, and does not block or potentiate the propensity of metrifonate to improve cognitive performance of rats.

We investigated the effects of acute p.o. pretraining treatment with an indirect acetylcholinesterase inhibitor, metrifonate, on water maze spatial navigation and passive avoidance behavior. Metrifonate (10-100 mg/kg, orally, p.o.) did not improve the water maze or passive avoidance performance of young intact rats. However, in young rats metrifonate over a broad dosage range (10-100 mg/kg, p.o.) was able to alleviate the adverse effects of scopolamine (a muscarinic acetylcholine receptor antagonist; 0.4 and 2.0 mg/kg in water maze and passive avoidance study, respectively) and medial septum-lesioning on spatial reference and working memory and passive avoidance performance. In old (23-month-old) rats, a defect of water maze and passive avoidance behavior was observed. In old rats, metrifonate improved spatial reference memory function in the water maze and also passive avoidance at 10-30 mg/kg, but the 3 mg/kg dose was ineffective. Very old (27-month-old) rats had a more severe impairment of water maze performance than old rats, and metrifonate 3-30 mg/kg did not improve their spatial navigation. These results show that metrifonate may over a wide range of doses stimulate cognitive functioning, but during advanced aging neurobiological defects develop that may mask some of the therapeutic effects of metrifonate in rats.

Data of health surveillance from 1970 to 1989 for the workers employed in dipterex packaging were collected and statistically analyzed with a microcomputer by scoring their whole blood choline esterase activities and symptoms and signs. Results showed working environment improved and air pollution and absorption of pollutants via skin decreased with technology innovation and longterm hygienic supervision, monitoring and health surveillance in the factory. Incidence of dipterex poisoning in workers employed in the department of pesticide packaging lowered significantly, from 25.26% in the early 1970's to 5.17% in late 1970's and to 1.85% during 1980's, with periodical physical examinations for them, and timely management of the patients with poisoning and the cases at high risk.

The blood dwelling stages of schistosomes have acetylcholinesterase (AChE) and nicotinic-like acetylcholine receptors (nAChR) on their teguments. Both AChE and nAChR are concentrated on the dorsal surface of the adult male, a major surface for nutrient uptake for the worm pair. Exposure of tegumental AChE and nAChR to acetylcholine (ACh), the natural ligand of these molecules, has a consequence for the transporting function of this membrane in some schistosome species. The rate of glucose uptake in vitro by Schistosoma haematobium and Schistosoma bovis adult worm pairs was enhanced by approximately 60% at blood concentrations of ACh. Schistosoma mansoni did not show a similar response. The specificity of the ACh interaction with nAChR and AChE was shown by ablation of the effect with specific antagonists of nAChR (d-tubocurarine and alpha-bungarotoxin) and an inhibitor of AChE (BW284C51). The primary effect occurs on the tegument since alpha-bungarotoxin and BW284C51 do not penetrate the schistosome tegument. The species differences in reliance on this mechanism are consistent with their relative sensitivities to the AChE inhibitory drug, metrifonate.

One consistent finding in senile dementia of the Alzheimer's type is that the brain has reduced ability to synthesize acetylcholine. This has been related, in part, to memory dysfunctions. Although a cholinergic deficit is not singularly responsible for symptoms of dementia, treatment strategies have been designed to facilitate cholinergic activity by inhibiting acetylcholinesterase (AChE). To minimize toxicity, however, a cholinesterase inhibitor selective for only AChE would be an ideal treatment. The purpose of this study was to determine the selectivity of physostigmine, metrifonate, methanesulfonyl fluoride and tetrahydroaminoacridine (tacrine) toward AChE as compared with butyrylcholinesterase (BChE) in human cortex. The results show that methanesulfonyl fluoride is selective as an inhibitor of AChE as compared with BChE. Physostigmine inhibited AChE more than BChE. Metrifonate was found to inhibit BChE more than AChE. Tetrahydroaminoacridine inhibited both enzymes in a complex way.

Acetylcholinesterase (AChE) is present in all stages of the life-cycle of schistosomes and is located in muscle and on the surface of the parasite. Metrifonate is a drug that inhibits AChE. We compared the AChEs from three schistosome species (Schistosoma mansoni, Schistosoma haematobium and Schistosoma bovis) that have different susceptibilities to metrifonate in vivo. Sensitivities to AChE inhibitors were similar. The subunits of AChE were 110 kDa and 76 kDa and the dominant molecular form of AChE was a G2 form in all three species. This was the major form on the tegument while additional molecular forms were associated with the internal tissues. Differences in relative amounts of AChE activity between these species were found in the adults but not in the schistosomula. At the adult stage the major difference between species lay in the relative amounts of AChE activity in their teguments. S. haematobium teguments carried 20 times and S. bovis 6.9 times the activity present on S. mansoni teguments. These quantitative differences associate with the relative sensitivities of these species to metrifonate.

Transdermal delivery of cholinesterase inhibitors (ChEI) for treatment of dementia would have advantages associated with continuous dosing and enhanced compliance, but feasibility depends on achieving desired levels of central nervous system enzyme inhibition. We developed a patch technique for assessing delivery of ChEI in rats and examined two organophosphate compounds, metrifonate and DDVP, and a carbamate, heptylphysostigmine, for production of peripheral and central nervous system ChE inhibition at target levels. With DDVP, a log-dose/percent brain AChE inhibition was obtained over a range of 10-65% inhibition within a 10-fold concentration of inhibitor in the patch. Brain cholinesterase was inhibited up to seven days after a 24-h patch application. Long-term inhibition was greater than that attained after intramuscular injection, but without the rapid initial inhibition peak seen with the latter route. In contrast to DDVP, sustained high levels of brain enzyme inhibition could not be produced by transdermal delivery of metrifonate or heptylphysostigmine. Apparently DDVP has features, i.e., liquid state in pure form and high inhibitor potency, which make it particularly suitable for patch administration.

Various organophosphorus compounds with low acute toxicity levels are widely used as insecticides. Human acute poisoning by organophosphates has often occurred accidentally. We determined the activity and isoenzyme patterns of serum cholinesterase (ChE) obtained from 13 human patients who attempted suicide with various organophosphates, i.e. Fenitrothion, Malathion, Isoxathion, Pyridaphenthion and Trichlorfon, and studied on the changes in the activity and isoenzyme patterns of serum ChE after ingestion. The following results were obtained. 1) Twenty ChE isoenzyme bands from normal human serum were detected by electrophoretic separation on polyacrylamide gradient gel. The main bands in the ChE isoenzyme pattern in normal serum were bands 4 and 5 which had the highest activity of acetylcholinesterase (AChE) with a molecular weight of 600,000-800,000, and bands 7, 12, 14, 17 and 18. 2) Inhibition of serum ChE activity was more severe as the amount ingested increased in patients who took Fenitrothion and Malathion. Reactivation of serum ChE activity was very slow in patients treated with PAM (2-pyridine aldoxime methiodide) in the late stage of ingestion or whose symptoms reappeared. 3) There were no differences in the patterns of serum ChE isoenzyme by organophosphorus compound. Band 7 disappeared in the serum ChE isoenzyme of almost every patient, and bands 12, 18, 14 and 17 of the serum ChE isoenzyme disappeared successively with the decline of serum ChE activity. Only band 5 of the isoenzyme remained in cases who had serum ChE activity lower than 5% of normal. 4) All 13 patients were treated with PAM and atropine immediately after being admitted to hospitals. We could not clearly determine the efficacy of PAM on reactivation of serum ChE activity and isoenzyme, because it was impossible in human poisoning to compare PAM efficacy with no treatment and with pre- and post-PAM treatment. 5) The activity and isoenzyme patterns of serum ChE recovered rapidly after combined hemoperfusion and hemodialysis treatment (HP-HD treatment) of the patients poisoned with Malathion. But HP-HD treatment had no effect on poisoning by Fenitrothion and Isoxathion. These findings demonstrated the changes in the activity and isoenzyme pattern of serum ChE in patients poisoned with several organophosphates after PAM and HP-HD treatment.

The effect of eight different acetylcholinesterase inhibitors (AChEIs) on the activity of acetylcholinesterase (AChE) molecular forms was investigated. Aqueous-soluble and detergent-soluble AChE molecular forms were separated from rat brain homogenate by sucrose density sedimentation. The bulk of soluble AChE corresponds to globular tetrameric (G4), and monomeric (G1) forms. Heptylphysostigmine (HEP) and diisopropylfluorophosphate were more selective for the G1 than for the G4 form in aqueous-soluble extract. Neostigmine showed slightly more selectivity for the G1 form both in aqueous- and detergent-soluble extracts. Other drugs such as physostigmine, echothiophate, BW284C51, tetrahydroaminoacridine, and metrifonate inhibited both aqueous- and detergent-soluble AChE molecular forms with similar potency. Inhibition of aqueous-soluble AChE by HEP was highly competitive with Triton X-100 in a gradient, indicating that HEP may bind to a detergent-sensitive non-catalytic site of AChE. These results suggest a differential sensitivity among AChE molecular forms to inhibition by drugs through an allosteric mechanism. The application of these properties in developing AChEIs for treatment of Alzheimer disease is considered.

Pigs in a herd infected with Sarcoptes scabiei var suis were treated with 20% phosmet at a dose rate of 20 mg/kg body weight. In one of the groups the environment was also treated using 2% trichlorphon. Post-treatment clinical examination and parasitological evaluation of skin scrapings revealed complete recovery by the end of the second month after initial treatment with no evidence of reinfection for a period of five months.

To determine whether cholinesterase inhibition by an organophosphate would influence atracurium's neuromuscular blockade, six horses were anesthetized and paralyzed with atracurium (total of five injections per horse) on experimental Day 1, then were given trichlorfon (64 mg/kg per os) 6 days later. On Day 7, horses were anesthetized and paralyzed in the same manner as on experimental Day 1. Blood was taken to measure serum cholinesterase activity prior to anesthesia on Days 1 and 7. No significant difference was noted in atracurium's neuromuscular blocking activity between the 2 experimental days (P less than 0.05), despite Day 7 cholinesterase activity that was 16% of pre-trichlorfon values. For atracurium Injections 1 and 2-5, 85 and 43 micrograms/kg of atracurium, respectively, were required to produce a 95-99% reduction in hoof twitch. The time from injection to maximum twitch reduction was approximately 9 min after Injection 1 and 5 min after subsequent injections. Time from injection to maximum twitch reduction was significantly longer for Injection 1 than Injections 2-5 on both experimental days. The time from maximum twitch reduction until 10% recovery was approximately 8 min, with no significant difference between experimental days. The time for twitch recovery from 10 to 75% was approximately 17 min for all injections. Antagonism of atracurium with edrophonium caused the twitch height to return to pre-atracurium strength in approximately 7 min. Edrophonium caused a significant increase in arterial blood pressure. Heart rate change was variable after edrophonium

Data have been compiled concerning insecticide-resistance in native populations of Musca domestica from 10 districts of the GDR for the period between 1976 and 1988. The insecticides tested were trichlorfon, dimethoate, bromophos, and trichlophenidine, i.e. those insecticides commonly used in house fly control on animal production farms. Pyrethrum and permethrin were also tested. Since the test strains were frequently selected in connection with control failures, the data may not be representative for all populations. But they allow some conclusions concerning the development of resistance. A stage of development has been reached which required a departure from the exclusive use of conventional procedures (residual spraying of surfaces with persistent contact pesticides). Intensified research on non-chemical or special chemical methods is urgently needed.

The authors found that the phosphororganic pesticide neguvon in a dose of 200 mg/kg of body weight diminished the content of dopamine in the striatum of white rats. This effect was increased after daily administration of the same dose for a period of 5 days. The reactivator of cholinesterase TMB-4(Trimedoxime) and H-choline blocker tropacine prevented this effect. They discussed the connection between the changes in the content of dopamine and modulating role of presynaptic choline receptors of H- and M- types in the dopaminergic terminals of the striatum.

The authors found that the contractile activity of isolated myofibers as-well as superprecipitation of myosin B were reduced after intoxication of white rats with 1/2 of dose of LD50 of neguvon. The reaktivator of cholinesterase TMB-4(Trimedoxime) (20 mg/kg) recovered the contractile capability of myofibres even on the third day after treatment, but superprecipitation of myosin B-after the twentieth day.

Spraying of spring wheat plants during vegetation with herbicides of 2,4-D group and ethrel increased chromosome aberrations in meiosis 2.8-5, 3.6-4.1 times. The insecticides, chlorophos and phtalophos, in a little higher dosage than it is usually practised increased the spontaneous level of chromosome aberrations 3.1-5.5 times. Phtalophos also induced chlorophyll mutations. These results testify to the necessity of keeping strictly to the established normative doses, because of overdosage is of potential genetic danger to spring wheat crops.

The cytogenetic action of three phosphoorganic pesticides, imidan, dipterex and gardona, in cultured human lymphocytes is studied. It is found that all the sunstances induce the signficant increase in the frequency of metaphases with chromosomes aberrations as compared with the control level. Maximal effects in vitro qere 6,0; 4,8 and 4,0% of aberrant cells for imidan, dipterex and gardona respectively. All investigated substances had a weak mutagenic activity in human lymphocyte culture.

Selecting of strain a Musca domestica resistant to trichlorphon with Bi 58 (active ingredient: dimethoate) resulted in an only slight (1.33-fold) increase of the resistance to dimethoate between 1974 and 1975. In the same time the resistance to trichlorphon rose to 5.28 times the original degree. The resistance extends to some other organophosphates, especially malathion, dichlorvos and bromophos, but not to diazinon. Among the insecticides registered for control of flies in pig-sties Bi 58 has the greatest chance, though there exists neither a certainty for the decrease of the resistance to trichlorphon nor for the non-appearance of a resistance to dimethoate. To overcome the problem of flies in stables and sties, particularly in plants of industrial cattle-breeding, complex measures must be taken.

Experiments conducted on pregnant Wistar rats show that chlorophos (Dipterex) has embroyotoxic and teratogenic effects after oral introduction in a 80 mg/kg dose during a critical period of embryogenesis. Embryotoxic and teratogenic effects are absent during the introduction of 8 mg/kg of the pesticide. The oral introduction of phthalophos (Imidan) in a 30 mg/kg dose once on day 9 of pregnancy and the introduction of a 1.5 mg/kg dose daily throughout the course of pregnancy caused increased postimplantation mortality of embryos. A dose of 30 mg/kg of phthalophos on day 9 or day 13 of pregnancy causes developmental abnormalities, including hyponathia and hydrocephaly. A 0.06 mg/kg phthalophos dose does not affect the course of embryogenesis in white rats. Thus the organophosphate pesticides Dipterex and Imidan exhibit embryotoxic and teratogenic effects at doses which significantly exceed the acutal amounts of the pesticide that can enter the human organism.

The effectiveness of four inorganic compounds and six preparations of insecticides was examined on larvae of Musca domestica in pig manure. The activity of sodiumfluoride and chlorinated hydrocarbons was low. Carbaryl indicated better effect. Good to very good results showed trichlofon and bromophos and the inorganic compounds sodium hexafluorsilicate, sodium tetraborate and calcium cyanamid. The influence of calcium cyanamid and sodium tetraborate led to considerable morphological deformations of pupae. With the latter compound and with fluorides a distinct prolongation of period of development was established in comparison with control. The effect of trichlorfon and bromophos decomposes rapily in pig manure. With trichlorfon an indirect ovicide effect was observed.

The structural development of fetuses was altered when Dipterex was administered by diet to pregnant rats from days 6 through 15 of gestation. Major external and skeletal alterations occurred after consumption of 432 or 519 mg/kg body weight per day, only minor skeletal changes occurred in the 375 mg/kg dose group and the incidence of alterations in the 145 mg/kg dose group was not significantly different from that in the pair-fed controls. The malformations seen at the two highest doses did not result directly from the associated decrease in food consumed. Dipterex was not shown to have teratogenic potential when given for the same time span, once daily by gavage, even at levels that produced maternal lethality. Imidan was not teratogenic when similarly given, either by diet at concentrations that resulted in a 45% reduction in food consumption, or by gavage at dose levels that resulted in some maternal lethality. Data collected from pair-fed control females revealed that limitation of food consumption to 13--15 g/rat per day from days 6 through 15 of gestation did not result in increased fetal mortality or stunting. However, fetal weight was reduced slightly, and the incidence of minor skeletal changes was approximately three to four times that among fetuses of control dams that were not pair-fed.

A detailed examination on a group of oxen naturally infested with Parafilaria bovicola suggested a prepatent period varying from 238 to 250 days. In these animals 54% of all lesions bled only once, 22% a second time and 24% more than twice. Of all active lesions 42% occurred in the shoulder region and decreased from this area both cranially and caudally. The same tendency was noticeable on carcasses after slaughter. During the observation period May 1974 to February 1975 the number of positive animals increased, reaching peak values during September - October 1974, after which a decline was noticed. The filaricidal effects of nine compounds were tested. These were suramin (used in combination with diethylcarbamazine citrate), thiacetarsamide sodium, fenchlorphos, phosmet, mebendazole, fenbendazole, levamisole hydrochloride and trichlorphon.

The development of chemical control of Musca domestica on Danish farms 1945--72 is outlined. It has been strongly influenced by successive development of resistance and failure of control by one insecticide after another. The chlorinated hydrocarbons used as residual sprays failed 1947--51. Organophosphorus compounds (OPC) were widely used from 1953, first as strips impregnated with parathion and residual sprays with diazinon. Resistance to OPC was first found in 1955, diazinon was given up in 1957--59 and parathion strips failed in the early '60's. Trichlorfon paint-on baits were widelyused 1958--64 and serious resistance did not appear until 1967, induced by selective pressure of fenthion and dimethoate used as residual sprays. High resistance to the contact effect of trichlorfon now occurs everywhere in Denmark. However, trichlorfon baits are still able to kill many flies. Residual sprays with fenthion, ronnel and fenitrothion were used to some extent 1960--70, but increased resistance reducing the residual effect developed in 2--3 years. Dimethoate was used on the majority of farms 1965--72. It was very effective the first years and resistance increased slowly until 1971--72, when high to extreme dimethoate-resistance became general on Danish farms. This was associated with high resistance to other OPC for fly control, e.g. fenthion, fenitrothion, bromophos, and tetrachlorvinphos, and to carbamates, with the result that no generally effective residual sprays were available. In 1971--72 frequent treatments with synergized pyrethroids have been tried. However, the method is often expensive, and serious resistance problems have appeared on a few farms. In this situation preventive, sanitary measures to eliminate or reduce fly breeding in manure are becoming decisive again, but difficult to practise due to lack of farm labour. The extreme Danish situation is compared with those in other areas, and probable reasons for differences in resistance and control problems are discussed, as well as possibilities for strategies to reduce resistance development.