cgp-20376 and amocarzine

An intense global collaborative effort under the leadership of the Steering Committee of the Filariasis Scientific Working Group of the Tropical Diseases Research Programme, World Health Organization, has brought together researchers, pharmaceutical chemists and clinicians in the development and search for antifilarial compounds which are more effective and more convenient to administer than diethylcarbamazine citrate, the current drug of choice for lymphatic filariasis. The Brugia spp.-rodent model has been used extensively for the primary screening and B. pahangi infections in the dog or cat for the secondary screening, of potential filaricides. Recently, the leaf-monkey (Presbytis spp.) infected with subperiodic B. malayi or Wuchereria kalimantani has been used for the tertiary evaluation and pharmacokinetic studies of compounds which have shown effectiveness in the primary and secondary screens. Both P. cristata and P. melalophos are extremely susceptible to subperiodic B. malayi infection, but the former is a better host as a higher peak microfilaremia and adult worm recovery rate were obtained. Although more than 30 potential filaricides have been evaluated in the tertiary screen, only a few compounds have shown some promise against lymphatic filariasis. CGP 20376, a 5-methoxyl-6-dithiocarbamic-S-(2-carboxy-ethyl) ester derivative of benzothiazole, had complete adulticidal and microfilaricidal activities against the parasite at a single oral dose of 20 mg kg-1. However, as the compound or its metabolites caused hepatotoxicity, its clinical use in the present formulation is not recommended.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cats; Disease Models, Animal; Dogs; Elephantiasis, Filarial; Filaricides; Humans; Ivermectin; Organic Chemicals; Piperazines; Thiazoles

Three recently introduced anthelmintic agents, the macrocyclic lactone, ivermectin, the amoscanate derivative CGP 6140 and the benzothiazole compound CGP 20376, were investigated for their in vitro modulatory effects on eosinophilic effector cells. The investigation comprised studies on the generation of the toxic oxygen intermediates superoxide anion and hydrogen peroxide which are major effector products of granulocytes. Eosinophils were obtained from 19 patients infected with the filarial parasite Onchocerca volvulus. Inhibitory effects on the generation of toxic oxygen intermediates were demonstrated for ivermectin and CGP 20376 at concentrations higher than 200 ng/ml (0.5 microM) and for CGP 6140 at concentrations higher than 1000 ng/ml (2.7 microM). An increased production of the reactive oxygen metabolites was demonstrated at low doses of ivermectin (20-40 ng/ml; corresponding to 0.02-0.04 microM) and CGP 6140 (40-100 ng/ml; 0.1-0.3 microM), respectively. The results reveal a dual, dose-dependent modulatory in vitro effect of the investigated anthelmintic drugs on the respiratory burst of eosinophilic effector cells indicating that these compounds may modulate host defense in vivo.

Topics: Eosinophils; Filaricides; Humans; Hydrogen Peroxide; In Vitro Techniques; Ivermectin; Piperazines; Respiratory Burst; Superoxides; Thiazoles

Alterations in the fine structures of female Brugia spp. and Litomosoides carinii were investigated after in vivo treatment with curative doses of 4 compounds: CGP 20376 [2-tert-butyl-benzothiazole-5-methoxy-6-dithiocarbamic-S-(2- carboxyethyl)-ester], CGP 21833 (2-tert-butyl-benzothiazole-5-methyl-6- N-methylamino-piperazinylthiocarbonylamide), CGP 6140 [4-Nitro-4'-(N-methyl-piperazinylthiocarbonylamido)-diphenylamine] and amoscanate (4-isothiocyanato-4'-nitrodiphenylamine). All compounds caused early alterations in the somatic muscle cells. These alterations usually appeared within 24 h after treatment; they occurred later only after treatment of L. carinii with amoscanate. In Brugia spp., swelling of the muscle cells occurred in which the glycogen deposits considerably increased in size. The electron density of the cytoplasm surrounding the myofilaments in the fibrillar portion of the muscle cells increased, and light zones appeared between the fibrils. The muscle cell mitochondria swelled, particularly their inner matrix, which became more electron-lucent, with some dense spots. In L. carinii the muscle cells were not increased in size, but their mitochondria were considerably swollen before disintegration; this was followed by disintegration of the myofilaments and vacuolization of the cytoplasm. Vacuolization before mitochondrial swelling was observed only after treatment with CGP 6140. Other tissues of this species were not altered before the 2nd day after treatment. In Brugia spp., electron-lucent appeared in the hypodermis either simultaneously with the alterations in the muscle cells or a few hours later. At 24 h after treatment with amoscanate, blebs were formed on the luminal side of the intestinal membrane.

Topics: Aniline Compounds; Animals; Anthelmintics; Benzothiazoles; Brugia; Diphenylamine; Elephantiasis, Filarial; Female; Filariasis; Filaricides; Filarioidea; Isothiocyanates; Microscopy, Electron; Muridae; Piperazines; Thiazoles; Thiocyanates

Electron microscopic examination was used to assess the effects of CGP 6140 (proposed generic name: amocarcine) and CGP 20376 (proposed generic name: metobethiamide) on the fine structure of microfilariae and third stage larvae of Onchocerca volvulus after in vitro exposure to different concentrations of these compounds. The range of concentrations was selected according to the expected plasma levels that may be reached with these drugs. The microfilariae showed distinct effects on the muscle cells as myelin figures, vacuolisation and disintegration of the cytoplasm after both compounds. The third stage larvae showed the same effects on the muscle cells including changes in the mitochondria and the hypodermal tissue. In addition, the glandular esophagus demonstrated degenerative alterations of the cytoplasm and loosening of the cuticular lining after exposure to CGP 6140. CGP 20376 led to severe damage of the fibres of the nerve ring. No degenerative changes of the microtubuli were observed. Comparison of the morphological effects observed by electron microscopy with the reduction rates of motility and moulting recorded in the in vitro tests showed good correlation between these different parameters. The observed effect on the fine structure was the most sensitive parameter.

Topics: Animals; Anthelmintics; Filaricides; Microfilariae; Microscopy, Electron; Onchocerca; Piperazines; Thiazoles

It is suggested that the recently developed benzothiazole and amoscanate derivatives with antifilarial activity exert their action in vitro by an inhibition of mitochondrial-derived respiration. It was confirmed that the drugs CGP 20376, 21835, 20308, 21306, and 6140 cause a rapid immobilization in vitro of the adult filarial worm, Litomosoides carinii, the time required being similar to rotenone at the same concentration. The other drugs investigated, CGPs 20309, 21833, 24589, 23518, and 13231, were also effective; however, they required much longer incubation times. Submitochondrial particles (SMP) were prepared from Ascaris muscle and rat liver. The concentration of drug causing 50% inhibition of respiration (IC50) was calculated. It was found that the drugs most rapidly inhibiting respiration have IC50s for NADH oxidase of less than 25 microM in both Ascaris and rat liver SMP. This effect on SMP respiration could be overcome by using succinate as a substrate, indicating the site of inhibition to be within complex I of the mitochondrial respiratory chain. Further experiments showed that whereas the respiratory chain's NADH:ferricyanide reductase was unaffected by these drugs, there were pronounced effects on both Ascaris and rat liver NADH:quinone reductase activity. This suggests that the inhibition within complex I occurs after the flavoprotein dehydrogenase, but before the site of the quinone reduction. The other compounds examined, which had a slower effect on motility, also showed inhibition of the NADH oxidase, but not to as great an extent as the aforementioned compounds. The compounds most active against motility were also most effective at inhibiting respiration in intact adult L. carinii. Analysis of the aerobic end products produced by L. carinii showed that acetate production was greatly reduced even in the presence of low concentrations of the drugs. There was also a slight decrease in lactate production. However, a direct effect on the glycolytic pathway was ruled out by two observations. One, that the production of lactate from cell-free extracts of L. carinii is unaffected by the presence of the drugs, and secondly, that a protozoan, Giardia lamblia, reliant on glycolysis for energy production, can survive for long periods of time in the presence of high concentrations of the drugs. A correlation can be observed between the time for immobilization of the filarial worm and the strength of inhibition of mitochondrial respiration. Therefor

Topics: Anaerobiosis; Aniline Compounds; Animals; Anthelmintics; Benzothiazoles; Diphenylamine; Energy Metabolism; Female; Filaricides; Filarioidea; Hydrogen-Ion Concentration; Isothiocyanates; Movement; Oxygen Consumption; Piperazines; Rotenone; Thiazoles; Thiocyanates; Time Factors

The effect of four new antifilarial compounds, (CGP 6140, 20376, 20309 and 21833, Ciba Geigy Ltd. Basle) on the viability of third and fourth stage larvae of Onchocerca volvulus was tested in vitro and in vivo. The motility of the larval stages and the moulting process from the third to the fourth stage were used as parameters for the drug activity in vitro. All four compounds showed good effects in vitro on the moulting rates compared to the controls after incubation at different concentrations in culture medium with serum. After an exposure for three hours to different concentrations of the compounds in medium without serum, only CGP 20376 had a clear inhibitory effect on the developing larvae. The moulting rate was reduced by this compound to 20% of the control values with 1 microgram/ml, a concentration that is comparable to plasma levels reported from chimpanzees. In both experimental situations, with serum and without serum, the motility of the developing larvae was only affected at very high concentrations of the compounds. The results obtained with fourth stage larvae were comparable. CGP 6140 and CGP 20376 were tested in vivo, using a diffusion chamber technique for the implantation of infective larvae into the peritoneum of Mastomys natalensis and assessing the survival of the implanted parasites. High doses of CGP 6140 had only unsatisfactory effects on the larvae in this model system whereas CGP 20376 had inhibited moulting and killed most larvae with a daily dose of 25 mg/kg for five consecutive days.

Topics: Animals; Anthelmintics; Benzothiazoles; Culture Media; Filaricides; Movement; Onchocerca; Piperazines; Thiazoles

An in vivo drug screen for identifying new compounds with activity against Onchocerca macrofilariae was developed using male Onchocerca gibsoni implanted subcutaneously in outbred mice. There were several similarities (Mel W, CGP 20376, CGP 6140, levamisole) and two differences (suramin, furapyrimidone) between levels of drug efficacy in this model and activity against natural infections of O. gibsoni and O. volvulus. There was considerable variation in the mouse reaction. This mouse model is a potentially useful primary screen for macrofilaricidal drugs against Onchocerca.

Topics: Animals; Anthelmintics; Arsenicals; Filaricides; Male; Mice; Onchocerca; Onchocerciasis; Piperazines; Random Allocation; Thiazoles

The new compounds CGP 6140 and CGP 20376 were tested for their anthelmintic activity against adult Paragonimus uterobilateralis in experimentally infected Sigmodon hispidus. Both drugs were effective at a dosage of 2 x 100 mg/kg b.w. given orally with an interval of six hours between the doses. This regimen killed 80% of the flukes collected on Days 7 and 11 after treatment with CGP 6140 and 100% of the flukes collected on Days 5, 7, and 11 after therapy with CGP 20376. Both compounds showed no effect at single doses of 25, 50 and 100 mg/kg or with 12.5 and 25 mg/kg/day given on four consecutive days.

Topics: Animals; Anthelmintics; Arvicolinae; Dose-Response Relationship, Drug; Female; Male; Paragonimiasis; Paragonimus; Piperazines; Thiazoles

The effect of two new filaricidal compounds, CGP 6140 and CGP 20376, on the microfilariae of O. volvulus was tested in vitro. Culture medium consisted of a 1:1 mixture of IMDM/NCTC 135 supplemented with 20% heat-inactivated foetal calf serum and gentamycin. For pulse experiments the microfilariae were exposed to the drugs for one or three hours in medium without serum and then replaced to serum supplemented medium. In the continuous cultures the microfilariae were exposed to the drugs fur up to 72 hours without a change of medium. The effect of the drugs was assessed by their ability to reduce the larval motility. A reduction of motility of 98-100% after 24 hours of exposure occurred at levels of 1 micron/ml CGP 20376 or 10 mu mg/ml CGP 6140. After exposure for one hour CGP 20376 immobilized 100% of the microfilariae irreversibly at a drug level of 1 microgram/ml. Whereas no significant immobilization was seen with CGP 6140 after one hour. After exposure for three hours CGP 6140 immobilized up to 100% of the microfilariae at drug levels of 10 and 5 micrograms/ml.

Topics: Animals; Anthelmintics; Dose-Response Relationship, Drug; Humans; Microfilariae; Onchocerca; Piperazines; Thiazoles

An in vitro system for chemotherapeutic research using adult male Onchocerca gutturosa has been developed as a model for O. volvulus. Using a culture system consisting of medium MEM + 10% heat inactivated foetal calf serum (IFCS) + LLCMK2 (monkey kidney) feeder cells in an atmosphere of 5% CO2 in air, we examined the effects of a range of antiparasitic drugs on worm motility. Ivermectin, levamisole, furapyrimidone, Mel W, chloroquine, metrifonate, flubendazole, amoscanate and the Ciba-Geigy compounds CGP 6140, CGP 20'376 and CGI 17658 either immobilized or significantly reduced motility levels at a concentration of 5 X 10(-5) M or less within a 7-day period. Worms were affected at very low concentrations by ivermectin (effective conc. to reduce motility levels to 50% of controls, 3.14 X 10(-8) M), levamisole (7.95 X 10(-8) M), CGP 6140 (8.87 X 10(-9) M) and CGP 20'376 (2.78 X 10(-8) M). Difficulties were experienced in accurately repeating the immotile endpoint for levamisole due to an inconsistent partial recovery of motility. Over a 7-day period diethylcarbamazine had little effect on motility levels, while suramin caused a slight increase in activity compared to controls at some timepoints. Subsequent experiments demonstrated some differences in drug efficacy depending on the presence or absence of serum and feeder cells in the culture system probably because of drug avidly binding to serum proteins. However, serum and cells were found to be essential ingredients of the culture system to maintain worms in good condition, indicating that new drugs should be evaluated both in the presence and absence of serum and cells. Comparisons were made between the responses of O. gutturosa and Brugia pahangi to certain drugs and these species were found to significantly differ in their sensitivities to ivermectin and a novel compound (Wellcome), indicating that Onchocerca parasites should be used wherever possible for compound identification and development intended for the treatment of onchocerciasis. The in vitro system described here, using male O. gutturosa, provides a basis for further research and a practical alternative to O. volvulus.

Topics: Animals; Anthelmintics; Brugia; Filaricides; Ivermectin; Levamisole; Male; Movement; Onchocerca; Onchocerciasis; Piperazines; Thiazoles

A primary in vitro screen was developed to screen for drug activity against isolated Onchocerca gibsoni. The assay estimates variation in motility through the use of a motility meter. Of the seven compounds tested in the screen; ivermectin, CGP 6140, CGP20376, Mel W and furapyrimidone gave MI50 concentrations (the concentration at which the motility was reduced to 50% of the control value at 72 hours) below 10(-4) M, whereas suramin gave variable results depending on the varying susceptibility of individual worms and levamisole at 10(-4) M had no significant effect on the worms. The effects of these drugs were not reversible as removal of the worms into drug-free medium caused no increase in motility. Thus the reduction in motility is regarded as indicating significant metabolic damage. The results compared favourably with reported in vivo tertiary screens for activity against Onchocerca species. This is a quantitative, inexpensive and reproducible method for assessing the effectiveness of drugs against Onchocerca and could be included into the primary screens for activity against filarial worms.

Topics: Animals; Anthelmintics; Arsenicals; Drug Evaluation, Preclinical; Filaricides; Ivermectin; Levamisole; Male; Movement; Nitrofurans; Onchocerca; Piperazines; Suramin; Thiazoles

The drug response of Dipetalonema viteae and B. pahangi under various culture conditions have been evaluated. B. pahangi female worms proved to be more susceptible to CGP 6140 (4-nitro-4'-(N-methyl)piperazinyl thiocarbonylido-diphenylamine), CGP 20376 (N-(2-tert-butyl-5-methoxy-benzothiazol-6-yl)dithiocarbonic acid 5-(1-carboxyethyl)-ether) and amoscanate when glucose availability was restricted. This increased potency may be related to effects on glycogen metabolism by CGP 20376 and amoscanate. Using the parameters of parasite motility, survival, glucose consumption and microfilarial output, Eagles Minimum Essential Medium supplemented with 10% inactivated foetal calf serum plus either a 95% air:5% CO2 or a 95% N2:5% CO2 gas phase was shown to be highly suitable for short term maintenance (5 days). Examination of 12 drugs selected on their in vivo activity against B. pahangi and D. viteae demonstrated little difference between the drug susceptibilities of male and female worms. However, there were intrinsic differences between worm species. The relationship between these disparate susceptibilities and the reported in vivo efficacies of these drugs and the importance of selecting appropriate conditions for in vitro drug assays are discussed. Ivermectin at 10(-5) M caused a rapid flaccid paralysis and a complete suppression of microfilarial output by D. viteae. Despite continued paralysis of the worms they continued to utilise as much glucose as untreated worms over a 4 day period.

Topics: Animals; Anthelmintics; Brugia; Dipetalonema; Diphenylamine; Female; Filaricides; Glucose; Isothiocyanates; Ivermectin; Male; Piperazines; Thiazoles; Thiocyanates