J. Pellegrino

Experimental Chemotherapy of Schistosomiasis mansoni

SummaryIn mice experimentally infected with Schistosoma mansoni, praziquantel (2-cyclohexylcarbonyl-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]isoquinoline-4-one), administered orally at the levels of 100 and 50 mg/kg, for 5 consecutive days, produced oogram changes in all animals and a pronounced hepatic shift of schistosomes (97.1 and 89.1, respectively). At lowest levels (12.5 and 6.3 mg/kg), alterations in the oogram could still be detected, although hepatic shift of schistosomes was no more evident. After a single intramuscular injection, the results obtained paralleled those observed with a single-dose oral treatment. The hepatic shift was only moderate at 200 and 100 mg/kg and the percentages of worms retained in the liver, after perfusion, were particularly low. When nasal route in a 1-day regimen was used, the results obtained were slightly less evident as compared with those observed by oral route (5-day schedule).Considering the percentage of oogram changes, the degree of hepatic shift of schistosomes and the percentage of worms fixed in the liver, the antischistosomal activity of praziquantel was greater in hamsters than in mice. Actually, a daily dose as low as 12.5 mg/kg, administered for 5 consecutive days, was sufficient to shift 60.4% of the worms towards the liver and to produce alterations of the oogram in 60% of the animals.In Cebus monkeys orally treated with 10 and 20 mg/kg of praziquantel, given 3 times within a single day (total doses of 30 and 60 mg/kg, respectively), a remarkable reduction in worm burden was observed. A single oral or intramuscular dose of 100 mg/kg was found to be curative. One Cebus dosed with 100 mg/kg, by nasal spray, was found to harbor only female worms at autopsy performed 69 days after treatment.ZusammenfassungVerabreichung von Praziquantel (2-Cyclohexylcarbonyl-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]isochinolin-4-on) an experimentell mit Schistosoma mansoni infizierte Mäuse in oralen Dosen von 100 bzw. 50 mg/kg an jeweils fünf aufein and erfolgenden Tagen verursachte bei allen Tieren Oogrammveränderungen und eine deutliche Verschiebung der Schistosomen (97,1 bzw. 89,1%) in die Leber. Auch bei den niedrigsten Dosen (12,5 und 6,3 mg/kg) wurden noch Oogrammveränderungen beobachtet, aber keine “liver shift” mehr. Nach einmaliger intramuskulärer Injektion ergaben sich vergleichbare Ergebnisse wie bei einmaliger oraler Gabe. Die “liver shift” nach Gabe von 200 bzw. 100 mg/kg war nur mäßig und der Anteil der nach Perfusion in der Leber zurückbleibenden Schistosomen besonders niedrig. Bei einmaliger nasaler Behandlung waren die Ergebnisse nicht ganz so gut wie nach oraler Gabe an fünf Tagen.Gemessen am Anteil der Tiere mit Oogrammveränderungen, dem Ausmaß der “liver shift” der Schistosomen und dem Prozentsatz der in der Leber fixierten Würmer ist Praziquantel im Hamster wirksamer gegen Schistosomen als in der Maus. Schon eine an fünf Tagen gegebene Dosis von 12,5 mg/kg bewirkte eine “liver shift” von 60,4% der Würmer und Oogrammveränderungen bei 60% der Tiere.Im Cebus-Affen wurde nach dreimaliger Gabe von 10 bzw. 20 mg/kg an einem Tag (Gesamtdosis 30 bzw. 60 mg/kg) eine bemerkenswerte Reduktion der Wurmzahl gefunden. Eine einmalige orale oder intramuskuläre Gabe von 100 mg/kg bewirkte eine Heilung. Ein Affe zeigte nach Behandlung mit 100 mg/kg in Form eines Nasensprays bei der Sektion nach 69 Tagen nur weibliche Schistosomen.In mice experimentally infected with Schistosoma mansoni, praziquantel (2-cyclohexylcarbonyl-1,3,4,6,7,11b-hexahydro-2H-pyrazino[2,1-a]isoquinoline-4-one), administered orally at the levels of 100 and 50 mg/kg, for 5 consecutive days, produces oogram changes in all animals and a pronounced hepatic shift of schistosomes (97.1 and 89.1, respectively). At lowest levels (12.5 and 6.3 mg/kg), alterations in the oogram could still be detected, although hepatic shift of schistosomes was no more evident. After a single intramuscular injection, the results obtained paralleled those observed with a single-dose oral treatment. The hepatic shift was only moderate at 200 and 100 mg/kg and the percentages of worms retained in the liver, after perfusion, were particularly low. When nasal route in a 1-day regimen was used, the results obtained were slightly less evident as compared with those observed by oral route (5-day schedule). Considering the percentage of oogram changes, the degree of hepatic shift of schistosomes and the percentage of worms fixed in the liver, the antischistosomal activity of praziquantel was greater in hamsters than in mice. Actually, a daily dose as low as 12.5 mg/kg, administered for 5 consecutive days, was sufficient to shift 60.4% of the worms towards the liver and to produce alterations of the oogram in 60% of the animals. In Cebus monkeys orally treated with 10 and 20 mg/kg of praziquantel, given 3 times within a single day (total doses of 30 and 60 mg/kg, respectively), a remarkable reduction in worm burden was observed. A single oral or intramuscular dose of 100 mg/kg was found to be curative. One Cebus doses with 100 mg/kg, by nasal spray, was found to harbor only female worms at autopsy performed 69 days after treatment.

Schistosomiasis mansoni: blockage of cercarial skin penetration by chemical agents: I. naphthoquinones and derivatives

Skin penetration by Schistosoma mansoni cercariae may be blocked by 1,4-and 1,2-naphthoquinones applied topically. Of 23 naphthoquinone derivatives synthesized, 15 afforded almost complete protection when applied in solution to the tails of mice 24 hours before exposure to cercariae. On the basis of present evidence the 5-carbon atom side chain present in naturally occurring lapachol seems to be important for activity, and this is in accord with the previous observation that effective protective agents are liposoluble. Topically applied cercarial penetration inhibitors are presently the only prophylactic agents available for use in the field.

Immunoglobulins in human schistosomiasis mansoni.

Immunoglobulin levels were determined by the reverse immunodiffusion technique in 3 groups of subjects: (A) 20 patients at the early stage of schistosomiasis mansoni infection; (B) 20 patients at the chronic stage of schistosomiasis; (C) 20 healthy controls. Group A showed significant increase of serum IgG (2,731 ? 608 mg/100 ml) and IgM (144 ? 92 mg/100 ml). The mean immunoglobulin levels in group C (control) were: IgG: 1,437 ? 224 mg/100 ml; IgM: 89 ? 16 mg/100 ml; IgA: 222 ? 93 mg/100 ml. The mean value of IgG, IgA, and IgM in group B did not differ significantly from the control group (1,705 317 mg/100 ml, 223 ? 120 mg/100 ml, 97 ? 15 mg/100 ml, respectively). The reverse immunodiffusion technique proved to be a simple and reliable method for the quantitative measurement of serum immunoglobulins. Human schistosomiasis mansoni presents different clinical and laboratory aspects according to the stage of the infection. During the early stage, high blood eosinophilia and positive precipitin tests are more frequent than in the chronic stage (Fiorillo et al., 1955; Ferreira et al., 1966; Reis et al., 1970). At the chronic stage (hepatointestinal and hepatosplenic forms) schistosome patients present blood eosinophilia and negative precipitin reaction (Dodin et al., 1966). Immunoelectrophoresis of sera from patients during the chronic stage shows specific antibodies against schistosome antigens in the IgG and IgM immunoglobulins (Silva and Ferri, 1965a, b). Hillyer (1969), using the conventional immunoplate technique, found increases of IgG and IgM in chronic schistosome patients. In this paper the immunoglobulin levels at the early and chronic stages of schistosomiasis were determined and an economical and very simple technique for quantitation of serum immunoglobulin is described. MATERIALS AND METHODS Schistosome patients and control Group A: Sera of 20 patients in the early stage (31 to 75 days of infection) of active schistosomiasis mansoni. Clinical diagnosis was based on the evidence provided by epidemiological data, clinical Received for publication 20 May 1970. * This work was supported by grants from the WHO, Geneva, Switzerland, and the Research Office of the U. S. Army (DAHC 19-69-G-0021). Contribution number 9 from the Schistosomiasis Research Unit. Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais. t Address: C. Postal 1404. Belo Horizonte, Brazil. symptoms, and laboratory tests as published elsewhere (Ferreira et al., 1966). All patients passed eggs in the stools, within 7 to 8 weeks after expo-

Experimental chemotherapy of Schistosomiasis III: laboratory and clinical trials with trichlorphone, an organophosphorus compound

Oogram studies have been carried out on mice, hamsters, and Cebus morikeys experimentally infected with Schistosoma mansoni and treated with trichlorphone (0,0-dimethyl 1-hydroxy-2, 2, 2-trichloroethylphosphonate). In mice, despite a slight hepatic shift of schistosomes, all animais presented oogram changes when dosed, per os, at the schedules of 200, and 100 mg/kg/day × 7. In hamsters, antischistosomal activity could be detected only at toxic leveis. In monkeys, trichlorphone showed insignificant action even after oral administration of 30 mg/kg/day for 10 consecutive days. In 5 volunteers, a sharp drop in cholinesterase plasma level was observed 24 hours after a single oral dose of 7.5 mg/kg. However, cholinesterase levels returned to the initial values within a period of 11 to 27 days. Trichlorphone was then administered to 12 schistosome patients (7.5 mg/kg/day, every fort- night, × 5). One month after therapy, interruption of egg laying was observed in 6 patients. Late parasitological control showed that all treated patients continued to pass viable S. mansoni eggs with their stools.

Activity of known antischistosomal agents on early-developing forms of Schistosoma mansoni.

Sixteen compounds, including several series of antischistosomal agents, were tested in mice (subcutaneous, oral, and intramuscular routes), previously injected intraperitoneally with 175 plus or minus 25 Schistosoma mansoni cercariae. Most drugs were given in a 5-day regimen, first dose being administered 3 hr before cercarial inoculation. Ten days after infection the animals were killed, the schistosomules collected from peritoneal washings, and counted under a dissecting microscopy (free organisms and larvae surrounded by macrophages). A high degree of activity (abscess of free larvae) was observed with Hoechst S-616 and S668, Ciba 17,581, Oxamniquine (Pfizer), and RD 12,869 compounds. A significant reduction (P less than 0.05) in the number of larvae was observed with A-16,612, Sb-EDTA, Hycanthone, Schistomide, Trichlorphone, and Antiomaline. No activity on nearly developing forms of S. mansoni was observed after treatment with Mirasan, Hoechst S-201, Schistocide T-109, Lucanthone, and Wellcome 153C51. In conclusion, from 16 compounds taken at random, all of which are active on mature schistosome infections, 11 displayed prophylactic activity. This indicates a high sensitivity of the technique using peritoneal schistosomules.

Infection of baby mice with Schistosoma mansoni; some biological aspects in connection with experimental chemotherapy.

Groups of mice 2–6 days old were exposed by paddling to 35 or 50 cercariae of Schistosoma mansoni per animal. The mean worm burden from 115 baby mice exposed to 35 cercariae was 20·7 (59·2% of worm return). In 400 adult mice, weighing about 20 g. and exposed to 120 cercariae, worm return was only 18·6% (a mean worm burden of 22·3 schistosomes). 8–10 weeks after exposure of baby mice to S. mansoni cercariae, 70–85% of the schistosomes were found in the mesenteric veins, 5–10% in the liver, and 10–20% in the portal vein. The mortality rate among 662 baby mice exposed to 50 cercariae was found to increase sharply after the 6th week of infection. The percentages of the various schistosome elements (oogram) in the intestinal wall of infected baby mice, 53–60 days after exposure to S. mansoni cercariae, did not differ from the pattern already described for mice infected when adult. The therapeutic activity of 14 antischistosomal compounds was compared in groups of baby mice and adult animals experimentally infected with S. mansoni. No significant differences were found when the hepatic shift of worms and oogram changes were used for the assessment of activity. The infection of baby mice with S. mansoni cercariae represents a simple and reliable working model for chemotherapeutic studies.

Ensaio laboratorial e clínico com Hycanthone, nôvo agente esquistossomicida

Starting from Miracil D (Lucanthone), a hydroxymethyl derivative (Hycanthone) can be obtained through the biological activity of the microorganism Aspergillus sclerotiorum. This compound was claimed to be the antischistosomal metabolite of Miracil D and was found very active when administered to mice, hamsters and Cebus monkeys experimentally infected with Schistosoma mansoni. Clinical trials with Hycanthone were performed on 52 patients with active schistosomiasis mansoni. The drug was administered, per os, at the dose leveis of 2 and 3 mg/kg/day with an antacid preparation, twice a day, for 5 consecutive days. In all but 2 patients treatment could be completed. Nausea and/or vomiting, anorexia, vertigo and headache were the commonest side effects. Therapeutic activity toas evaluated by repeated stool examinations (4 to 6) and one rectal biopsy performed at least 4 months after treatment. The percentages of patients considered as cured were 83.3 and 80.0 for the schedules of 2 and 3 mg/kg, respectively. Laboratory and clinical data on the antischistosomal activity of Hycanthone obtained so far emphasize need for further clinical trials with this compound.

Intradermal test with histamine in schistosomiasis mansoni.

Histamine diphosphate was injected intradermally, at dilutions of 10(-3), 10(-4), and 10(-5), into 25 boys (10--12 yr old), and 25 men (20--30 yr old) with active schistosomiasis mansoni. A linear relationship between the mean area of the wheals and the logarithm of histamine concentrations was determined. Both equations (men and boys) were significant (P less than 0.05) but showed no difference between their regression coefficients. These findings contrast with those obtained with the intradermal test performed with Schistosoma mansoni antigens, in which skin response in men is significantly greater than in boys.