Evaluation of Microfilaricidal Effects in the Cornea Trials with Levamisole and Mebendazole

Br J Ophthalmol: first published as 10.1136/bjo.62.7.440 on 1 July 1978. Downloaded from

British Journal of Ophthalmology, 1978, 62, 440-444

Evaluation of microfilaricidal effects in the cornea from topically applied drugs in ocular onchocerciasis: trials with levamisole and mebendazole

BARRIE R. JONES,' JOHN ANDERSON,2 AND HARALD FUGLSANG2 From the 'Department of Clinical Ophthalmology, Institute of Ophthalmology, Moorfields Eye Hospital, London, and 2MRC/WHO Grantholders, 77 Pennard Road, Shepherd's Bush, London

SUMMARY Increasing concentrations of levamisole and of mebendazole were applied to 1 eye in groups of 4 patients with ocular onchocerciasis in northern Cameroon. No effects resulted from up to 3 0% mebendazole suspensions, but 3 0% levamisole solutions rapidly caused entry of microfilariae, straightening out and subsequent opacification of previously curled-up living microfilariae, the rapid formation of typical limbal globular infiltrates, and the subsequent formation of fluffy opacities around the microfilariae. These changes are typical of all other drugs so far studied that have a microfilaricidal action on 0. volvulus-diethylcarbamazine citrate (DEC), suramin, and metrifonate. The efficacy of 3 0 % levamisole approximated to that of 0 03 % DEC. This is in keeping with published observations on the filaricidal activity of these 2 compounds. It is suggested that

this system of drug testing should be considered for systematic use in the search for more effective copyright. and safer drugs for onchocerciasis.

The treatment of ocular onchocerciasis is still un- applied topically to 1 eye, preparations of levamisole satisfactory (Anderson and Fuglsang, 1977), and and mebendazole (Fig. 1) were instilled in a small the World Health Organisation is urging the search number of patients with ocular onchocerciasis. for new drugs and optimum utilisation of existing http://bjo.bmj.com/ drugs alone or in combinations. Materials and methods The clinically observable effects of diethylcarbamazine citrate (DEC) on microfilariae (MFS) of The trial was carried out at Toubourou in north Onchocerca volviulus are well defined, whether the Cameroon. All the patients were infected with the drug is administered systemically (Anderson et al., savanna strain of 0. volvulus. Both levamisole and 1976a) or topically (Ben-Sira et al., 1970; Aviel and mebendazole have low water solubilities, so that a

David, 1972; Anderson and Fuglsang, 1973). The range of concentrations could not be prepared in on September 29, 2021 by guest. Protected last authors have shown that drug effect can be simple aqueous solution. Antifungal imidazole drugs judged by the entry of MFS into the cornea, the of low water solubility have been successfully straightening out of previously curled-up living formulated for use in the eye by dissolving the MFS, the formation of typical limbal globular compound in chloroform, diluting in arachis oil infiltrates, and the subsequent formation of fluffy opacities around the MFS (Anderson and Fuglsang, H 1973; Anderson et al., 1976a). These effects have also been observed during systemic treatment with -H N S II suramin (Anderson et al., 1976b) and with metrifonate (Fuglsang and Anderson, 1977), both of which 0 drugs have a microfilaricidal action. (S)- (-)-HCL To test the value of observing effects from drugs

1 a') LEVAMISOLE Ib) MEBENDAZOLE Address for reprints: Professor Barrie R. Jones, Depart- ment of Clinical Ophthalmology, Moorfields Eye Hospital, Fig. 1 Structuralformula of(a) levamisole and (b) City Road, London EC1V 2PD mebendazole 440 Br J Ophthalmol: first published as 10.1136/bjo.62.7.440 on 1 July 1978. Downloaded from

Evaluation of microfilaricidal effects in the cornea from topically applied drugs in ocular onchocerciasis 441

(BP) to the desired concentration and driving off these patients, but no changes were observed in the the chloroform by heat or vacuum (Jones, 1975). left control eyes. This method was used for preparing levamisole drops 001, 0-03, 01, 03, 1 0, and 30%. EXPERIMENT 2 Mebendazole is not sufficiently soluble in either In order to compare the effects of levamisole with water or arachis oil for simple formulation. It was those of DEC 4 other patients were given 3 % dissolved in dimethyl sulphoxide (DMSO) and levamisole to the right eye and 1 % DEC to the left diluted in arachis oil to give suspensions of eye. The drops were instilled at midday and again mebendazole (001, 003, 0 1, 0 3, 1 0, and 3 0%) in the afternoon on the first day. When the patients each with a final concentration of 1 % DMSO. were examined the following morning drug effect After testing to exclude immediate irritancy and was obvious in both eyes of all 4 patients. However, toxicity in the rabbit eye by the Draize method, 8 the DEC effects were considerably more pronounced patients were given levamisole and 4 mebendazole. than those of levamisole, inasmuch as there were The drops were instilled in the right eyes only, while 10 to 20 times as many new globular infiltrates in the left eyes served as controls. The patients were the left eyes, which also showed far more inflam- examined with a Haag-Streit 900 slit lamp before matory response at the limbus on the second and treatment and as far as possible before each instilla- third days of therapy. tion. Drug effect was judged by the above-mentioned changes, which in the case of DEC therapy are EXPERIMENT 3 usually obvious within 6 hours of the initial applica- Mebendazole was given to 4 other patients at the tion of a 0-01 % solution (Jones et al., 1978). same dilutions and at the same time intervals as Photographs of corneal and limbal lesions were described for levamisole in experiment 1; 1 % taken with a portable photographic system (Jones, aureomycin ointment was given to the left control Sheen, and Minassian, in preparation) using a eyes. There were no visible drug effects even after reversed Canon FL 35 mm f2-8 automatic diaphragm 3 days of treatment by the 3% suspension. Subse- lens (Fig. 2) and a preset Leitz UM 10 /0 22 geo- quent administration of 1 % DEC drops to both copyright. logical microscope lens (Fig. 3), in each case with eyes of 2 of these patients was rapidly followed by an extension tube to give the magnification indicated. corneal changes typical of microfilaricidal effect. Diffuse oblique illumination was from a modified Vivitar 283 flash unit. Positive transparencies Discussion taken on Kodachrome 64 were enlarged on Cibachrome. Levamisole (Fig. Ia) is a wide-spectrum anthelmin-

thic given at doses ranging from 2 5 to 5*0 mg/kg http://bjo.bmj.com/ Results daily for 1 to 3 days (Davis, 1973). It has also been shown to have immunopotentiating activity (Janssen, EXPERIMENT 1 1976) and antifilarial activity against both MFS and At midday 4 patients were given a drop of 0-01 % adult Dirofilaria immitis in dogs (Tulloch and levamisole to the right eye and 1 % aureomycin Anderson, 1971); and also against MFS and at ointment to the left. Six hours later there was no higher dose levels against adult Breinlia sergenti in

detectable effect, and the levamisole solution was the slow loris (Zaman and Natarajan, 1973). on September 29, 2021 by guest. Protected increased to 0 3 % (1 drop). The next morning there Lammler et al. (1971; see also World Health was still no visible effect, so the strength of levamisole Organisation, 1974) found that the minimum was increased to 3 0%, which was repeated at mid- effective dose against the microfilariae of Litomo- day without re-examination. At 1500 h the previously soides carinii infection in the multimammate rat, described drug effects were obvious, and in particular Mastomys natalensis, was 25 mg/kg for levamisole 10 to 30 new discrete globular limbal infiltrates and 60 to 125 mg/kg for DEC, each given 5 times were present in the right eyes of each patient (Fig. 2). daily by mouth. The respective maximum tolerated At the cessation of therapy 3 days later, most of doses were 160 mg/kg x 5 for levamisole and these globular infiltrates had largely diffused away, 2000 mg/kg x 5 for DEC. Levamisole also had but in 1 patient, a boy of 14, 1 still well-defined activity against the adult worms at high dose levels, infiltrate showed a haemorrhage within it (Fig. 3). whereas DEC had none. Maertens and Wery (1975) Photomicrographs with magnification x 64 revealed found that 100 mg mebendazole given orally twice that erythrocytes had broken up the creamy, rather daily for 14 days had no effect on Dipetalonema solid-looking exudate within the globule. Straighten- perstans in a series of approximately 10 patients, ing of corneal MFS and subsequent development of whereas this dosage with 50 mg levamisole daily in fluffy opacities were seen in the right eyes of all addition had a significant microfilaricidal effect. Br J Ophthalmol: first published as 10.1136/bjo.62.7.440 on 1 July 1978. Downloaded from

442 2Barrie R. Jones, John Anderson, and Harald Fuglsang

Fig. 2 Peripheral corneal and limbal globular infiltrates after 7 hours of topical treatment with levamisole (30%) (x 18-8: primary magnification x 4-7, enlarged x 4) copyright. http://bjo.bmj.com/

Fig. 3 Limbal globular infiltrate after 3 days of treatment with topical levamisole (3 %). Haemorrhage into the on September 29, 2021 by guest. Protected globular infiltrate has broken up the rather solid exudate within the globule ( x 64: primary magnification x 16, enlarged x 4) Br J Ophthalmol: first published as 10.1136/bjo.62.7.440 on 1 July 1978. Downloaded from

Evaluation of microfilaricidal effects in the cornea from topically applied drugs in ocular onchocerciasis 443

However, the same combined dosage with both dazole tablets 27 mg/kg daily for 7 days to an levamisole and mebendazole had no demonstrable experimentally infected chimpanzee. Duke suggested effect on microfilaria counts in 9 patients with that the lack of action against 0. volvulus was onchocerciasis; nor did 400 mg mebendazole com- partly due to poor absorption of drug from the gut: bined with 300 mg levamisole daily for 6 weeks have only 5 to 10% is absorbed. However, the absorption any effect on microfilaria counts, nor any side effects from similar oral dosing (400 to 600 mg 3 times in a further 5 patients. Duke (1974a) treated a daily) for 21 to 30 days gave unequivocal ultrasonic chimpanzee infected with 0. volvulus with levamisole echotomographic, immunological, and clinical evi- at a dose of 10 mg/kg intramuscularly daily for dence of destruction of Echinococcus granulosus 15 days, and showed that it was both micro- and cysts in hepatic hydatid disease in each of 4 patients macrofilaricidal at this high dosage, which was too treated. This effect was apparently mediated by toxic to be considered suitable for man. The maxi- damage to the germinal layer of the cysts (Bekhti mum dose tolerated by a single human volunteer was et al., 1977). 2 mg/kg intramuscularly daily for 15 days, but at The present investigation gave no evidence of this level the drug had no action on MFS or adult effect on 0. volvulus microfilariae in the cornea from worms (Duke, 1975). 3% mebendazole applied topically as a suspension The observation that 0 01 and 0 3 % solutions of in arachis oil with I % DMSO. It is possible that levamisole gave no effect when applied directly to poor penetration into the cornea may have contri- the eye and that the 3 % preparation was less buted to or been responsible for the lack of effect. effective than 1 % DEC, together with other Although 40 mg/kg mebendazole has been given observations on the effect of various concentrations daily for up to 24 years in treatment of alveolar of DEC (Jones et al., 1978), suggest that the topical hydatid disease without adverse side effects (D. efficacy of 3 % levamisole approximates to that of Thienpont, personal communication) there is no 0-03 to 0-1 % DEC. This ratio of therapeutic activity clear evidence that this drug affects the adult

is in keeping with the observations of Duke in the 0. volvulus. copyright. chimpanzee. There was strikingly close similarity in Since the microfilaricidal effects of DEC, suramin, all respects between the changes in MFS and the metrifonate, and now levamisole can be easily associated inflammatory reactions induced by demonstrated in the cornea, it is suggested that this corresponding concentrations of either levamisole model should be considered for systematic drug or DEC applied topically. This suggests that the testing in onchocerciasis. When absence of topical levamisole effect is initiated by a direct action on irritancy and toxicity, and the solubility and

the MFS comparable with that ofDEC rather than by absorption allow, the drug should ideally be applied http://bjo.bmj.com/ an immunomodulation of host defence mechanisms. topically to 1 eye, ascending cautiously up a half-log Depending on the pharmacokinetic behaviour of dilution series, leaving the fellow eye to act as a levamisole, it is possible that the adverse sequelae control. In this way the threshold of effective drug to killing MFS in the eye might be easier to concentration can be determined and the dose- regulate than is the case with DEC, but further response relation defined (Jones et al., 1978). It is investigation is necessary to elucidate the possibilities also practicable to observe and measure unwanted of useful therapy of ocular onchocerciasis with side effects or sequelae to killing MFS and to study levamisole. ways of minimising these. on September 29, 2021 by guest. Protected Mebendazole (Fig. lb) is also a wide-spectrum The way in which the above drugs act on the MFS anthelminthic given at a dose of 100 mg twice daily is still not understood, but it is a help to be able to for 3 days (Davis, 1973). Maertens and Wery (1975) observe the sequence of events in the cornea. The observed reduction in filaraemia due to Dipetalonema appearance of the typical globular infiltrates at the perstans in a group of 6 patients given mebendazole limbus which occur in microfilaria-loaded corneas 100 mg twice daily for 30 days in a randomised after the administration of microfilaricidal drugs placebo-controlled coded trial, but no effect was requires further studies with immunopathological seen in another group treated for only 14 days. examination. Detailed examination of patients with There was complete and apparently permanent ocular onchocerciasis sometimes reveals the presence clearing in a further patient given 7 weeks' treatment. of occasional globular infiltrates in scanty numbers On the other hand Aguilar and Manzo (1973) were in untreated persons. It may be that they are related unable to detect any effect on either microfilarial or to the spontaneous death of microfilariae or to the adult forms of 0. volvulus in 19 patients treated immunopathological processes which are involved with 400 mg of mebendazole daily for 10 days. in the killing or absorption of the MFS. The Similarly, Duke (1974b) found neither micro- nor question remains whether or not they are unique to macrofilaricidal effect when he administered meben- onchocerciasis. Br J Ophthalmol: first published as 10.1136/bjo.62.7.440 on 1 July 1978. Downloaded from

444 4Barrie R. Jones, John Anderson, and Harald Fuglsang

Mr R. Watkins, chief pharmacist, Moorfields Eye of ocular onchocerciasis. American Journal of Ophthal- Hospital, prepared the ophthalmic formulations of mology, 70, 741-743. Davis, A. (1973). Drug Treatment in intestinal helminthiases. levamisole and mebendazole donated by Janssen World Health Organisation: Geneva. Pharmaceuticals; Mr M. G. Falcon carried out the Duke, B. 0. L. (1974a). The effect of levamisole on Oncho- preliminary topical toxicity experiments in the cerca volvulus. Transactions ofthe Royal Society of Tropical rabbit eyes. The Department of Audio-Visual Medicine and Hygiene, 68, 71. Duke, B. 0. L. (1974b). Mebendazole-ineffective against Communications kindly printed the illustrations. Onchocerca volvulus. Transactions of the Royal Society of Tropical Medicine and Hygiene, 68, 172. We acknowledge the help of our assistants in the Duke, B. 0. L. (1975). Further trial of levamisole against United Cameroon Republic, Mr P. Houlbai and Onchocerca volvulus. Transactions of the Royal Society of Tropical Medicine and Hygiene, 69, 287-288. Mr Antoine. The Medical Research Council and Fuglsang, H., and Anderson, J. (1977). Effects of a single the World Health Organisation supported this study. dose of metrifonate on the forest strain of Onchocerca volvulus in Cameroon. Tropenmedizin und Parasitologie, 28, 439-446. Janssen, P. A. J. (1976). The levamisole story. Progress References Research, 20, 347-383. Jones, B. R. (1975). Principles in the management of Anderson, J., and Fuglsang, H. (1973). Topical diethyl- oculomycocis. American Journal of Ophthalmology, 79, carbamazine in ocular onchocerciasis. Transactions of the 719-751. Royal Society of Tropical Medicine and Hygiene, 67, Jones, B. R., Anderson, J., and Fuglsang, H. (1978). Effects 710-717. of various concentrations of diethylcarbamazine citrate Anderson, J., and Fuglsang, H. (1977). Ocular onchocerciasis. applied as eye drops in ocular onchocerciasis, and the Tropical Diseases Bulletin, 74, 257-272. possibilities of improved therapy from continuous, non- Anderson, J., Fuglsang, H., and Marshall, T. F. de C. pulsed delivery. British Journal of Ophthalmology, 62, (1976a). Effects of diethylcarbamazine on ocular oncho- 428-439. cerciasis. Tropenmedizin und Parasitologie, 27, 263-278. Lammier, G., Herzog, H., and Schutze, H. R. (1971). Anderson, J., Fuglsang, H., and Marshall, T. F. de C. Chemotherapeutic studies on Litomosoides carinii infection (1976b). Effects of suramin on ocular onchocerciasis. of Mastomys natalensis. Bulletin of the World Health Tropenmedizin und Parasitologie, 27, 279-296. Organisation, 44, 757-763. copyright. Aguilar, F., and Manzo, A. G. (1973). Enfermedad de robles Maertens, K., and Wery, M. (1975). Effect of mebendazole (oncocercosis): ensayos terapeuticos con mebendazole. and levamisole on Onchocerca volvulus and Dipetalonema 9th International Congress on Tropical Medicine and perstans. Transactions of the Royal Society of Tropical Malaria, 3, 120. Medicine and Hygiene, 69, 359-360. Aviel, E., and David, R. (1972). Topical diethylcarbamazine Tulloch, G. S., and Anderson, R. A. (1971). Acceptability in ocular onchocerciasis. IsraeliJournal ofMedical Sciences, of daily L-tetramisole by pound dogs infected with 8, 1166-1168. Dirofilaria immitis. Nature, 232, 644. Bekhti, A., M., World Health Organisation Expert Committee on Filariasis: Schaaps, J-P., Capron, Dessaint, J-P., http://bjo.bmj.com/ Santoro, F., and Capron, A. (1977). Treatment of hepatic Third Report. Technical Report Series No. 352 (1974). hydatid disease with mebendazole: preliminary results in Zaman, V., and Natarajan, P. N. (1973). Antifilarial activity four cases. British Medical Journal, 2, 1047-1051. of levamisole on Breinlia sergenti and its comparison with Ben-Sira, I., Aviel, E., Lazar, M., Lieberman, T. W., and diethylcarbamazine. Journal of Tropical Medicine and Leopold, I. H. (1970). Topical hetrazan in the treatment Hygiene, 76, 126. on September 29, 2021 by guest. Protected