Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Evaluation of schistosomicides against experimentally established schistosoma mansoni infections in mice and hamsters Allen Yarinsky To cite this article: Allen Yarinsky (1975) Evaluation of schistosomicides against experimentally established schistosoma mansoni infections in mice and hamsters, Journal of Toxicology and Environmental Health, 1:2, 229-242, DOI: 10.1080/15287397509529324 To link to this article: http://dx.doi.org/10.1080/15287397509529324
Published online: 20 Oct 2009.
Submit your article to this journal
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh19 Download by: [University of Florida]
Date: 14 November 2015, At: 09:39
EVALUATION OF SCHISTOSOMICIDES AGAINST EXPERIMENTALLY ESTABLISHED Schistosoma mansoni INFECTIONS IN MICE AND HAMSTERS Allen Yarinsky Sterling Winthrop Research Institute, Rensselaer,
Downloaded by [University of Florida] at 09:39 14 November 2015
New York
The procedures employed at the Sterling Winthrop Research Institute for the maintenance of Schistosoma mansoni, method of infection, and measurement of efficacy are discussed. Data are presented illustrating the activities of a number of known antischistosomal agents in mice and hamsters against a Puerto Rican strain of the parasite with a well-documented origin.
INTRODUCTION A number of techniques for infection, medication, and evaluation of antischistosomal agents in rodents have been reviewed by Schubert (1948a,b), Standen (1963), and Pellegrino and Katz (1968, 1969a). Recently, Lammler and Petranyi (1971) have shown the multimammate mouse, Mastomys natalensis, to be a suitable host for S. mansoni. In establishing a drug-testing program, attention must be given to the target for chemotherapeutic attack. A consideration of the life cycle of S. mansoni in relation to the mammalian host shows that it can be conveniently separated into at least three stages, each of which is potentially vulnerable to chemotherapy. The first stage is the young schistosomulum. In order for a therapeutic agent with prophylactic properties to be detected, its effect must serve to abrogate early infection. Medication should be initiated immediately prior to cercarial exposure of the animal and continued during the first few days of development of the parasite. A second phase of the infection that is amenable to attack occurs during the period of growth of the sexually immature schistosomes. Conditions for therapeutic evaluation would be limited to weeks 2-5 when the parasites are maturing in the hepatic portal system. In this case suppression of the infection would be highly desirable. The third phase of the infection against which chemotherapy may be applied occurs after maturation of the parasites when deposition of eggs takes place by the female worms. Requests for reprints should be sent to Allen Yarinsky, Sterling Winthrop Research Institute, Rensselaer, New York 12144.
229 Journal of Toxicology and Environmental Health, 1:229-242, 1975 Copyright © 1975 by Hemisphere Publishing Corporation
230
A. YARINSKY
Downloaded by [University of Florida] at 09:39 14 November 2015
The stage of the life cycle selected for chemotherapeutic attack depends upon the goal of the investigator. Since millions of people are infected with schistosomes and many exhibit serious pathology associated with the disease, it seems logical to employ a test system in the laboratory that reasonably mimics the situation in humans so that a drug can be evaluated for curative properties; that is, it should kill mature worms and thereby rid the host of the source of eggs. In the event a successful curative drug is discovered, its properties would then be characterized against other stages of the parasite so that a complete chemotherapeutic picture will emerge. Fortunately, the three schistosomes that are infective for man can be successfully maintained in the laboratory. However, initial screening of chemical compounds is usually directed against S. mansoni. If significant activity against this species is obtained and confirmed, supplementary tests can be conducted with S. haematobium and S. japonicum so that the spectrum of the compound's activity can be determined. Methods of infecting mammalian hosts and the preferred routes and regimens of medication vary from laboratory to laboratory. Cercariae may be given subcutaneously, percutaneously, or intraperitoneally. The route of infection does not appear to influence the assessment of chemotherapeutic agents against adult worms residing in the mesenteric veins and hepatic portal system. For example, in two separate laboratories no significant differences were noted in susceptibility of the Sterling Winthrop Research Institute Puerto Rican strain of 5. mansoni in mice to intramuscular injections of hycanthone and a chloroindazole analog IA-4, although in one laboratory the mice were infected percutaneously (Bueding et a!., 1973) and in the other, intraperitoneally (Table 2). Furthermore, to the author's knowledge there is no evidence that the pathology produced in murine schistosomiasis is dependent on the route of infection. Available information strongly implicates the sexually mature parasites in initiating the series of events leading to formation of granulomas in the tissues. Elegant work by Warren and von Lichtenberg and their colleagues have characterized the pathogenesis of experimental schistosomiasis mansoni principally as a immunological phenomenon involving a delayed hypersensitivity response by the host to the schistosome egg and soluble antigenic substances secreted therefrom (Warren, 1972; Dunsford et al., 1974). Test compounds may be administered orally by intubation or inclusion in the diet and parenterally by injection. The period of medication usually varies from 1 to 5 or 10 days, depending on the choice of the route of administration. Criteria for drug efficacy include reduction in the numbers of eggs deposited in tissues, oogram changes, increase in survival time of medicated animals, the hepatic shift of schistosomes, and reduction in live-worm burden. In addition, alterations in the worm's
Downloaded by [University of Florida] at 09:39 14 November 2015
EVALUATION OF SCHISTOSOMICIDES
231
biochemical pathways induced by a test drug may provide useful information (Bueding and Fisher, 1969). Because of the variety of model systems, standardization of methodology by pharmaceutical, university, and government laboratories is difficult if not impossible to attain, nor is it necessarily desirable. For example, if a pharmaceutical firm discovers a novel synthetic to be orally curative in mice, as judged by high mortality of worms of an Egyptian strain of S. mansoni, the compound may be requested by N. Katz for testing in his laboratory in Brazil. In this case, Katz might choose to administer the drug by another route, possibly with a different medication regimen, against a Brazilian strain of 5. mansoni, probably in another strain of mouse, and he may select the oogram technique to quantitate efficacy. Should the compound again prove to be antischistosomal, the original work has been confirmed and a great deal of supplemental information is obtained. It should be emphasized that the in vivo testing of schistosomicidal agents is not a simple matter and the results are not necessarily meaningful in relation to chemotherapy of the naturally acquired infection of humans. This report contains a description of the procedures employed at the Sterling Winthrop Research Institute (SWRI) for the schistosome infection of small animals and also a measurement of chemotherapeutic efficacy. Data are presented illustrating the activity profiles of a number of experimentally verified, as well as several clinically proven, antischistosomals tested at SWRI against a Puerto Rican strain of 5. mansoni with a well-documented origin. MATERIALS AND METHODS Rodent models have been used for more than 25 yr at SWRI for in vivo screening and evaluation of synthetic chemical agents designed as antischistosomal drugs. The strain of S. mansoni was obtained from infected hamsters received from M. Schubert of the New York University College of Medicine in 1948. The strain was originally brought from Puerto Rico by W. Wright of the National Institutes of Health and given to H. Stunkard of New York University in 1943, who supplied it to Schubert. The intermediate snail host, Biomphalaria glabrata, was obtained by SWRI in 1947 from Wright. The progeny of these snails, which were raised at SWRI, were infected in 1948. Subsequently, the infection has been continuously passaged through this strain of B. glabrata and Syrian hamsters. Details for breeding and rearing the snails and the methodology for infecting them with miracidia and later harvesting cercariae to infect animals have been published (Archer and Yarinsky, 1972). Since different geographical strains of a given species of schistosome or even substrains from the same general locale may exhibit varying
Downloaded by [University of Florida] at 09:39 14 November 2015
232
A. YARINSKY
susceptibilities to a particular drug (Gonnert and Vogel, 1955; Hsu et al., 1963; Taylor and Nelson, 1971; Lee et al., 1971; Foster et al., 1971; Bueding et al., 1973; Foster and Cheetham, 1973; SWRI, unpublished data), it is.difficult to compare directly the results of evaluations of antischistosomal drugs from different laboratories. Even within a single laboratory, to ensure long-term reliability of the test system, it is important to safeguard the purity of separate strains of schistosomes that are on hand. At SWRI about 300 18-22-g female Swiss-Webster mice or 70-80-g male Syrian hamsters are routinely infected at a time. It is imperative that the animals be infected within a brief period so that viability of the cercariae will not be reduced. For this reason the cercariae are injected intraperitoneally; each mouse receives 200 organisms and each hamster receives 125. The infection procedure is completed within 2 hr after cercarial emergence from the snails and gives rise to approximately 10 adult worm pairs per animal. Test medications to groups of 5-10 animals begin 46 days postinfection, which is after the appearance of eggs in the tissues. Compounds to be administered orally are suspended or dissolved in 10% autoclaved gelatin, or in some cases distilled water, and given in two equally subdivided daily dosages, spaced 8 hr apart, for 5 consecutive days. The total daily dose, calculated to base, is 400 mg/kg for mice and 200 mg/kg for hamsters. When desirable, medications are also prepared in distilled water or a suitable oil and injected parenterally, usually as a single dose. Animals are killed with chloroform or by cervical dislocation 3 wk after the initiation of medication and the liver and intestine with adhering mesenteries are clamped off and extirpated. The liver is crushed between rectangular glass plates and examined under a dissecting microscope for living and dead adult schistosomes. The intestine, cut into loops so that the mesenteric veins converge to the center of the loop, is similarly pressed between glass plates and examined for the presence of living and dead worms. Occasionally, as a consequence of intraperitoneal inoculation, small numbers of immature stages of the parasite can be found in the peritoneal cavity. They are easily recognized by their small size and lack of development and are not included in the chemotherapeutic assessment. Experience has shown that within a 3-wk period after medication, an active drug will cause schistosomes to relocate from the mesenteric veins to the liver where they will be observed to be immobile and partially depigmented with early signs of host cell infiltration and digestion. Worms in this condition are classified as dead. If at least 30% of the total number of schistosomes observed are dead, the test is repeated. If efficacy is confirmed, a search is initiated among metabolites and analogs of the test compound in the hope of finding increased activity. Eventually the most effective compound is titrated in terms of an ED 50 , which is the dose required to kill 50% of the schistosomes. The EDS0 is calculated for oral as well as parenteral medications. Infected, nonmedicated control animals are included in all tests.
EVALUATION OF SCHISTOSOMICIDES
233
RESULTS AND COMMENTS
Downloaded by [University of Florida] at 09:39 14 November 2015
As early as 1906 antimonials were employed for the treatment of trypanosomiasis, cutaneous leishmaniasis, and kala azar. However, it was not until 1918 that Christopherson introduced tartar emetic (potassium antimony tartrate) for the treatment of schistosomiasis (Schmidt and Peter, 1938). The intravenous route of medication, the length of treatment, and the incidence and severity of numerous side effects including cardiovascular damage were considered objectionable. Although substitution of sodium for the potassium ion increased tolerance to the drug, a high incidence of side effects still occurs. A search for a less toxic and more easily administrable schistosomicide led to stibophen, which was introduced in 1929 (Schmidt and Peter, 1938). The drug, injected intramuscularly about 15 times over a period of 3 wk, has proven to be better tolerated than tartar emetic, although the cure rate is not as high. Freidheim et al. (1954) reported a novel antimony-containing compound, potassium antimony a,a'-dimercaptosuccinate, to be antischistosomal. In clinical trials both intramuscular and intravenous administration of the sodium salt, stibocaptate, resulted in variable effectiveness. This and other considerations have limited the usefulness of the drug (Standen, 1963). Table 1 illustrates the activity of the three antimony-containing drugs in mice and hamsters. They were evaluated at SWRI by a series of intraperitoneal . injections given for 5 days beginning on the 39th postinfection day (Berberian and Freele, 1964). Noteworthy is that greater sensitivity to the antimonials by the worms was observed in the hamster. A breakthrough in chemotherapy occurred in the late 1930s at Farbenfabriken Bayer where a series of xanthenone compounds were synthesized (Mauss, 1948) and tested (Kikuth and Gonnert, 1948). Although one of the series, Miracil D (lucanthone), an orally administered nonantimonial, was demonstrated to be clinically effective against S. mansoni and 5. haematobium, consistently high cure rates were not achieved with the drug (Blair, 1958). In testing a large number of Miracil-like compounds with variations in the substituents on the rings, the German workers concluded that best activity was obtained with the dialkylaminoalkylamino side chain in the 1-position and a methyl in the 4-position. It was further believed, on the basis of products recovered from the urine of medicated animals, that a metabolite of Miracil D might be responsible for schistosomicidal activity. However, extensive research failed to isolate any substance that was more potent than the parent compound (Archer and Yarinsky, 1972). An investigation at SWRI to isolate the active moiety of Miracil D proceeded in parallel with that of the Germans. In 1963 a fermentation medium containing the organism Aspergillus scleratorium produced a number of products from Miracil D including a 4-hydroxymethyl homolog,
Downloaded by [University of Florida] at 09:39 14 November 2015
TABLE 1. Activity of Representative Antimoniais against Schistosoma mansoni in Mice and Hamsters ED50 ± SE (mg/kg) Name
Structure
o=c
Antimony potassium tartrate
r°\
Mice0
Hamsters0
17.2 ±5.5
4.3 ± 0.4
190 ± 12
24.0 ± 1.2
>200
56.0 ± 23
H-C-O-Sb -S H-C-O / O=C-O-K
Stibophen
SO3Na
NaOjS
SO3Na
Sodium antimony dimercaptosuccinate (Stibocaptate)
SO3Na
NaOoc
COONa
1 j ; Sb-S S-Sb? HC-S/ HC-CH NaOOC I
.S-CH C COONa
NaOOC COONa
"Intraperitoneal medication was administered once daily for 5 consecutive days beginning on the 39th postinfection day.
Downloaded by [University of Florida] at 09:39 14 November 2015
EVALUATION OF SCHISTOSOMICIDES
235
hycanthone, which was found to be a more potent schistosomicide in mice and hamsters when given orally than the parent compound. In addition, the homoiog was active in both rodents by intramuscular injection1 (Rosi et al., 1965; Berberian et al., 1967a,b). It was subsequently demonstrated that between 6 and 24 hr after mice were given a high single intramuscular injection of 80 mg/kg, a distinct shift of worms from the mesenteric veins to the liver occurred. Dead worms were observed as early as 24 hr after treatment with a marked increase in their number between days 4 and 7 (Table 3). The results of clinical trials have confirmed the efficacy of hycanthone against 5. mansoni and 5. haematobium following a single intramuscular injection of 3 me/kg (Dennis, 1970). Hycanthone is ineffective in mice against the Philippine and Japanese strains of 5. japonicum (Yarinsky et al., 1972). Enhancement of schistosomicidal activity by conversion of a parent compound to a more active "metabolite" has also been accomplished with other drugs. An 8-quinolinemethanol synthesized by the Boots Pure Drug Co. was reported to be antischistosomal in mice (Bristow et al., 1967). Activity against S. mansoni was confirmed in this host, but no effect was obtained in the hamster. Microbial conversion of the 8-methyl to an 8-hydroxymethyl by chemists at SWRI led to an increase in activity, particularly in the hamster (Bailey et al., 1970). No activity was demonstrated against 5. japonicum. Pfizer workers achieved fermentative conversion at the 6-methyl position of an aminomethyltetrahydroquinoline, UK 3883, to a more potent 6-hydroxymethyl analog, UK 4271 (oxamniquine) (Baxter and Richards, 1971; Foster and Cheetham, 1973; Foster et al., 1973). In limited studies at SWRI, the 6-hydroxymethyl derivative was more active than the parent compound when given orally to the hamster but not by intramuscular injection in the mouse. Owing to differences in experimental techniques, a direct comparison of the LD5Os for oxamniquine and its parent compound, UK 3883, cannot be made with the data reported by the Pfizer group (Foster et al., 1971; Foster and Cheetham, 1973). Niridazole (Ambilhar, manufactured by Ciba) displayed activity at SWRI against 5. mansoni in the mouse and hamster following oral administration of the compound. Its efficacy against 5. japonicum in mice was also confirmed. A study of the structural essentials required of the nitroheterocyclic niridazole for antischistosomal activity led to an investigation of a conformationally related nitrofuran derivative, SQ 18,506 (Robinson et al., 1970). Reportedly S Q 1 8 , 5 0 6 is effective against experimentally induced infections of 5. mansoni and 5. japonicum (Bueding et al., 1971; 1
Data on antischistosomal efficacy of this and other compounds mentioned in the text are given in Table 2.
Downloaded by [University of Florida] at 09:39 14 November 2015
iTABLE 2. Activity of Selected Antischistosomal Agents against Schistosoma mansoni Infections of Mice and Hamsters ED50
±SE(mg/kg) Hamsters
Mice Structure
Name
O
po"
im 6
po"
im*
NHCH 2 CH 2 N(CjH 5 ) 2
Miracil D (lucanthone)
CH,
46.0 ± 2 . 4
> 150
8.0 ± 1.0
> 50
Hycanthone
CH,OH
14.2 ±0.1
12.0 ± 1 . 3
0.93 ± 0.1
1.5 ± 0.2
CH,
23.0 ± 8 . 5
32.0 ± 4 . 8
> 200
> 200
8-Hydroxymethyl derivative of Boots compound
CHjOH
17.0 ± 2 . 6
18.0 ± 2 . 8
approx. 70
approx. 100
UK 3883
CH,
< 12.5
12.5-25
6.25-50
> 100
CH,OH
< 12.5
> 25
< 6.25
> 6.25
to
w 6-Chloro-50-diethylaminoethylamino-8methylquinoline (Boots Pure Drug Co.)
UK 4271 (oxamniquine)
NHCH 2 CH 2 N(C 2 H s )j
CH2NHCH(CHj)2
Niridazole
N
?H* •N
?H*
126 + 26.3
> 150
100-200
> 50
205 ± 73
> 800
>100
not tested
not tested
24.0 ±5.7
not tested
13.0 ±4.8
> 400
not tested
> 200
not tested
>200
>100
> 200
>100
NH II O
SQ 18,506
Downloaded by [University of Florida] at 09:39 14 November 2015
N
IA-4
1 rr -CH=CH-
N
N(CH 2 ) 2 N(C 2 H 5 ) 2
CH 2 OH
Nicarbazin
O2N
Metrifonate (trichlorphon) CH 3 O
"Oral medication was given in equally subdivided daily dosages spaced 8 hr apart, for 5 consecutive days initiated on the 46th postinfection day. ^"Intramuscular medication was given as a single dose on the 46th postinfection day.
238
A. YARINSKY
TABLE 3. Mortality of Sch/stosoma mansoni in Mice Given a Single Intramuscular Injection of 80 mg/kg Hycanthone 46 Days after Infection Living and dead worms (%)°
Necropsy period, postmedication
Downloaded by [University of Florida] at 09:39 14 November 2015
2hr
6 hr 1 day 2 days 4 days 1 wk 2 wk 3wk 4 wk 8 wk 12 wk
Total
Mesenteric veins
Liver Live
Dead
Live
Dead
Live
9.3
0 0 5.0 9.6
90.7 88.2 65.8 45.8 31.2
0 0 1.2 4.6 3.6 5.8 0.5
100.0 100.0 93.8 85.8 72.7
11.8 28.0 40.0 41.5 0.6 0 0 0 0 0
23.7 89.5 95.0 95.0 100.0 99.0 98.3
4.1 4.5 2.5 0 1.0
1.7
2.5 0 0 0
4.7 4.5 2.5 0 1.0 1.7
Dead 0 0 6.2
14.2 27.3 95.3 95.5 97.5 100.0 99.0 98.3
°No dead worms were found in control infected nonmedicated mice at the various necropsy periods; 91-100% of the worms were in the mesenteric veins.
Erickson et al., 1971; Lennox and Bueding, 1972). In our laboratory the compound showed modest activity when given orally to mice with a 5. mansoni infection. Poor absorption of SQ 18,506, possibly related to particle size, may be a factor in determining antischistosomal effectiveness (Bueding et al., 1971). Recently, interest has been shown in a chloroindazole, IA-4 (Bueding et al., 1973). Some efficacy was observed with a single intramuscular injection to mice and hamsters infected with 5. mansoni. Interestingly, IA-4 (as well as hycanthone) exhibited poor activity against another Puerto Rican strain of S. mansoni maintained at the Parke Davis laboratory (Elslager, 1970). The relative insensitivity of this strain to thioxanthenes underscores the need for testing a variety of known antischistosomals belonging to different chemical classes before a laboratory embarks on a chemotherapeutic program with synthetic agents having unproven qualities. Nicarbazin, an equimolar complex of 4,4'-dinitrocarbanilide and 2-hydroxy-4,6-dimethylpyrimidine, was found to suppress egg production in female worms in mice under continuous drug pressure (Campbell and Cuckler, 1967; Pellegrino and Katz, 1969b). This was not achieved in hamsters (Pellegrino and Katz, 1969b). When the complex was tested by oral means at 400 mg/kg/day X 5 days in mice and at 200 mg/kg/day X 5 days in hamsters, dead worms were not observed in both rodents.
EVALUATION OF SCHISTOSOMICIDES
239
Downloaded by [University of Florida] at 09:39 14 November 2015
Of the organic phosphates explored for antischistosomal properties, metrifonate (trichlorphon) has received the greatest attention. Although the compound produced oogram changes in mice infected with 5. mansoni, it is essentially ineffective against this species in the hamster and in man (Katz et al., 1968; James et al., 1972). Infections with S. haematobium respond to treatment (Cerf et al., 1962; Hanna et al., 1966; Talaat et al., 1966; Forsyth and Rashid, 1967a,b; Davis and Bailey, 1969; Plestina et al., 1972; James et al., 1972). In tests conducted at SWRI, metrifonate was not lethal to S. mansoni in mice and hamsters following oral medication of 200 mg/kg/day X 5 days and by a single intramuscular injection of 100 mg/kg.
DISCUSSION Although in vivo screening tests are time-consuming and restrictive in regard to the number of compounds that can be put through the system during any period, they offer an inestimable advantage in providing a dynamic environment for a synthetic agent in which the activity of a nonmetabolized drug as well as the products of metabolism can be realized. Procedures employed at SWRI utilizing the mouse and hamster and a long-established Puerto Rican strain of 5. mansoni have proven valuable in detecting activity of experimental as well as clinically established antischistosomal drugs. Of 14 antimonial and nonantimonial reference compounds tested in vivo, only two were without activity. Of the latter, metrifonate is ineffective against 5. mansoni infection in humans and nicarbazin has not been shown to be a clinically useful drug. Despite the introduction of new chemotherapeutic agents, the worldwide incidence of schistosomiasis is on the increase. Factors such as population growth, dam-building in conjunction with extensive irrigation projects, and the lack of recognition of the insidious nature of the disease are contributing to its spread. Implementation of control measures including chemotherapy, mollusciciding, and ways to better manage water resources have been and will continue to be difficult to accomplish. Although notable achievements have taken place during the past decade, problems related to laboratory and field investigations require resolution. In encouraging research to characterize the influence of the disease on disability and socioeconomic development, it is significant that the World Health Organization (WHO, 1974) acknowledges that "it is not merely lack of manpower and money that has inhibited progress, but also insufficient confidence among the decision-makers in such control measures as are available and a lack of conviction that the disease deserves a high priority."
240
A. YARINSKY
Downloaded by [University of Florida] at 09:39 14 November 2015
REFERENCES Archer, S. and Yarinsky, A. 1972. Recent developments in the chemotherapy of schistosomiasis. In Progress in drug research, ed. E. Jucker, vol. 16, pp. 11-66. Basel: Birkhäeuser. Bailey, D. M., Archer, S., Wood, D., Rosi, D. and Yarinsky, A. 1970. Synthesis and schistosomicidal activity of 6-chloro-5-{[2-(diethylamino)ethyl]amino]-8-quinolinemethanol. J. Med. Chem. 13:598-601. Baxter, C. A. R. and Richards, H. C. 1971. Schistosomicides. 1. Derivatives of 2-aminomethyl1,2,3,4-tetrahydroquinoline. J. Med. Chem. 14:1033-1042. Berberian, D. A. and Freele, H. 1964. Chemotherapeutic effect of antischistosomal drugs in experimentally induced Schistosoma mansoni infections in Swiss mice and Syrian hamsters. J. Parasitol. 50:435-440. Berberian, D. A., Freele, H., Rosi, D., Dennis, E. W. and Archer, S. 1967a. Schistosomicidal activity of lucanthone hydrochloride, hycanthone and their metabolites in mice and hamsters. J. Parasitol. 53:306-311. Berberian, D. A., Freele, H., Rosi, D., Dennis, E. W. and Archer, S. 1967b. A comparison of oral and parenteral activity of hycanthone and lucanthone in experimental infections with Schistosoma mansoni. Amer. J. Trop. Med. Hyg. 16:487-491. Blair, D. M. 1958. Lucanthone hydrochloride, a review. Bull. World Health Org. 18:989-1010. Bristow, N. W., Lessel, B., Richards, H. C. and Williams, G. A. H. 1967. New aminoquinoline with schistosomicidal activity. Nature 216:282-283. Bueding, E. and Fisher, J. 1969. Biochemical effects of schistosomicides. Ann. N.Y. Acad. Sci. 160:5 36-543. Bueding, E., Náquira, C., Bouwman, S. and Rose, G. 1971. The antischistosomal activity of a nitrovinylfuran derivative (SQ 18,506) in mice and hamsters. J. Pharmacol. Exp. Ther. 178:402-410. Bueding, E., Fisher, J. and Bruce, J. 1973. The antischistosomal activity of a chloroindazole analog of hycanthone in mice infected with Schistosoma mansoni. J. Pharmacol. Exp. Ther. 186:402-407. Campbell, W.C. and Cuckler, A. S. 1967. Inhibition of egg production of Schistosoma mansoni in mice treated with nicarbazin. J. Parasitol. 53:977-980. Cerf, J., Lebrun, A. and Dierichx, J. 1962. A new approach to helminthiasis control: The use of an organophosphorous compound. Amer. J. Trop. Med. Hyg. 11:514-517. Davis, A. and Bailey, D. R. 1969. Metrifonate in urinary schistosomiasis. Bull. World Health Org. 41:209-224. Dennis, E. W. 1970. Chemotherapy of schistosomiasis (past and present) with special reference to hycanthone. A summary. In Proc. II Simposio Sobre Esquistossomose, July 2-6, 1969, pp. 23-48. Publ. by A. Prata and E. Aboim, Salvador, Brazil. Dunsford, H. A., Lucia, H. L., Doughty, B. L. and von Lichtenberg, F. 1974. Artificial granulomas from bentonite and latex carrier particles. Amer. J. Trop. Med. Hyg. 23:203-217. Elslager, E. F. 1970. Schistosomiasis chemotherapy-Progress at a snail's pace. Presented at Twelfth National Medicinal Chemical Symposium, American Chemical Society, University of Washington, Seattle, June 22-25. Erickson, D. G., Bourgeois, J. G., Sadun, E. H. and Bueding, E. 1971. Antischistosomal activity of a nitrovinylfuran derivative (SQ 18,506) in Rhesus monkeys. J. Pharmacol. Exp. Ther. 178:411-416. Forsyth, D. M. and Rashid, C. 1967a. Treatment of urinary schistosomiasis. Practice and theory. Lancet 1:130-133. Forsyth, D. M. and Rashid, C. 1967b. Treatment of urinary schistosomiasis with trichlorophone. Lancet 2:909-912. Foster, R. and Cheetham, B. L. 1973. Studies with the schistosomicide oxamniquine (UK-4271) I. Activity in rodents and in vitro. Trans. Roy. Soc. Trop. Med. Hyg. 67:674-684. Foster, R., Cheetham, B. L., King, D. F. and Mesmer, E. T. 1971. The action of UK 3883, a novel 2-aminomethyltetrahydroquinoline derivative, against mature schistosomes in rodents and primates. Ann. Trop. Med. Parasitol. 65:59-70.
Downloaded by [University of Florida] at 09:39 14 November 2015
EVALUATION OF SCHISTOSOMICIDES
241
Foster, R., Cheetham, B. L. and King, D. F. 1973. Studies with the schistosomicide oxamniquine (UK-4271) II. Activity in primates. Trans. Roy. Soc. Trop. Med. Hyg. 67:685-693. Friedheim, E. A. H., da Silva, J. R. and Martins, A. V. 1954. Treatment of schistosomiasis mansoni with antimony-α,α'-dimercaptopotassium succinate (TWSb). Amer. J. Trop. Med. Hyg. 3:714-727. Gönnert, R. and Vogel, H. 1955. Über die Abhängigkeit des Therapieerfolges von Wirts und Parasitenstamm bei der experimentellen Schistosomiasis. Z. Tropenmed. Parasitol. 6:193-198. Hanna, S., Basmy, K., Selim, O., Shoeb, S. M. and Awny, A. Y. 1966. Effects of administration of an organo-phosphorus compound as an antibilharzial agent, with special reference to plasma cholinesterase. Brit Med. J. 1:1390-1392. Hsü, S. Y. Li, Chu, K. Y. and Hsü, H. F. 1963. Drug susceptibility of geographic strains of Schistosoma japonicum. Z. Tropenmed. Parasitol. 14:37-40. James, C., Webbe, G. and Preston, J. M. 1972. A comparison of the susceptibility to metrifonate of Schistosoma haematobium, S. mattheei and 5. mansoni in hamsters. Ann. Trop. Med. Parasitol. 66:461-414. Katz, N., Pellegrino, J. and Pereira, J. P. 1968. Experimental chemotherapy of schistosomiasis. III–Laboratory and clinical trials with trichlorphone, an organophosphorus compound. Rev. Soc. Brasil. Med. Trop. 2:237-245. Kikuth, W. and Gönnert, R. 1948. Experimental studies on the therapy of schistosomiasis. Ann. Trop. Med. Parasitol. 42:256-267. Lämmler, G. and Petranyi, G. 1971. Chemotherapeutic studies on experimental Schistosoma mansoni infection of Mastomys natalensis. Bull. World Health Org. 44:739-750. Lee, H. G., Cheever, A. W. and Fairweather, W. R. 1971. Influence of parasite strain on chemotherapy of murine infections with Schistosoma mansoni. Bull. World Health Org. 45:147-155. Lennox, R. W. and Bueding, E. 1972. The relative chemotherapeutic efficacy of a nitrovinylfuran (SQ 18,506) against immature and mature stages of Schistosoma mansoni. Amer. J. Trop. Med. Hyg. 21:302-306. Mauss, H. 1948. Miracil, a new chemotherapeutic agent. Berichte 81:19-31. Pellegrino, J. and Katz, N. 1968. Experimental chemotherapy of schistosomiasis mansoni. In Advances in parasitology, ed. B. Dawes, vol. 6, pp. 233-290. New York: Academic Press. Pellegrino, J. and Katz, N. 1969a. Laboratory evaluation of antischistosomal agents. Ann. N.Y. Acad. Sci. 160:429-460. Pellegrino, J. and Katz, N. 1969b. Experimental chemotherapy of schistosomiasis IV–Oogram studies with nicarbazin, an egg-suppressive agent. Rev. Inst. Med. Trop. Sao Paulo 11:215-221. Plestina, R., Davis, A. and Bailey, D. R. 1972. Effect of metrifonate on blood cholinesterases in children during the treatment of schistosomiasis. Bull. World Health Org. 46:747-759. Robinson, C. H., Bueding, E. and Fisher, J. 1970. Relationship between structure, conformation, and antischistosomal activity of nitroheterocyclic compounds. Mol. Pharmacol. 6:604-616. Rosi, D., Peruzzotti, G., Dennis, E. W., Berberian, D. A., Freele, H. and Archer, S. 1965. A new, active metabolite of "Miracil D." Nature 208:1005-1006. Schmidt, H. and Peter, F. M. 1938. Advances in the therapeutics of antimony, pp. 85-118. Leipzig: Georg Thieme. Schubert, M. 1948a. Conditions for drug testing in experimental schistosomiasis mansoni in mice. Amer. J. Trop. Med. 28:121-136. Schubert, M. 1948b. Screening of drugs in experimental schistosomiasis mansoni in mice. Amer. J. Trop. Med. 28:137-156. Standen, O. D. 1963. Chemotherapy of helminthic infections. In Experimental chemotherapy, ed. R. J. Schnitzer and F. Hawking, vol. 1, pp. 701-892. New York: Academic Press. Talaat, S. M., Amin, N. and El Masry, B. 1966. A comparative study of dipterex and tartar emetic in the treatment of urinary schistosomiasis. Trans. Roy. Soc. Trop. Med. Hyg. 60:579-584. Taylor, M. G. and Nelson, G. S. 1971. A comparison of the susceptibility to niridazole of two geographical strains of Schistosoma mansoni in mice with a note on the susceptibility of S. mattheei. Trans. Roy. Soc. Trop. Med. Hyg. 65:169-174.
242
A. YARINSKY
Warren, K. S. 1972. The ¡mmunopathogenesis of schistosomiasis: A multidisciplinary approach. Trans. Roy. Soc. Trop. Med. Hyg. 66:417-434. WHO. 1974. Annual Report of the Director-General to the World Health Assembly and to the United Nations. Official Records of the World Health Organization, no. 213, p. 41. The Work of WHO, 1973. Geneva. Yarinsky, A., Hernandez, P., Ferrari, R. A. and Freele, H. W. 1972. Effects of hycanthone against two strains of Schistasoma japonicum in mice. Jap. J. Parasitol. 21:101-108.
Downloaded by [University of Florida] at 09:39 14 November 2015
Received April 2, 1975 Accepted April 7, 1975
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Evaluation of schistosomicides against experimentally established schistosoma mansoni infections in mice and hamsters Allen Yarinsky To cite this article: Allen Yarinsky (1975) Evaluation of schistosomicides against experimentally established schistosoma mansoni infections in mice and hamsters, Journal of Toxicology and Environmental Health, 1:2, 229-242, DOI: 10.1080/15287397509529324 To link to this article: http://dx.doi.org/10.1080/15287397509529324
Published online: 20 Oct 2009.
Submit your article to this journal
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh19 Download by: [University of Florida]
Date: 14 November 2015, At: 09:39
EVALUATION OF SCHISTOSOMICIDES AGAINST EXPERIMENTALLY ESTABLISHED Schistosoma mansoni INFECTIONS IN MICE AND HAMSTERS Allen Yarinsky Sterling Winthrop Research Institute, Rensselaer,
Downloaded by [University of Florida] at 09:39 14 November 2015
New York
The procedures employed at the Sterling Winthrop Research Institute for the maintenance of Schistosoma mansoni, method of infection, and measurement of efficacy are discussed. Data are presented illustrating the activities of a number of known antischistosomal agents in mice and hamsters against a Puerto Rican strain of the parasite with a well-documented origin.
INTRODUCTION A number of techniques for infection, medication, and evaluation of antischistosomal agents in rodents have been reviewed by Schubert (1948a,b), Standen (1963), and Pellegrino and Katz (1968, 1969a). Recently, Lammler and Petranyi (1971) have shown the multimammate mouse, Mastomys natalensis, to be a suitable host for S. mansoni. In establishing a drug-testing program, attention must be given to the target for chemotherapeutic attack. A consideration of the life cycle of S. mansoni in relation to the mammalian host shows that it can be conveniently separated into at least three stages, each of which is potentially vulnerable to chemotherapy. The first stage is the young schistosomulum. In order for a therapeutic agent with prophylactic properties to be detected, its effect must serve to abrogate early infection. Medication should be initiated immediately prior to cercarial exposure of the animal and continued during the first few days of development of the parasite. A second phase of the infection that is amenable to attack occurs during the period of growth of the sexually immature schistosomes. Conditions for therapeutic evaluation would be limited to weeks 2-5 when the parasites are maturing in the hepatic portal system. In this case suppression of the infection would be highly desirable. The third phase of the infection against which chemotherapy may be applied occurs after maturation of the parasites when deposition of eggs takes place by the female worms. Requests for reprints should be sent to Allen Yarinsky, Sterling Winthrop Research Institute, Rensselaer, New York 12144.
229 Journal of Toxicology and Environmental Health, 1:229-242, 1975 Copyright © 1975 by Hemisphere Publishing Corporation
230
A. YARINSKY
Downloaded by [University of Florida] at 09:39 14 November 2015
The stage of the life cycle selected for chemotherapeutic attack depends upon the goal of the investigator. Since millions of people are infected with schistosomes and many exhibit serious pathology associated with the disease, it seems logical to employ a test system in the laboratory that reasonably mimics the situation in humans so that a drug can be evaluated for curative properties; that is, it should kill mature worms and thereby rid the host of the source of eggs. In the event a successful curative drug is discovered, its properties would then be characterized against other stages of the parasite so that a complete chemotherapeutic picture will emerge. Fortunately, the three schistosomes that are infective for man can be successfully maintained in the laboratory. However, initial screening of chemical compounds is usually directed against S. mansoni. If significant activity against this species is obtained and confirmed, supplementary tests can be conducted with S. haematobium and S. japonicum so that the spectrum of the compound's activity can be determined. Methods of infecting mammalian hosts and the preferred routes and regimens of medication vary from laboratory to laboratory. Cercariae may be given subcutaneously, percutaneously, or intraperitoneally. The route of infection does not appear to influence the assessment of chemotherapeutic agents against adult worms residing in the mesenteric veins and hepatic portal system. For example, in two separate laboratories no significant differences were noted in susceptibility of the Sterling Winthrop Research Institute Puerto Rican strain of 5. mansoni in mice to intramuscular injections of hycanthone and a chloroindazole analog IA-4, although in one laboratory the mice were infected percutaneously (Bueding et a!., 1973) and in the other, intraperitoneally (Table 2). Furthermore, to the author's knowledge there is no evidence that the pathology produced in murine schistosomiasis is dependent on the route of infection. Available information strongly implicates the sexually mature parasites in initiating the series of events leading to formation of granulomas in the tissues. Elegant work by Warren and von Lichtenberg and their colleagues have characterized the pathogenesis of experimental schistosomiasis mansoni principally as a immunological phenomenon involving a delayed hypersensitivity response by the host to the schistosome egg and soluble antigenic substances secreted therefrom (Warren, 1972; Dunsford et al., 1974). Test compounds may be administered orally by intubation or inclusion in the diet and parenterally by injection. The period of medication usually varies from 1 to 5 or 10 days, depending on the choice of the route of administration. Criteria for drug efficacy include reduction in the numbers of eggs deposited in tissues, oogram changes, increase in survival time of medicated animals, the hepatic shift of schistosomes, and reduction in live-worm burden. In addition, alterations in the worm's
Downloaded by [University of Florida] at 09:39 14 November 2015
EVALUATION OF SCHISTOSOMICIDES
231
biochemical pathways induced by a test drug may provide useful information (Bueding and Fisher, 1969). Because of the variety of model systems, standardization of methodology by pharmaceutical, university, and government laboratories is difficult if not impossible to attain, nor is it necessarily desirable. For example, if a pharmaceutical firm discovers a novel synthetic to be orally curative in mice, as judged by high mortality of worms of an Egyptian strain of S. mansoni, the compound may be requested by N. Katz for testing in his laboratory in Brazil. In this case, Katz might choose to administer the drug by another route, possibly with a different medication regimen, against a Brazilian strain of 5. mansoni, probably in another strain of mouse, and he may select the oogram technique to quantitate efficacy. Should the compound again prove to be antischistosomal, the original work has been confirmed and a great deal of supplemental information is obtained. It should be emphasized that the in vivo testing of schistosomicidal agents is not a simple matter and the results are not necessarily meaningful in relation to chemotherapy of the naturally acquired infection of humans. This report contains a description of the procedures employed at the Sterling Winthrop Research Institute (SWRI) for the schistosome infection of small animals and also a measurement of chemotherapeutic efficacy. Data are presented illustrating the activity profiles of a number of experimentally verified, as well as several clinically proven, antischistosomals tested at SWRI against a Puerto Rican strain of 5. mansoni with a well-documented origin. MATERIALS AND METHODS Rodent models have been used for more than 25 yr at SWRI for in vivo screening and evaluation of synthetic chemical agents designed as antischistosomal drugs. The strain of S. mansoni was obtained from infected hamsters received from M. Schubert of the New York University College of Medicine in 1948. The strain was originally brought from Puerto Rico by W. Wright of the National Institutes of Health and given to H. Stunkard of New York University in 1943, who supplied it to Schubert. The intermediate snail host, Biomphalaria glabrata, was obtained by SWRI in 1947 from Wright. The progeny of these snails, which were raised at SWRI, were infected in 1948. Subsequently, the infection has been continuously passaged through this strain of B. glabrata and Syrian hamsters. Details for breeding and rearing the snails and the methodology for infecting them with miracidia and later harvesting cercariae to infect animals have been published (Archer and Yarinsky, 1972). Since different geographical strains of a given species of schistosome or even substrains from the same general locale may exhibit varying
Downloaded by [University of Florida] at 09:39 14 November 2015
232
A. YARINSKY
susceptibilities to a particular drug (Gonnert and Vogel, 1955; Hsu et al., 1963; Taylor and Nelson, 1971; Lee et al., 1971; Foster et al., 1971; Bueding et al., 1973; Foster and Cheetham, 1973; SWRI, unpublished data), it is.difficult to compare directly the results of evaluations of antischistosomal drugs from different laboratories. Even within a single laboratory, to ensure long-term reliability of the test system, it is important to safeguard the purity of separate strains of schistosomes that are on hand. At SWRI about 300 18-22-g female Swiss-Webster mice or 70-80-g male Syrian hamsters are routinely infected at a time. It is imperative that the animals be infected within a brief period so that viability of the cercariae will not be reduced. For this reason the cercariae are injected intraperitoneally; each mouse receives 200 organisms and each hamster receives 125. The infection procedure is completed within 2 hr after cercarial emergence from the snails and gives rise to approximately 10 adult worm pairs per animal. Test medications to groups of 5-10 animals begin 46 days postinfection, which is after the appearance of eggs in the tissues. Compounds to be administered orally are suspended or dissolved in 10% autoclaved gelatin, or in some cases distilled water, and given in two equally subdivided daily dosages, spaced 8 hr apart, for 5 consecutive days. The total daily dose, calculated to base, is 400 mg/kg for mice and 200 mg/kg for hamsters. When desirable, medications are also prepared in distilled water or a suitable oil and injected parenterally, usually as a single dose. Animals are killed with chloroform or by cervical dislocation 3 wk after the initiation of medication and the liver and intestine with adhering mesenteries are clamped off and extirpated. The liver is crushed between rectangular glass plates and examined under a dissecting microscope for living and dead adult schistosomes. The intestine, cut into loops so that the mesenteric veins converge to the center of the loop, is similarly pressed between glass plates and examined for the presence of living and dead worms. Occasionally, as a consequence of intraperitoneal inoculation, small numbers of immature stages of the parasite can be found in the peritoneal cavity. They are easily recognized by their small size and lack of development and are not included in the chemotherapeutic assessment. Experience has shown that within a 3-wk period after medication, an active drug will cause schistosomes to relocate from the mesenteric veins to the liver where they will be observed to be immobile and partially depigmented with early signs of host cell infiltration and digestion. Worms in this condition are classified as dead. If at least 30% of the total number of schistosomes observed are dead, the test is repeated. If efficacy is confirmed, a search is initiated among metabolites and analogs of the test compound in the hope of finding increased activity. Eventually the most effective compound is titrated in terms of an ED 50 , which is the dose required to kill 50% of the schistosomes. The EDS0 is calculated for oral as well as parenteral medications. Infected, nonmedicated control animals are included in all tests.
EVALUATION OF SCHISTOSOMICIDES
233
RESULTS AND COMMENTS
Downloaded by [University of Florida] at 09:39 14 November 2015
As early as 1906 antimonials were employed for the treatment of trypanosomiasis, cutaneous leishmaniasis, and kala azar. However, it was not until 1918 that Christopherson introduced tartar emetic (potassium antimony tartrate) for the treatment of schistosomiasis (Schmidt and Peter, 1938). The intravenous route of medication, the length of treatment, and the incidence and severity of numerous side effects including cardiovascular damage were considered objectionable. Although substitution of sodium for the potassium ion increased tolerance to the drug, a high incidence of side effects still occurs. A search for a less toxic and more easily administrable schistosomicide led to stibophen, which was introduced in 1929 (Schmidt and Peter, 1938). The drug, injected intramuscularly about 15 times over a period of 3 wk, has proven to be better tolerated than tartar emetic, although the cure rate is not as high. Freidheim et al. (1954) reported a novel antimony-containing compound, potassium antimony a,a'-dimercaptosuccinate, to be antischistosomal. In clinical trials both intramuscular and intravenous administration of the sodium salt, stibocaptate, resulted in variable effectiveness. This and other considerations have limited the usefulness of the drug (Standen, 1963). Table 1 illustrates the activity of the three antimony-containing drugs in mice and hamsters. They were evaluated at SWRI by a series of intraperitoneal . injections given for 5 days beginning on the 39th postinfection day (Berberian and Freele, 1964). Noteworthy is that greater sensitivity to the antimonials by the worms was observed in the hamster. A breakthrough in chemotherapy occurred in the late 1930s at Farbenfabriken Bayer where a series of xanthenone compounds were synthesized (Mauss, 1948) and tested (Kikuth and Gonnert, 1948). Although one of the series, Miracil D (lucanthone), an orally administered nonantimonial, was demonstrated to be clinically effective against S. mansoni and 5. haematobium, consistently high cure rates were not achieved with the drug (Blair, 1958). In testing a large number of Miracil-like compounds with variations in the substituents on the rings, the German workers concluded that best activity was obtained with the dialkylaminoalkylamino side chain in the 1-position and a methyl in the 4-position. It was further believed, on the basis of products recovered from the urine of medicated animals, that a metabolite of Miracil D might be responsible for schistosomicidal activity. However, extensive research failed to isolate any substance that was more potent than the parent compound (Archer and Yarinsky, 1972). An investigation at SWRI to isolate the active moiety of Miracil D proceeded in parallel with that of the Germans. In 1963 a fermentation medium containing the organism Aspergillus scleratorium produced a number of products from Miracil D including a 4-hydroxymethyl homolog,
Downloaded by [University of Florida] at 09:39 14 November 2015
TABLE 1. Activity of Representative Antimoniais against Schistosoma mansoni in Mice and Hamsters ED50 ± SE (mg/kg) Name
Structure
o=c
Antimony potassium tartrate
r°\
Mice0
Hamsters0
17.2 ±5.5
4.3 ± 0.4
190 ± 12
24.0 ± 1.2
>200
56.0 ± 23
H-C-O-Sb -S H-C-O / O=C-O-K
Stibophen
SO3Na
NaOjS
SO3Na
Sodium antimony dimercaptosuccinate (Stibocaptate)
SO3Na
NaOoc
COONa
1 j ; Sb-S S-Sb? HC-S/ HC-CH NaOOC I
.S-CH C COONa
NaOOC COONa
"Intraperitoneal medication was administered once daily for 5 consecutive days beginning on the 39th postinfection day.
Downloaded by [University of Florida] at 09:39 14 November 2015
EVALUATION OF SCHISTOSOMICIDES
235
hycanthone, which was found to be a more potent schistosomicide in mice and hamsters when given orally than the parent compound. In addition, the homoiog was active in both rodents by intramuscular injection1 (Rosi et al., 1965; Berberian et al., 1967a,b). It was subsequently demonstrated that between 6 and 24 hr after mice were given a high single intramuscular injection of 80 mg/kg, a distinct shift of worms from the mesenteric veins to the liver occurred. Dead worms were observed as early as 24 hr after treatment with a marked increase in their number between days 4 and 7 (Table 3). The results of clinical trials have confirmed the efficacy of hycanthone against 5. mansoni and 5. haematobium following a single intramuscular injection of 3 me/kg (Dennis, 1970). Hycanthone is ineffective in mice against the Philippine and Japanese strains of 5. japonicum (Yarinsky et al., 1972). Enhancement of schistosomicidal activity by conversion of a parent compound to a more active "metabolite" has also been accomplished with other drugs. An 8-quinolinemethanol synthesized by the Boots Pure Drug Co. was reported to be antischistosomal in mice (Bristow et al., 1967). Activity against S. mansoni was confirmed in this host, but no effect was obtained in the hamster. Microbial conversion of the 8-methyl to an 8-hydroxymethyl by chemists at SWRI led to an increase in activity, particularly in the hamster (Bailey et al., 1970). No activity was demonstrated against 5. japonicum. Pfizer workers achieved fermentative conversion at the 6-methyl position of an aminomethyltetrahydroquinoline, UK 3883, to a more potent 6-hydroxymethyl analog, UK 4271 (oxamniquine) (Baxter and Richards, 1971; Foster and Cheetham, 1973; Foster et al., 1973). In limited studies at SWRI, the 6-hydroxymethyl derivative was more active than the parent compound when given orally to the hamster but not by intramuscular injection in the mouse. Owing to differences in experimental techniques, a direct comparison of the LD5Os for oxamniquine and its parent compound, UK 3883, cannot be made with the data reported by the Pfizer group (Foster et al., 1971; Foster and Cheetham, 1973). Niridazole (Ambilhar, manufactured by Ciba) displayed activity at SWRI against 5. mansoni in the mouse and hamster following oral administration of the compound. Its efficacy against 5. japonicum in mice was also confirmed. A study of the structural essentials required of the nitroheterocyclic niridazole for antischistosomal activity led to an investigation of a conformationally related nitrofuran derivative, SQ 18,506 (Robinson et al., 1970). Reportedly S Q 1 8 , 5 0 6 is effective against experimentally induced infections of 5. mansoni and 5. japonicum (Bueding et al., 1971; 1
Data on antischistosomal efficacy of this and other compounds mentioned in the text are given in Table 2.
Downloaded by [University of Florida] at 09:39 14 November 2015
iTABLE 2. Activity of Selected Antischistosomal Agents against Schistosoma mansoni Infections of Mice and Hamsters ED50
±SE(mg/kg) Hamsters
Mice Structure
Name
O
po"
im 6
po"
im*
NHCH 2 CH 2 N(CjH 5 ) 2
Miracil D (lucanthone)
CH,
46.0 ± 2 . 4
> 150
8.0 ± 1.0
> 50
Hycanthone
CH,OH
14.2 ±0.1
12.0 ± 1 . 3
0.93 ± 0.1
1.5 ± 0.2
CH,
23.0 ± 8 . 5
32.0 ± 4 . 8
> 200
> 200
8-Hydroxymethyl derivative of Boots compound
CHjOH
17.0 ± 2 . 6
18.0 ± 2 . 8
approx. 70
approx. 100
UK 3883
CH,
< 12.5
12.5-25
6.25-50
> 100
CH,OH
< 12.5
> 25
< 6.25
> 6.25
to
w 6-Chloro-50-diethylaminoethylamino-8methylquinoline (Boots Pure Drug Co.)
UK 4271 (oxamniquine)
NHCH 2 CH 2 N(C 2 H s )j
CH2NHCH(CHj)2
Niridazole
N
?H* •N
?H*
126 + 26.3
> 150
100-200
> 50
205 ± 73
> 800
>100
not tested
not tested
24.0 ±5.7
not tested
13.0 ±4.8
> 400
not tested
> 200
not tested
>200
>100
> 200
>100
NH II O
SQ 18,506
Downloaded by [University of Florida] at 09:39 14 November 2015
N
IA-4
1 rr -CH=CH-
N
N(CH 2 ) 2 N(C 2 H 5 ) 2
CH 2 OH
Nicarbazin
O2N
Metrifonate (trichlorphon) CH 3 O
"Oral medication was given in equally subdivided daily dosages spaced 8 hr apart, for 5 consecutive days initiated on the 46th postinfection day. ^"Intramuscular medication was given as a single dose on the 46th postinfection day.
238
A. YARINSKY
TABLE 3. Mortality of Sch/stosoma mansoni in Mice Given a Single Intramuscular Injection of 80 mg/kg Hycanthone 46 Days after Infection Living and dead worms (%)°
Necropsy period, postmedication
Downloaded by [University of Florida] at 09:39 14 November 2015
2hr
6 hr 1 day 2 days 4 days 1 wk 2 wk 3wk 4 wk 8 wk 12 wk
Total
Mesenteric veins
Liver Live
Dead
Live
Dead
Live
9.3
0 0 5.0 9.6
90.7 88.2 65.8 45.8 31.2
0 0 1.2 4.6 3.6 5.8 0.5
100.0 100.0 93.8 85.8 72.7
11.8 28.0 40.0 41.5 0.6 0 0 0 0 0
23.7 89.5 95.0 95.0 100.0 99.0 98.3
4.1 4.5 2.5 0 1.0
1.7
2.5 0 0 0
4.7 4.5 2.5 0 1.0 1.7
Dead 0 0 6.2
14.2 27.3 95.3 95.5 97.5 100.0 99.0 98.3
°No dead worms were found in control infected nonmedicated mice at the various necropsy periods; 91-100% of the worms were in the mesenteric veins.
Erickson et al., 1971; Lennox and Bueding, 1972). In our laboratory the compound showed modest activity when given orally to mice with a 5. mansoni infection. Poor absorption of SQ 18,506, possibly related to particle size, may be a factor in determining antischistosomal effectiveness (Bueding et al., 1971). Recently, interest has been shown in a chloroindazole, IA-4 (Bueding et al., 1973). Some efficacy was observed with a single intramuscular injection to mice and hamsters infected with 5. mansoni. Interestingly, IA-4 (as well as hycanthone) exhibited poor activity against another Puerto Rican strain of S. mansoni maintained at the Parke Davis laboratory (Elslager, 1970). The relative insensitivity of this strain to thioxanthenes underscores the need for testing a variety of known antischistosomals belonging to different chemical classes before a laboratory embarks on a chemotherapeutic program with synthetic agents having unproven qualities. Nicarbazin, an equimolar complex of 4,4'-dinitrocarbanilide and 2-hydroxy-4,6-dimethylpyrimidine, was found to suppress egg production in female worms in mice under continuous drug pressure (Campbell and Cuckler, 1967; Pellegrino and Katz, 1969b). This was not achieved in hamsters (Pellegrino and Katz, 1969b). When the complex was tested by oral means at 400 mg/kg/day X 5 days in mice and at 200 mg/kg/day X 5 days in hamsters, dead worms were not observed in both rodents.
EVALUATION OF SCHISTOSOMICIDES
239
Downloaded by [University of Florida] at 09:39 14 November 2015
Of the organic phosphates explored for antischistosomal properties, metrifonate (trichlorphon) has received the greatest attention. Although the compound produced oogram changes in mice infected with 5. mansoni, it is essentially ineffective against this species in the hamster and in man (Katz et al., 1968; James et al., 1972). Infections with S. haematobium respond to treatment (Cerf et al., 1962; Hanna et al., 1966; Talaat et al., 1966; Forsyth and Rashid, 1967a,b; Davis and Bailey, 1969; Plestina et al., 1972; James et al., 1972). In tests conducted at SWRI, metrifonate was not lethal to S. mansoni in mice and hamsters following oral medication of 200 mg/kg/day X 5 days and by a single intramuscular injection of 100 mg/kg.
DISCUSSION Although in vivo screening tests are time-consuming and restrictive in regard to the number of compounds that can be put through the system during any period, they offer an inestimable advantage in providing a dynamic environment for a synthetic agent in which the activity of a nonmetabolized drug as well as the products of metabolism can be realized. Procedures employed at SWRI utilizing the mouse and hamster and a long-established Puerto Rican strain of 5. mansoni have proven valuable in detecting activity of experimental as well as clinically established antischistosomal drugs. Of 14 antimonial and nonantimonial reference compounds tested in vivo, only two were without activity. Of the latter, metrifonate is ineffective against 5. mansoni infection in humans and nicarbazin has not been shown to be a clinically useful drug. Despite the introduction of new chemotherapeutic agents, the worldwide incidence of schistosomiasis is on the increase. Factors such as population growth, dam-building in conjunction with extensive irrigation projects, and the lack of recognition of the insidious nature of the disease are contributing to its spread. Implementation of control measures including chemotherapy, mollusciciding, and ways to better manage water resources have been and will continue to be difficult to accomplish. Although notable achievements have taken place during the past decade, problems related to laboratory and field investigations require resolution. In encouraging research to characterize the influence of the disease on disability and socioeconomic development, it is significant that the World Health Organization (WHO, 1974) acknowledges that "it is not merely lack of manpower and money that has inhibited progress, but also insufficient confidence among the decision-makers in such control measures as are available and a lack of conviction that the disease deserves a high priority."
240
A. YARINSKY
Downloaded by [University of Florida] at 09:39 14 November 2015
REFERENCES Archer, S. and Yarinsky, A. 1972. Recent developments in the chemotherapy of schistosomiasis. In Progress in drug research, ed. E. Jucker, vol. 16, pp. 11-66. Basel: Birkhäeuser. Bailey, D. M., Archer, S., Wood, D., Rosi, D. and Yarinsky, A. 1970. Synthesis and schistosomicidal activity of 6-chloro-5-{[2-(diethylamino)ethyl]amino]-8-quinolinemethanol. J. Med. Chem. 13:598-601. Baxter, C. A. R. and Richards, H. C. 1971. Schistosomicides. 1. Derivatives of 2-aminomethyl1,2,3,4-tetrahydroquinoline. J. Med. Chem. 14:1033-1042. Berberian, D. A. and Freele, H. 1964. Chemotherapeutic effect of antischistosomal drugs in experimentally induced Schistosoma mansoni infections in Swiss mice and Syrian hamsters. J. Parasitol. 50:435-440. Berberian, D. A., Freele, H., Rosi, D., Dennis, E. W. and Archer, S. 1967a. Schistosomicidal activity of lucanthone hydrochloride, hycanthone and their metabolites in mice and hamsters. J. Parasitol. 53:306-311. Berberian, D. A., Freele, H., Rosi, D., Dennis, E. W. and Archer, S. 1967b. A comparison of oral and parenteral activity of hycanthone and lucanthone in experimental infections with Schistosoma mansoni. Amer. J. Trop. Med. Hyg. 16:487-491. Blair, D. M. 1958. Lucanthone hydrochloride, a review. Bull. World Health Org. 18:989-1010. Bristow, N. W., Lessel, B., Richards, H. C. and Williams, G. A. H. 1967. New aminoquinoline with schistosomicidal activity. Nature 216:282-283. Bueding, E. and Fisher, J. 1969. Biochemical effects of schistosomicides. Ann. N.Y. Acad. Sci. 160:5 36-543. Bueding, E., Náquira, C., Bouwman, S. and Rose, G. 1971. The antischistosomal activity of a nitrovinylfuran derivative (SQ 18,506) in mice and hamsters. J. Pharmacol. Exp. Ther. 178:402-410. Bueding, E., Fisher, J. and Bruce, J. 1973. The antischistosomal activity of a chloroindazole analog of hycanthone in mice infected with Schistosoma mansoni. J. Pharmacol. Exp. Ther. 186:402-407. Campbell, W.C. and Cuckler, A. S. 1967. Inhibition of egg production of Schistosoma mansoni in mice treated with nicarbazin. J. Parasitol. 53:977-980. Cerf, J., Lebrun, A. and Dierichx, J. 1962. A new approach to helminthiasis control: The use of an organophosphorous compound. Amer. J. Trop. Med. Hyg. 11:514-517. Davis, A. and Bailey, D. R. 1969. Metrifonate in urinary schistosomiasis. Bull. World Health Org. 41:209-224. Dennis, E. W. 1970. Chemotherapy of schistosomiasis (past and present) with special reference to hycanthone. A summary. In Proc. II Simposio Sobre Esquistossomose, July 2-6, 1969, pp. 23-48. Publ. by A. Prata and E. Aboim, Salvador, Brazil. Dunsford, H. A., Lucia, H. L., Doughty, B. L. and von Lichtenberg, F. 1974. Artificial granulomas from bentonite and latex carrier particles. Amer. J. Trop. Med. Hyg. 23:203-217. Elslager, E. F. 1970. Schistosomiasis chemotherapy-Progress at a snail's pace. Presented at Twelfth National Medicinal Chemical Symposium, American Chemical Society, University of Washington, Seattle, June 22-25. Erickson, D. G., Bourgeois, J. G., Sadun, E. H. and Bueding, E. 1971. Antischistosomal activity of a nitrovinylfuran derivative (SQ 18,506) in Rhesus monkeys. J. Pharmacol. Exp. Ther. 178:411-416. Forsyth, D. M. and Rashid, C. 1967a. Treatment of urinary schistosomiasis. Practice and theory. Lancet 1:130-133. Forsyth, D. M. and Rashid, C. 1967b. Treatment of urinary schistosomiasis with trichlorophone. Lancet 2:909-912. Foster, R. and Cheetham, B. L. 1973. Studies with the schistosomicide oxamniquine (UK-4271) I. Activity in rodents and in vitro. Trans. Roy. Soc. Trop. Med. Hyg. 67:674-684. Foster, R., Cheetham, B. L., King, D. F. and Mesmer, E. T. 1971. The action of UK 3883, a novel 2-aminomethyltetrahydroquinoline derivative, against mature schistosomes in rodents and primates. Ann. Trop. Med. Parasitol. 65:59-70.
Downloaded by [University of Florida] at 09:39 14 November 2015
EVALUATION OF SCHISTOSOMICIDES
241
Foster, R., Cheetham, B. L. and King, D. F. 1973. Studies with the schistosomicide oxamniquine (UK-4271) II. Activity in primates. Trans. Roy. Soc. Trop. Med. Hyg. 67:685-693. Friedheim, E. A. H., da Silva, J. R. and Martins, A. V. 1954. Treatment of schistosomiasis mansoni with antimony-α,α'-dimercaptopotassium succinate (TWSb). Amer. J. Trop. Med. Hyg. 3:714-727. Gönnert, R. and Vogel, H. 1955. Über die Abhängigkeit des Therapieerfolges von Wirts und Parasitenstamm bei der experimentellen Schistosomiasis. Z. Tropenmed. Parasitol. 6:193-198. Hanna, S., Basmy, K., Selim, O., Shoeb, S. M. and Awny, A. Y. 1966. Effects of administration of an organo-phosphorus compound as an antibilharzial agent, with special reference to plasma cholinesterase. Brit Med. J. 1:1390-1392. Hsü, S. Y. Li, Chu, K. Y. and Hsü, H. F. 1963. Drug susceptibility of geographic strains of Schistosoma japonicum. Z. Tropenmed. Parasitol. 14:37-40. James, C., Webbe, G. and Preston, J. M. 1972. A comparison of the susceptibility to metrifonate of Schistosoma haematobium, S. mattheei and 5. mansoni in hamsters. Ann. Trop. Med. Parasitol. 66:461-414. Katz, N., Pellegrino, J. and Pereira, J. P. 1968. Experimental chemotherapy of schistosomiasis. III–Laboratory and clinical trials with trichlorphone, an organophosphorus compound. Rev. Soc. Brasil. Med. Trop. 2:237-245. Kikuth, W. and Gönnert, R. 1948. Experimental studies on the therapy of schistosomiasis. Ann. Trop. Med. Parasitol. 42:256-267. Lämmler, G. and Petranyi, G. 1971. Chemotherapeutic studies on experimental Schistosoma mansoni infection of Mastomys natalensis. Bull. World Health Org. 44:739-750. Lee, H. G., Cheever, A. W. and Fairweather, W. R. 1971. Influence of parasite strain on chemotherapy of murine infections with Schistosoma mansoni. Bull. World Health Org. 45:147-155. Lennox, R. W. and Bueding, E. 1972. The relative chemotherapeutic efficacy of a nitrovinylfuran (SQ 18,506) against immature and mature stages of Schistosoma mansoni. Amer. J. Trop. Med. Hyg. 21:302-306. Mauss, H. 1948. Miracil, a new chemotherapeutic agent. Berichte 81:19-31. Pellegrino, J. and Katz, N. 1968. Experimental chemotherapy of schistosomiasis mansoni. In Advances in parasitology, ed. B. Dawes, vol. 6, pp. 233-290. New York: Academic Press. Pellegrino, J. and Katz, N. 1969a. Laboratory evaluation of antischistosomal agents. Ann. N.Y. Acad. Sci. 160:429-460. Pellegrino, J. and Katz, N. 1969b. Experimental chemotherapy of schistosomiasis IV–Oogram studies with nicarbazin, an egg-suppressive agent. Rev. Inst. Med. Trop. Sao Paulo 11:215-221. Plestina, R., Davis, A. and Bailey, D. R. 1972. Effect of metrifonate on blood cholinesterases in children during the treatment of schistosomiasis. Bull. World Health Org. 46:747-759. Robinson, C. H., Bueding, E. and Fisher, J. 1970. Relationship between structure, conformation, and antischistosomal activity of nitroheterocyclic compounds. Mol. Pharmacol. 6:604-616. Rosi, D., Peruzzotti, G., Dennis, E. W., Berberian, D. A., Freele, H. and Archer, S. 1965. A new, active metabolite of "Miracil D." Nature 208:1005-1006. Schmidt, H. and Peter, F. M. 1938. Advances in the therapeutics of antimony, pp. 85-118. Leipzig: Georg Thieme. Schubert, M. 1948a. Conditions for drug testing in experimental schistosomiasis mansoni in mice. Amer. J. Trop. Med. 28:121-136. Schubert, M. 1948b. Screening of drugs in experimental schistosomiasis mansoni in mice. Amer. J. Trop. Med. 28:137-156. Standen, O. D. 1963. Chemotherapy of helminthic infections. In Experimental chemotherapy, ed. R. J. Schnitzer and F. Hawking, vol. 1, pp. 701-892. New York: Academic Press. Talaat, S. M., Amin, N. and El Masry, B. 1966. A comparative study of dipterex and tartar emetic in the treatment of urinary schistosomiasis. Trans. Roy. Soc. Trop. Med. Hyg. 60:579-584. Taylor, M. G. and Nelson, G. S. 1971. A comparison of the susceptibility to niridazole of two geographical strains of Schistosoma mansoni in mice with a note on the susceptibility of S. mattheei. Trans. Roy. Soc. Trop. Med. Hyg. 65:169-174.
242
A. YARINSKY
Warren, K. S. 1972. The ¡mmunopathogenesis of schistosomiasis: A multidisciplinary approach. Trans. Roy. Soc. Trop. Med. Hyg. 66:417-434. WHO. 1974. Annual Report of the Director-General to the World Health Assembly and to the United Nations. Official Records of the World Health Organization, no. 213, p. 41. The Work of WHO, 1973. Geneva. Yarinsky, A., Hernandez, P., Ferrari, R. A. and Freele, H. W. 1972. Effects of hycanthone against two strains of Schistasoma japonicum in mice. Jap. J. Parasitol. 21:101-108.
Downloaded by [University of Florida] at 09:39 14 November 2015
Received April 2, 1975 Accepted April 7, 1975
