Journal of Helminthology (1979) 53, 175-187
SUPPLEMENTARY REVIEW ARTICLE A review of methods for testing compounds for filaricidal activity D. A. DENHAM External Staff Member of the Medical Research Council, Department of Medical Helminthology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC\E 1HT
There is an undoubted need for a new drug for the treatment of the various types of human filariasis. Very few compounds have been used to treat these infections and, of these, only diethylcarbamazine (DEC) has been used on a wide scale against bancroftian and brugian filariasis (Santiago-Stevenson, Oliver-Gonzalez and Hewitt, 1947; Wilson, 1950). The filaricidal activity of DEC was discovered more than 30 years ago (Hewitt, Kushner, Stewart, White, Wallace and Subbarow, 1947). Coincidentally, the onchocercicidal activity of suramin was described in the same year but its activity was detected by testing it in human patients with onchocerciasis without a preliminary trial in an animal model (van Hoof, Henrard, Peel and Wanson, 1947). It is generally accepted (World Health Organization, 1974) that a macrofilaricidal compound is needed to treat Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus infections. DEC is microfilaricidal against W. bancrofti, B. malayi, and O. volvulus (Hawking 1978a). Suramin is mainly macrofilaricidal against O. volvulus but its toxicity is such as to preclude its use in bancroftian or brugian filariases (Hawking, 1978b) although it has been shown to be macrofilaricidal against W. bancrofti (Thooris, 1956). There are two quite different requirements of an anthelmintic. For an individual patient, the need is to alleviate clinical suffering under the care of a skilled physician. However, probably the greater need is for a compound which can be used in mass treatment to prevent transmission of the parasite although a very important side-effect would be a general improvement in the affected population after the use of the compound. For this purpose the drug must be suitable for use either without clinical supervision or with the help of semi-skilled personnel only. The situation, in which so few compounds have been used to treat important human diseases is very unusual. Whilst many of the reasons for this situation are politico-economic others are scientific and technical. The major scientific obstacle in the search for new filaricides has been the lack of suitable models which can be used to screen drugs intended for use in human or animal filariases. Litomosoides carinii has been used most widely as a screen and the major reasons for this are that it was used to demonstrate the filaricidal activity of DEC and that until comparatively recently, no other parasites have been available in the laboratory. The purpose of this paper is to review the ways in which different screens are used and to suggest how screening should be conducted. Characteristics of the ideal screen Before reviewing the different screening techniques it is worth considering the desirable characteristics of a model which could be used for the detection of filaricidal activity in a new compound. Firstly, the parasite used must be susceptible to the drugs already known to be active in human filariasis. A model which, despite any other attractions, does not show this characteristic, should be rejected, although as will be seen, later, the activity of DEC in 175
D. A. DENHAM
every screen is very limited. On the other hand the fact that a single drug, such as DEC, is active in a model, is not a sufficient reason to adopt it for screening purposes. Secondly, the host-parasite system should be easily and reliably maintained in the laboratory. This implies that the vertebrate host should be readily available and easy to handle and that the vector should be easily bred. Some, otherwise excellent, systems have to be rejected on these criteria. It would, for example, be difficult to justify using a parasite that is transmitted by mosquitoes of the genus Mansonia as a primary screen, as the laboratory culture of these mosquitoes is so complicated. Similarly, parasites which mature only in primates must be excluded as primary screens, although they might be used as a final screen before undertaking clinical trials in human patients. Thirdly, the parasite should be as closely related both in its anatomical position in the host and its biochemistry to the causative organisms of human filariases as possible. For a primary screen, this may have less significance, but if there were a choice between two models which, from other viewpoints, were about equal, it could be an important deciding factor. For example, the question might be whether L. carinii in cotton rats is more or less useful than B. malayi in jirds. On the criterion of relationship to a human parasite, the Brugia-yird system would be chosen. Fourthly, it would be advantageous to use a mammalian host whose biochemistry and response to toxicological agents is well understood. Often, a potentially useful drug has already been screened for activity in another pharmacological field using mice or rats. Thus, if one of these hosts could be used, considerable economy could be achieved when determining the levels of drug to use and the likely toxicological response. Fifthly, the screen should use as little of the test compound as possible as test compounds are frequently available in limited quantities. There has been no overall evaluation of the usefulness of the different systems which might be used as primary screens because the information required for such an evaluation is not readily available. Ideally, one would want to know the effect of all drugs active in human and animal filariases in each animal model before deciding which one was best as a primary screen. So far this information, in published form, is only available for L. carinii infections (Lammler and Hertzog, 1974a and b). The issue is further complicated because so few drugs have been used in the treatment of the human filariases, even on an experimental basis, although many more have been used in the treatment of Dirofilaria immitis infection in dogs. Since the discovery of DEC and, more especially, during the last twenty years, several filarial parasites have been introduced into the laboratory. I shall now consider the advantages and disadvantages of some of these models and review the progress which has been made with each system. Infections with L. carinii L. carinii infects a number of rodent species, including the natural host, Sigmodon hispidus (the Cotton rat), Meriones unguiculatus (the Mongolian jird), Rattus norvegicus (the laboratory rat), Praomys natalensis (the multimammate rat mouse) and Cricetulus griceus (the Chinese hamster). This parasite has been used extensively as a primary screen for filaricidal drugs. It is worth considering the actions of DEC and suramin on this parasite as the design and interpretation of any screen using this system is affected by the results obtained with these drugs. In the original work, on DEC, Hewitt et al. (1947) found an immediate reduction in the number of microfilariae in the blood after treatment. This has since been confirmed by several other workers and Hawking, Sewell and Thurston (1950) found that microfilarial levels fell within minutes of an intravenous injection of DEC into infected cotton rats. 176
Review of filaricide testing methods
However, there has been some controversy concerning the effect of DEC on adult L. carinii. Figure 1 shows the microfilarial counts in three groups of cotton rats treated by Hewitt et al. (1947) with different dose schedules. Even after treatment at 25 mg/kg three times a day for 30 days, microfilariae reappeared in the blood soon after treatment ceased. This is clear evidence that, at least, some adult worms were still alive after such long term treatment. However, Hewitt et al. (1947) claimed to have found more dead adult worms in treated than untreated cotton rats. They considered that the lethal effects of DEC on adult worms involved a number of variables; the most significant being the amount of drug given, the frequency of dosage and the time between treatment and autopsy. The best results were obtained by giving the compound three times a day for 30 days and killing the cotton rats 42 or 52 days after treatment. Unfortunately no counts of adult worms are given in this paper. Hawking, Sewell and Thurston (1950) said "the lethal action of hetrazan on adult female worms seems to be slight. The male worms are equally insusceptible". In fact, they found that after 14 days treatment with 250 mg/kg twice a day (i.e. 7 times the level used by Hewitt et al. (1947) 9.28% of female worms and 2.35% of male worms were dead compared with 0.76 % and 0.34 % for untreated, control rats. They killed their animals 8 or 28 days after treatment, which is too early according to Hewitt et al. (1947), but, even so, it is clear that DEC is not strongly macrofilaricidal in this system. These results raise an interesting question: if DEC were presented to a team currently running a screen for filaricidal activity, would it select this drug for further investigation? As will be seen this question may be asked profitably after consideration of each potential screen. If one accepts that the greatest need is for a drug which kills adult filarial worms in man, drugs with an activity similar to that of DEC against L. carinii are a disappointment. Examination of the literature (Lammler, Saupe, Hertzog and Schiitze, 1971a; Lammler, Hertzog and Schutze, 1971b), and various personal communications, suggests that many compounds will be found to have microfilaricidal activity against L. carinii. The results obtained with DEC suggest that any drug which shows such activity against L. carinii must be further investigated. The results obtained with suramin against L. carinii also have important implications. Suramin is an effective macrofilaricide against O. volvulus (van Hoof et al., 1947; Burch and Ashburn, 1951; Duke, 1968) but the original reports of its activity in cotton rats infected with L. carinii suggested that it was not effective against this species (Hawking, 1962). Lammler, Hertzog and Schutze (1971c) have shown that suramin is definitely macrofilaricidal against L. carinii in multimammate rats but that the worms do not die until some weeks after treatment. Stohler (1975) confirmed these observations and has shown that the drug had similar effects on L. carinii in cotton rats, jirds and Chinese hamsters. The important conclusion from these observations is that, unless a period of at least 35 days is left between treatment and autopsy, a macrofilaricidal effect of the type exhibited by suramin would not be detected. This would make the screen much more expensive to run and with any given amount of animal accommodation would reduce the number of drugs which could be tested. Thompson, Boche and Blair (1968) observed that jirds and cotton rats infected with L. carinii responded in different ways when treated with amodiaquine. Both hosts were treated with 100 mg/kg/day for 5 days and killed 10 days later. Amodiaquine killed 85.4% of adult worms in the thoracic cavity in jirds but only 45.3 % in the thoracic cavity of cotton rats; it was not microfilaricidal in either host. In view of this result Stohler (1975) compared the activity of 17 filaricides in cotton rats, multimammate rats, jirds and Chinese hamsters infected with L. carinii. The compounds fall into different groups according to their activity in different hosts. DEC and metrifonate were uniformly microfilaricidal in all four hosts but had no effect on macrofilariae. Trivalent antimony sodium gluconate and 177
D. A. DENHAM
furazolidone were both micro- and macro-filaricidal in all four hosts. Nitrofurantoin was similar but had only marginal microfilaricidal activity in Chinese hamsters. The remaining 11 compounds gave different results in different hosts. Fewest compounds were active in the Chinese hamster; all 17 compounds were active in cotton rats but 7 showed no effect on the parasite in Chinese hamsters. These results indicate that the phenomenon initially reported by Thompson et al. (1968) is not unique. Stohler's results also suggest that most compounds were active in the cotton rat but this conclusion must be treated with some suspicion because the drugs were selected for inclusion in the experiment because they had shown some activity in a L. carinii primary screen. If jirds or Chinese hamsters had been used as the host in the initial screen the results might have been different. The biggest technical disadvantage of screens using L. carinii is that hosts are normally infected by allowing infected mites to feed on the test animal with the result that infection levels are unpredictable. On the other hand, the maintenance of a mite colony and infection of animals is easy and requires little work time once the necessary routine has been established. McCall (1976 and personal communication) has reported that if animals are heavily infected by feeding infected mites on them larvae can be recovered from their skins 1-2 days later and are reliably infective if inoculated into other rodents but, probably, even more useful is to collect larvae from the pleural cavity and inoculate these into new animals. These systems could well revolutionize quantitative studies with this parasite.
m 100 1 2 3
LLJ
J
90
h-
--
1,2 and 3. 25 mg TID/Kg 14 days DEC at 25 mg BID/Kg for 28 days 25 mg TID/Kg 30 days
UJ
I" £ 70 of
60
0
50 -
1 40
SUPPLEMENTARY REVIEW ARTICLE A review of methods for testing compounds for filaricidal activity D. A. DENHAM External Staff Member of the Medical Research Council, Department of Medical Helminthology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC\E 1HT
There is an undoubted need for a new drug for the treatment of the various types of human filariasis. Very few compounds have been used to treat these infections and, of these, only diethylcarbamazine (DEC) has been used on a wide scale against bancroftian and brugian filariasis (Santiago-Stevenson, Oliver-Gonzalez and Hewitt, 1947; Wilson, 1950). The filaricidal activity of DEC was discovered more than 30 years ago (Hewitt, Kushner, Stewart, White, Wallace and Subbarow, 1947). Coincidentally, the onchocercicidal activity of suramin was described in the same year but its activity was detected by testing it in human patients with onchocerciasis without a preliminary trial in an animal model (van Hoof, Henrard, Peel and Wanson, 1947). It is generally accepted (World Health Organization, 1974) that a macrofilaricidal compound is needed to treat Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus infections. DEC is microfilaricidal against W. bancrofti, B. malayi, and O. volvulus (Hawking 1978a). Suramin is mainly macrofilaricidal against O. volvulus but its toxicity is such as to preclude its use in bancroftian or brugian filariases (Hawking, 1978b) although it has been shown to be macrofilaricidal against W. bancrofti (Thooris, 1956). There are two quite different requirements of an anthelmintic. For an individual patient, the need is to alleviate clinical suffering under the care of a skilled physician. However, probably the greater need is for a compound which can be used in mass treatment to prevent transmission of the parasite although a very important side-effect would be a general improvement in the affected population after the use of the compound. For this purpose the drug must be suitable for use either without clinical supervision or with the help of semi-skilled personnel only. The situation, in which so few compounds have been used to treat important human diseases is very unusual. Whilst many of the reasons for this situation are politico-economic others are scientific and technical. The major scientific obstacle in the search for new filaricides has been the lack of suitable models which can be used to screen drugs intended for use in human or animal filariases. Litomosoides carinii has been used most widely as a screen and the major reasons for this are that it was used to demonstrate the filaricidal activity of DEC and that until comparatively recently, no other parasites have been available in the laboratory. The purpose of this paper is to review the ways in which different screens are used and to suggest how screening should be conducted. Characteristics of the ideal screen Before reviewing the different screening techniques it is worth considering the desirable characteristics of a model which could be used for the detection of filaricidal activity in a new compound. Firstly, the parasite used must be susceptible to the drugs already known to be active in human filariasis. A model which, despite any other attractions, does not show this characteristic, should be rejected, although as will be seen, later, the activity of DEC in 175
D. A. DENHAM
every screen is very limited. On the other hand the fact that a single drug, such as DEC, is active in a model, is not a sufficient reason to adopt it for screening purposes. Secondly, the host-parasite system should be easily and reliably maintained in the laboratory. This implies that the vertebrate host should be readily available and easy to handle and that the vector should be easily bred. Some, otherwise excellent, systems have to be rejected on these criteria. It would, for example, be difficult to justify using a parasite that is transmitted by mosquitoes of the genus Mansonia as a primary screen, as the laboratory culture of these mosquitoes is so complicated. Similarly, parasites which mature only in primates must be excluded as primary screens, although they might be used as a final screen before undertaking clinical trials in human patients. Thirdly, the parasite should be as closely related both in its anatomical position in the host and its biochemistry to the causative organisms of human filariases as possible. For a primary screen, this may have less significance, but if there were a choice between two models which, from other viewpoints, were about equal, it could be an important deciding factor. For example, the question might be whether L. carinii in cotton rats is more or less useful than B. malayi in jirds. On the criterion of relationship to a human parasite, the Brugia-yird system would be chosen. Fourthly, it would be advantageous to use a mammalian host whose biochemistry and response to toxicological agents is well understood. Often, a potentially useful drug has already been screened for activity in another pharmacological field using mice or rats. Thus, if one of these hosts could be used, considerable economy could be achieved when determining the levels of drug to use and the likely toxicological response. Fifthly, the screen should use as little of the test compound as possible as test compounds are frequently available in limited quantities. There has been no overall evaluation of the usefulness of the different systems which might be used as primary screens because the information required for such an evaluation is not readily available. Ideally, one would want to know the effect of all drugs active in human and animal filariases in each animal model before deciding which one was best as a primary screen. So far this information, in published form, is only available for L. carinii infections (Lammler and Hertzog, 1974a and b). The issue is further complicated because so few drugs have been used in the treatment of the human filariases, even on an experimental basis, although many more have been used in the treatment of Dirofilaria immitis infection in dogs. Since the discovery of DEC and, more especially, during the last twenty years, several filarial parasites have been introduced into the laboratory. I shall now consider the advantages and disadvantages of some of these models and review the progress which has been made with each system. Infections with L. carinii L. carinii infects a number of rodent species, including the natural host, Sigmodon hispidus (the Cotton rat), Meriones unguiculatus (the Mongolian jird), Rattus norvegicus (the laboratory rat), Praomys natalensis (the multimammate rat mouse) and Cricetulus griceus (the Chinese hamster). This parasite has been used extensively as a primary screen for filaricidal drugs. It is worth considering the actions of DEC and suramin on this parasite as the design and interpretation of any screen using this system is affected by the results obtained with these drugs. In the original work, on DEC, Hewitt et al. (1947) found an immediate reduction in the number of microfilariae in the blood after treatment. This has since been confirmed by several other workers and Hawking, Sewell and Thurston (1950) found that microfilarial levels fell within minutes of an intravenous injection of DEC into infected cotton rats. 176
Review of filaricide testing methods
However, there has been some controversy concerning the effect of DEC on adult L. carinii. Figure 1 shows the microfilarial counts in three groups of cotton rats treated by Hewitt et al. (1947) with different dose schedules. Even after treatment at 25 mg/kg three times a day for 30 days, microfilariae reappeared in the blood soon after treatment ceased. This is clear evidence that, at least, some adult worms were still alive after such long term treatment. However, Hewitt et al. (1947) claimed to have found more dead adult worms in treated than untreated cotton rats. They considered that the lethal effects of DEC on adult worms involved a number of variables; the most significant being the amount of drug given, the frequency of dosage and the time between treatment and autopsy. The best results were obtained by giving the compound three times a day for 30 days and killing the cotton rats 42 or 52 days after treatment. Unfortunately no counts of adult worms are given in this paper. Hawking, Sewell and Thurston (1950) said "the lethal action of hetrazan on adult female worms seems to be slight. The male worms are equally insusceptible". In fact, they found that after 14 days treatment with 250 mg/kg twice a day (i.e. 7 times the level used by Hewitt et al. (1947) 9.28% of female worms and 2.35% of male worms were dead compared with 0.76 % and 0.34 % for untreated, control rats. They killed their animals 8 or 28 days after treatment, which is too early according to Hewitt et al. (1947), but, even so, it is clear that DEC is not strongly macrofilaricidal in this system. These results raise an interesting question: if DEC were presented to a team currently running a screen for filaricidal activity, would it select this drug for further investigation? As will be seen this question may be asked profitably after consideration of each potential screen. If one accepts that the greatest need is for a drug which kills adult filarial worms in man, drugs with an activity similar to that of DEC against L. carinii are a disappointment. Examination of the literature (Lammler, Saupe, Hertzog and Schiitze, 1971a; Lammler, Hertzog and Schutze, 1971b), and various personal communications, suggests that many compounds will be found to have microfilaricidal activity against L. carinii. The results obtained with DEC suggest that any drug which shows such activity against L. carinii must be further investigated. The results obtained with suramin against L. carinii also have important implications. Suramin is an effective macrofilaricide against O. volvulus (van Hoof et al., 1947; Burch and Ashburn, 1951; Duke, 1968) but the original reports of its activity in cotton rats infected with L. carinii suggested that it was not effective against this species (Hawking, 1962). Lammler, Hertzog and Schutze (1971c) have shown that suramin is definitely macrofilaricidal against L. carinii in multimammate rats but that the worms do not die until some weeks after treatment. Stohler (1975) confirmed these observations and has shown that the drug had similar effects on L. carinii in cotton rats, jirds and Chinese hamsters. The important conclusion from these observations is that, unless a period of at least 35 days is left between treatment and autopsy, a macrofilaricidal effect of the type exhibited by suramin would not be detected. This would make the screen much more expensive to run and with any given amount of animal accommodation would reduce the number of drugs which could be tested. Thompson, Boche and Blair (1968) observed that jirds and cotton rats infected with L. carinii responded in different ways when treated with amodiaquine. Both hosts were treated with 100 mg/kg/day for 5 days and killed 10 days later. Amodiaquine killed 85.4% of adult worms in the thoracic cavity in jirds but only 45.3 % in the thoracic cavity of cotton rats; it was not microfilaricidal in either host. In view of this result Stohler (1975) compared the activity of 17 filaricides in cotton rats, multimammate rats, jirds and Chinese hamsters infected with L. carinii. The compounds fall into different groups according to their activity in different hosts. DEC and metrifonate were uniformly microfilaricidal in all four hosts but had no effect on macrofilariae. Trivalent antimony sodium gluconate and 177
D. A. DENHAM
furazolidone were both micro- and macro-filaricidal in all four hosts. Nitrofurantoin was similar but had only marginal microfilaricidal activity in Chinese hamsters. The remaining 11 compounds gave different results in different hosts. Fewest compounds were active in the Chinese hamster; all 17 compounds were active in cotton rats but 7 showed no effect on the parasite in Chinese hamsters. These results indicate that the phenomenon initially reported by Thompson et al. (1968) is not unique. Stohler's results also suggest that most compounds were active in the cotton rat but this conclusion must be treated with some suspicion because the drugs were selected for inclusion in the experiment because they had shown some activity in a L. carinii primary screen. If jirds or Chinese hamsters had been used as the host in the initial screen the results might have been different. The biggest technical disadvantage of screens using L. carinii is that hosts are normally infected by allowing infected mites to feed on the test animal with the result that infection levels are unpredictable. On the other hand, the maintenance of a mite colony and infection of animals is easy and requires little work time once the necessary routine has been established. McCall (1976 and personal communication) has reported that if animals are heavily infected by feeding infected mites on them larvae can be recovered from their skins 1-2 days later and are reliably infective if inoculated into other rodents but, probably, even more useful is to collect larvae from the pleural cavity and inoculate these into new animals. These systems could well revolutionize quantitative studies with this parasite.
m 100 1 2 3
LLJ
J
90
h-
--
1,2 and 3. 25 mg TID/Kg 14 days DEC at 25 mg BID/Kg for 28 days 25 mg TID/Kg 30 days
UJ
I" £ 70 of
60
0
50 -
1 40
