Longterm follow-up of onchocerciasis patients in Latin America after treatment and retreatment with amocarzine. Preliminary results.

Amocarzine has been reported to have onchocercacidal effects. Four months posttherapy the majority of adult worms were dead or moribund. The effect of skin microfilariae lasted up to one year as reflected by markedly reduced microfilaridermia. Since the duration of the onchocercacidal effect of amocarzine beyond one year was unknown and since such an effect may influence the planning of future control strategies, efforts were made to follow-up the already treated amocarzine patients for a second year. The present study from Latin America showed that various amocarzine drug regimens produced a prolonged reduction of microfilaridermia at the end of the second year following the initial therapy, the best levels were about 7-17% of the initial parasite load in the skin for some three days amocarzine regimens. Such an effect occurring in a transmission area of onchocerciasis in Latin America provides additional, although indirect, evidence of a macrofilaricidal effect of amocarzine. Similar experiences of a prolonged amocarzine effect on skin microfilariae has also been observed in West Africa (Ghana, Mali). Preliminary results of retreatment schedules at the start of the third year post-initial therapy showed that simplified postprandial dose regimen of one or two days were well tolerated. It is premature at the time of this report to judge upon their ultimate efficacy, but they had significantly reduced levels of moderate microfilaridermia.

Antifilarial efficacy in vitro of 2-tert-butyl-benzothiazole derivatives on adult Litomosoides carinii.

Six 2-tert-butyl-benzothiazole derivatives (2-tert-butyl-6-isothiocyanato-5-methyl-benzothiazole (CGP 21306); 3-[(2-tert-butyl-5-methyl-benzothiazole-6-yl)aminothiocar-bonyl thiol] propionic acid (CGP 21835); 2-tert-butyl-5-methyl-6-(N-methyl-piperazinyl-thiocarbonylamino)- benzothiazole(CGP 21833); 2-tert-butyl-5-methyl-6-(4-dimethylamino-piperid-1-yl-thiocarbo nylamino)- benzothiazole (CGP 26702); CGP 20376, the 5-methoxy analogue to CGP 21835 and CGP 20309, the 5-methoxy analogue to CGP 21833) were tested in vitro against adult Litomosoides carinii. When exposed to the drugs in protein-free medium RPMI 1640 drug concentrations of 1 nmol/ml caused complete immobilization of female worms within 4 h (CGP 21306, 21835, 20376) or 20 h (CGP 21833, 26702, 20309). Short term exposure for 1 h had similar effects with CGP 21306, 21835, 21833 and 20376. However, the levels of CGP 26702 and 20309 (both thiourea derivatives) had to be increased to 10 nmol/ml to obtain complete immobilization after short term exposure. Male parasites were more resistant to the drugs than females under these experimental conditions. When the medium was supplemented with 10% foetal calf serum (FCS) the efficacy of all compounds was reduced. The effect of the compounds during permanent and short term exposure on the release of microfilariae was determined over an incubation period of 7 days (medium RPMI 1640/10% FCS). In general only drug concentrations which affected markedly the motility of the worms within 2 days reduced significantly the number of microfilariae released by treated worms.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro effects of 2-tert-butyl-benzothiazole derivatives on microfilariae of Litomosoides carinii, Brugia malayi and Acanthocheilonema viteae.

Six 2-tert-butyl-benzothiazole derivatives (2-tert-butyl-6-iso-thiocyanato-5-methyl-benzothiazole CGP 21306); 3-[(2-tert-butyl-5-methyl-benzothiazole-6-yl) aminothiocarbonylthiol] propionic acid (CGP 21835); 2-tert-butyl-5-methyl-6-(N-methyl-piperazinyl-thiocarbonylamino)-b enzothiazole (CGP 21833); 2-tert-butyl-5-methyl-6-(4-dimethylamino-piperid-1-yl-thiocarbo nylamino)- benzothiazole (CGP 26702); CGP 20376, the 5-methoxy analogue to CGP 21835 and CGP 20309, the 5-methoxy analogue to CGP 21833) with known, high filaricidal activity in vivo were tested for in vitro efficacy against microfilariae of L. carinii (Lc), B. malayi (Bm) and A. viteae (Av) in order to study intrinsic antifilarial activities. All drugs affected the motility of the microfilariae of the three species in a species, dose and time dependent fashion. Lc was the most sensitive, Av the most resistant species. CGP 20376 and 21835 were the most effective compounds followed by CGP 21306. Complete immobilization of microfilariae was observed after 20 h in protein-free medium RPMI 1640 at drug concentrations of 0.1 to 10 nmol/ml. Effects were still marked 2 when graded on a 4 (full motility) to 0 (immobile) scale at concentrations of 0.01-0.1 nmol/ml. In the case of the thiourea derivatives CGP 21833, 26702 and 20309 concentrations had to be increased 10-100 fold to obtain similar effects. When proteins were present in the incubation medium (10% foetal calf serum, 100% normal serum) the efficacy of the compounds was reduced, i.e. drug concentrations had to be increased up to 100 fold to produce similar effects as in protein-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Onchocercacidal effects of amocarzine (CGP 6140) in Latin America.

An open clinical trial of amocarzine was carried out in onchocerciasis patients in Ecuador and Guatemala. Administration after food was more effective than that during fasting. The most effective and best tolerated regimen, 3 mg/kg twice daily after food for 3 days (in 312 patients), killed 73% of 1477 female worms at nodulectomy 4 months after treatment. The mean microfilarial skin count was greatly reduced within a week (6-11% Of day 0 value on day 8) and it remained low at least 6 months (14-18% on day 180). Follow-up of a higher dose 3 day regimen taken while fasting showed microfilaridermia of 7-9% of the day 0 value 2 years after treatment.

Antifilarial activities of benzazole derivatives. 3. Effects of benzothiazoles on third stage larvae and preadult worms of Acanthocheilonema viteae, Brugia malayi and B. pahangi in Mastomys natalensis.

Ten structurally defined benzothiazoles (5-methyl and the analogous 5-methoxy derivatives) with known macrofilaricidal and microfilaricidal activities were tested for efficacy against third stage larvae and preadult worms in Acanthocheilonema viteae, Brugia malayi, and B. pahangi infected Mastomys natalensis. Drugs were administered in single oral doses of maximally 100 mg/kg. The benzothiazoles were active against the two stages of the three species. Generally the 5-methoxy derivatives displayed slightly higher activity than the 5-methyl compounds. 6-Isothiocyanates (CGP 21306, CGP 20308) and 6-dithiocarbamic-S-(2-carboxyethyl)esters (CGP 21835, CGP 20376) were more active than thiocarbonylamides (CGP 21833, CGP 20309, CGP 26702, CGP 24589). 6-Dithiocarbamic-S-(sulfomethylsodium)esters (CGP 26701, CGP 24588) showed intermediate efficacy. A. viteae was usually slightly more resistant than the Brugia spp. Minimum curative doses (greater than 95% reduction of worms) against the two stages of the various species were either identical or preadult worms were slightly more resistant than third stage larvae. When these curative doses were compared with curative adulticidal doses or effective doses against microfilariae the various doses were very similar and never differed from each other by more than the factor 2.

Antifilarial activity of CGP 20376 against subperiodic Brugia malayi in the leaf-monkey Presbytis cristata.

CGP 20376, a 5-methoxyl-6-dithiocarbamic-S- (2-carboxy-ethyl) ester derivative of benzothiazole was evaluated for its antifilarial properties and shown to be extremely effective against subperiodic Brugia malayi in the leaf-monkey, Presbytis cristata at oral doses of 20-100 mg/kg. The compound and/or its metabolites had complete micro- and microfilaricidal activities even when given at a single dose of 20 mg/kg. Lower doses had incomplete filaricidal action.

In vivo effect of benzothiazole and amoscanate derivatives on the fine structure of adult Brugia spp. and Litomosoides carinii.

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.

Antifilarial activities of benzazole derivatives. 2. Microfilaricidal effects against Litomosoides carinii, Acanthocheilonema. vitae, Brugia malayi and B. pahangi in Mastomys natalensis.

The spectrum of antimicrofilarial activities of eighteen 2-tertbutylbenzazole derivatives was evaluated comparatively in Mastomys natalensis infected with Litomosoides carinii, Acanthocheilonema viteae, Brugia malayi or B. pahangi. The minimal effective dose (DEM) against microfilariae (greater than 95% reduction of microfilariae counts in the peripheral blood) was determined on day 3 (DEM-3), on day 7, 14, 21, 28 and 42 (DEM-7, DEM-14, DEM-21, DEM-28 and DEM-42) after the first treatment. All compounds were effective against the microfilariae of all 4 species. The benzoxazole derivatives were invariably less potent than the corresponding benzothiazole analogues. Upon repeated oral treatment (once daily [o.d.] for five days) the DEM-7 of the benzoxazoles varied depending on the species and on the chemical structure between 25 mg/kg o.d. x 5 and greater than 100mg/kg o.d. x 5 days. Within the benzothiazole series the DEM-7 varied between 6.25 mg/kg o.d. x 5 and 100 mg/kg x 5. In all but 5 of the 40 parasite-compound combinations of the benzothiazoles the 5-methoxy-derivates were more effective than the 5-methyl analogues. Similar differences were found with the eight benz-oxazoles tested. The lowest DEM-7 was observed with compound CGP 20308 which is 2-tert-butyl-5-methoxy-6-isothio-cyanatobenzothiazole and with compound CGP 20376 which is 3-(2-tert-butyl-5-methoxy-benzothiazol-6-yl] amino-thiocabo-nylthio) propionic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Proposed form to evaluate some histological aspects of macrofilarial morphology, its age dependent alterations and drug related changes in nodules of Onchocerca volvulus and O. gibsoni.

Histological assessment of drug-related pathomorphological changes in macrofilariae of the two nodule forming species Onchocerca gibsoni and O. volvulus is hampered by the variability of age-dependent signs of natural degeneration and the virtual inexistence of clinically established reference medicaments. A specific form was designed to record systematically all characteristics observed in the various organs, tissues, cell types and developmental stages inside normal and drug-affected macrofilariae. Its tabular format permits the synoptic presentation of the findings in all female and male macrofilariae from a particular treated or untreated individual. The proposed form enumerates thirty eight morphological characteristics subdivided into the six subunits cuticle, hypodermis, longitudinal muscle, genital tract, pseudocoelomic cavity and intestinal tract (gut). The normal morphology of sexually mature and productive male and female O. volvulus is described and illustrated by photomicrographs. The cuticle of the male is wrinkled and forms regular striations strictly transverse to the body's longitudinal axis. The longitudinal muscles are well developed and cover almost 100% of the circumference at the anterior and posterior end. Towards the mid-body region the muscular portion diminishes to 50-60% and is replaced there by the very voluminous lateral hypodermic chords. The male genital tract originates with its distal end of the testis close to the anterior end and runs through almost the entire body very close to the posterior end. Spermatogenesis is clearly synchronized and the various development stages are arranged in strict sequential order inside the genital tract. Each type of gonosomal cell may thus serve as a marker of the respective region of the body. The same applies for females where the paired genital tubes run through almost the entire length of the body until the two uteri unite to form a short unpaired vagina. Each of the two reproductive tracts originates at different levels in the posterior part of the female. Because of this, two distinct stages of the sexual products are normally present in sections of the two uteri in the various parts of the female macrofilariae. Towards the anterior end identical stages of embryos may be found. The wide uteri narrow shortly before the vaginal junction to less than 40 microns in the very anterior part of the body. This part is characterized by the voluminous lateral chords of hypodermis and the well developed longitudinal muscles covering greater than 50% of the circumference. Drug-related changes come to light first in this part of the female's body.(ABSTRACT TRUNCATED AT 400 WORDS)

Antifilarial activities of benzazole derivatives. 1. Macrofilaricidal effects against Litomosoides carinii, Dipetalonema viteae, Brugia malayi, and B. pahangi in Mastomys natalensis.

Eighteen 2-tert-butyl-benzazole derivatives were evaluated comparatively as macrofilaricidal agents against L. carinii (L.c.), D. viteae (D.v.), B. malayi (B.m.) and B. pahangi (B.p.). Upon repeated treatment (once daily) for five consecutive days the eight benzoxazole derivatives were invariably less potent than the corresponding benzothiazole derivatives. The minimal curative dose (DCM) of the benzoxazoles varied depending on the species and on the chemical structure between 25 and 100 mg/kg p.o. once daily for five days. In the benzothiazole series the lowest DCMs were observed with compound CGP 20376 which is the 5-methoxy-6-dithiocarbamic-S-(2-carboxy-ethyl)ester derivative. This compound eliminated all macrofilariae of L.c., B.m. and Bp. at 6.25 mg/kg p.o. once a day for five days, whereas 12.5 mg/kg x 5 days were needed against D.v. For all other benzothiazole derivatives the DCMs varied between 6.25 mg/kg p.o. x 5 to 100 mg/kg x 5. Six of the most potent benzothiazoles were tested by single oral treatment. In general doses had to be increased 2-4 times to reach minimum curative effects. CGP 20376 was fully effective against B.m. and B.p. at 12.5 mg/kg p.o., against L.c. at 25 mg/kg p.o. and against D.v. at 50 mg/kg p.o.. This compound has been selected from this series of novel benzazoles as a first candidate for trials against human bancroftian filariasis.

Antifilarial activity of CGP 20,376 in chimpanzees (Pan t. troglodytes) naturally infected with Dipetalonema vanhoofi.

CGP 20,376, a benzthiazole and new antifilarial agent, was investigated at CIRMF in eight wild born chimpanzees naturally infected with Dipetalonema vanhoofi. Single oral doses (3.75, 7.5, 11 and 15 mg/kg) were administered. Drug levels during the first hour after administration were assessed in seven chimpanzees at 10 minute intervals in the blood. Levels of unchanged drug (CGP 20,376) were higher than those of its metabolite (CGP 20,308). However, there was considerable variation between individuals, although the results for each animal were consistent. Because of investigational limitations a complete drug profile could not be established. Unsheathed microfilariae of D. vanhoofi were monitored during the first hour following drug administration in seven chimpanzees. In five the microfilaraemia dropped to low counts within 10 minutes and remained below the initial values for the next 50 minutes while in two other chimpanzees it showed a more irregular reduction. Periodic microfilarial counts over the next 20 months, at roughly 30 day intervals, showed that three chimpanzees, treated with 7.5, 11 and 15 mg/kg respectively, remained free of circulating microfilariae from Day 1 to Day 600, the chimpanzee treated with 3.75 mg/kg remained microfilaremic and, in three chimpanzees low numbers of microfilariae reappeared within one year, whereas in the remaining ape they reappeared after one year. No major clinical adverse effects were observed, but liver function tests showed mild reversible changes at the 11 and 15 mg/kg doses. CGP 20,376 was therefore microfilaricidal, except for the lowest dose, and it was possibly macrofilaricidal in those chimpanzees which remained free of microfilariae for 600 days. Clinically CGP 20,376 was well tolerated.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological alterations in female litomosoides carinii after treatment with two benzthiazole derivatives in vivo.

Multimammate rats (Mastomys natalensis) infected with Litomosoides carinii were treated 65 days after infection with a single oral dose of 50 mg/kg of CGP 20308 or CGP 20376 (Ciba Geigy Limited, Basel). Autopsies were performed between 1 and 28 days after treatment. The numbers of worms and the numbers and condition of intrauterine developmental stages in the females were determined. Other female filariae were investigated by transmission electron microscopy (TEM). Female filariae were observed to be immobile on Day 2, later became encapsulated, and had almost disappeared on Day 28. The number of embryos had decreased on Day 6 and even more on Day 14, when only 5-10% appeared light-microscopically unaffected. TEM examination of the filarial midbody regions revealed, that the fine structures of all somatic muscle cells began to disintegrate as early as Day 4 after treatment with either drug. The breakdown of these cells was increased on Day 6 and Day 14. The cytoplasm of the nerve fibres was more electron-dense in treated than in untreated worms. No alterations were observed in the hypodermis, the cuticle, the intestine and the uterine epithelia. Sections from anterior and posterior regions of the parasites were studied after treatment with CGP 20308 only. Degenerating nerve fibres were found in the nerve rings of these filariae 4 days after treatment.

4-Isothiocyanato-4'-nitrodiphenylamine (C 9333-Go/CGP 4540), an anthelminthic with an unusual spectrum of activity against intestinal nematodes, filariae and schistosomes.

4-isothiocyanato-4'-nitrodiphenylamine was found to possess activity against intestinal nematodes in mice, against schistosomes in various hosts including primates and against two filarial species in the mongolian jird. Upon administration in a single oral dose it is equally effective against S. haematobium, S. mansoni and S. japonicum.