Topical treatment of New and Old World cutaneous leishmaniasis in experimental animals

734 TRANSACTIONS OF THE ROYALSocr~~uOF TROPICAI. MEDICINE+.NDHYGIENE(1987),81, 734-737

Topical

treatment of New and Old World cutaneous leishmaniasis in experimental animals JOSEPH EL-ON’ AND ALBERT D. HAMBURGER' and Immunology, Faculty of Health Sciences, Ben Gut-ion University of the Negev, Beer Sheva, Israel; 2 Teva Pharmaceutical Industries Ltd, Jerusalem, Israel

‘Department of Microbiology

Abstract The effect of topical treatment with 15% paromomycin sulphate and 12% methylbenzethonium chloride in white soft paraffin on cutaneous leishmaniasis in Balb/c mice was studied. The Saudi Arabian strain of Leishmania major, although being the most virulent to the mice, showed the highest susceptibility to this treatment. After 10 days treatment, parasites were totally eliminated from the treated lesion and healing was complete. 3 American strains were also tested: L. mexicana amazonensis; L. braziliensis panamensis and L. mexicana mexicana. The first was the most virulent and the most resistant to local treatment, L. b. panumensis displayed an intermediate response,and L. m. mexicana was highly susceptible. The diseaserelapsed in 50% of the infected treated mice within 135 days (L. major), 120 (L. m. mexicana), 25 (L. b. panamensis), and 14 days (L. m. amuzonensis) after the end of the treatment. Introduction No satisfactory treatment is available for cutaneous or mucocutaneous leishmaniasis. The available drugs of choice, the pentavalent antimonials (SEBAIet al., 1975; SCHEWACH-MILLET et al., 1981)and particularly pentamidine (STECK, 1974) and amphotericin B (CROFTS, 1976), may have adverseside effects and are frequently not effective (KERN, 1981). In our recent work, a topical treatment was developed and found td be highly effective against cutaneous leishmaniasis (CL) causedbv the Israeli L. mujor strain in experir&ntai animals TEL-ON et al., 1984). In addition, preliminary uncontrolled clinical studies suggest that- this ointment may be effective against CL in humans (EL-ON er al., 1985). Similar treatment was also eff&tive againsi recuirent CL caused by L. tropica (EL-ON er al., 1985). In the present study, the effectiveness of such treatment for other Leishmunia parasites was investigated. Materials and Methods Strains of Leishmania and their maintenance The Saudi Arabian strain L. major LRC-L318 and the American strain L. mexicana amuzonensisLRC-L309 were obtained from the World Health Organization Leishmania collection maintained in the Department of Parasitology of the Hebrew University in Jerusalem. Another 2 American strains, L. mexicana mexicana LV4, and L. braziliensis panunwnsisBoynton LV 186, were obtained by courtesy of Dr R. A. Neal of the London School of Hygiene and Tropical Medicine. All strains were stored in liquid nitrogen and maintained by continuous passage in inbred Balblc mice. The effect of drugs on Leishmania development The method of mouse infection, drug administration and drug efficacy scoring were similar to those described by Address for correspondence: Joseph El-On, Ph.D., Dept of Microbiology & Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva, Israel.

EL-ON et al. (1984). Briefly, infected animals were treated topically twice daily, for a period of 10 to 20 d, with ointment comprising 15% paromomycin sulphate and 12% methylbenzethonium chloride in soft white paraffin (Pointment). All ointment preparations (U.K. Patent Application Number 211723A) were manufactured and supplied by Teva Pharmaceutical Industries Ltd, Jerusalem. The effect of the drug on in vitro development of Leishmania was monitored in male, 8 to 12-week-old Balb/c mice following the development of a local dermal lesion caused by the parasite. The mice were inoculated in the base of the tail with either 5X106 (L. majar), 7.5X106 to 1X10’ (L. m. amuzonensis)or 1x IO’ (L. m. mexicana; L. b. panamensis) infective oromastieotes. isolated in culture from recentlv infected &ce: s&ples containing amastigotes from thi lesion were inoculated into diphasic blood agar medium, and the resulting promastigotes used to infect-the mice within approximately 4 weeks of cultivation. The development of the lesion was inspected macroscopically and the presenceof parasites in biopsy material was monitored microscopically in both smears and cultures. Cultures were considered negative only after 20 days observation. Lesion size was measured (in mm) in two diameters (D; d) at right angles to each other using a calliper gauge and the lesion size (S) was determined according to the formula:

S=D

x d 2

ProtozoaI examinations and lesion size measurementswere performed before treatment, at the end of the treatment and every 2 weeks thereafter.

Results

Development of cutaneous leishmaniasisin Balblc mice L. major LRC-L318 was highly virulent to Balb/c

mice. 5 to 6 weeks after infection a lesion with an averagesize (S) of 22.3 + 8.5 mm’ developed. After an ad&tional‘7’weeks, big lesions with an aierage size of 127.5 f 47.0 mm* and manv metastasesin the leas and the tail becameapparent. All the mice died withi;l a period of 2.5 - 3.5 months. L. m. amazonensis caused the development of local lesions 6-7 weeks

J.

EL-ON

AND

after infection. Metastasesdeveloped in all animals 12 to 16 weeks after infection with a lesion size of more than 252.7 + 54.7 mm*. The first mouse died 90 days after infection; most of the animals died between 20 and 28 weeks after parasite inoculation and all had died by day 220. Lesions caused by L. m. mexicana infection developed with a longer incubation period and a lower mortality rate. A very small lesion (2.7 + 0.9 mm*) developed in a period of 7.5 weeks after infection. 50% of the mice died during the 7th month after infection. L. b. panamensisdeveloped very slowly in Balbic mice with an incubation period of 6-8 weeks. During this period no sign of either a nodule or a lesion could be detected, although parasites were present at the site of inoculation. Average lesion size of 54.3 f 28.9 mm* developed 5 months after infection, reaching 83.9 + 32.1 mm* within an additional 2.5 months. The mice died 7-9 months after parasite inoculation. No spontaneous healing or recovery from infection was observed in any of these groups of mice, and the internal lymphatic organs became heavily infected with the parasites whilst the disease progressed.

A.

D.

735

HAMBURGER

the treatment. This percentagewas increasedgradually, giving a cure rate of 50% 120 days after the end of the treatment. L. m. amazonensis. This strain was the least susceptible to local treatment with P-ointment. Mice infected with 7.5 x lo6 promastigotes in the base of the tail were treated twice daily for a period of 10 days, starting 58 days after infection. No parasites were detected in the lesions of the treated mice 2 days after the end of the treatment. However, 3 out of 6 mice relapsed on day 14, and 2 relapsed on day 68. The remaining mouse relapsed 108 days after termination of treatment. Since the standard 10 days’ course did not eradicate the parasites,it was extended to 20 days. In this additional group, 24 mice were inoculated with 1 x 10’ promastigotes. Treatment was started after 44 days when 77% of the mice had nodules only and the remaining 30% had an average lesion size of 23.5 F 9.2 mm*. The results obtained in this experiment were similar to those obtained with the short standard course treatment (Fig. 3). Out of 24 infected

Effect of topical treatment L. major LRC-L318. This strain, though highly

virulent to Balb/c mice, was highly susceptible to topical treatment (Fig. 1;).Mice with an averagelesion size of 70.4 + 25.8 mm , that had developed 65 days after inoculation of 5 X IO6parasites, were completely cleared after 10 days of treatment with P-ointment. In thesemice, no parasiteswere detected 85 days after termination of treatment, and the parasites reappearedin the skin, mainly asmetastasesin the tail and in the feet, but not in the base of the tail. However, the parasitesreappearedalso in the site of inoculation in 38% of the treated mice, 110days after termination of treatment. L. m. mexicana. Mice having lesions with an average size of 20.9 + 9.09 mm*, treated with P-ointment starting 92 days after infection, showed 100% recovery after the end of a 10 day treatment period (Fig. 2). The parasiteswere totally eliminated from the treated lesions which healed within lo-20 days. The disease relapsed in 10% of the mice, 75 days after the end of

’

3 AFTER

4

II

,;

iNFCTlON

Fig. 2. The effect of P-ointment on L. m. mexicana development in Balbic mice infected in the base of the tail with 1 x IO’ promastigates; n and TR as in Fig. 1. *=7

--j

5

6

100

Zoo

0

I bUTHi

I

2 MONTHS

3 AFTER

4

5

6

INFECTION

Fig. 1, The effect of P-ointment on L. major LRGL318 development in Balbic mice infected in the base of the tail with 5 x IO6 promastigotes; n is the number of mice surviving and TR is the duration of the treatment.

2

I MONTHS

3 AFTER

4 INFECTION

Fig. 3. The effect of P-ointment on the development of L. m. antazaemis in Balbk mice infected with 1 x 10’ promastigotes; n and TR as in Fig. 1.

TOPICAL TREATMENT OF CUTANEOUS LEISHMANIASIS IN EXPERIMENTAL ANIMALS

736 14oF

r loo/

Leishmanio braziliensls panomensis .___

- 100

A TREATED 0 UNTREATED

3

4 MONTHS

5 AFTER

6

7

0

INFECTION

Fig. 4. The effectof P-ointmenton L. b. panamtmisdevelopmentin Balbic mice infectedin the baseof the tail with 1 x 10’ promastigates;n and TR as in Fig. 1.

mice, 70% showed the presenceof parasitesat the end of the treatment. An additional 17% relansed on dav 30 and the remaining 13%relapsedwithin-a further 21 days. L. b. panamensis.Infected mice with an averagelesion

size of 51.7 + 25.6 mm*, treated for a period of 10 days with P-ointment starting 144 days after infection, were completely cleared of parasitesat the end of the treatment (Fig. 4). However, within an additional 10 days, the percentageof cured mice was reduced to 88%, and to only 43% on day 30 after termination of treatment. Reappearance of the parasites in the remaining 43% was apparent only after an additional 70 days. Discussion The Saudi Arabian strain of L. major was the most virulent to Balb/c mice. Infection was generally accompanied by development of metastasesin the tails and foot-pads of the mice, not related to the parasite dose but rather a result of high susceptibility of the Balb/c mice to the strain. This hypothesis is further supported by the slow development and reduced mortality rate obtained with the American strains, of which 1.5 to 2 times larger inocula were used. L. m. amazontmsis, although showing the largest lesion size, was lessvirulent than L. major LRC-L318. These results are similar to those described by NEAL & HALE (1984) with L. m. amazonensis LV-81 in Balb/c mice, showing maximum lesion size development 146 days after parasite inoculation. The lesions developed in these mice were granulomatous, accompanied by thickening of the surrounding tissue. This phenomenon might be responsible for limited nenetration of the drug, resulting in the poor success bf the treatment; prordngation of treatment had no sienificant effect on the outcome of infection. These gdings might also result from resistanceof the strain to paromomycin. In contrast, the Saudi Arabian L. major strain, although highly virulent, was extremely susceptible to this treatment. However, the disease relapsed 85 days after ending treatment and metastases usually developed before the reappearance of

parasites at the site of the treated lesion. Such development might be correlated with the high virulence of this strain on the one hand and its high susceptibility to local treatment on the other hand. No significant difference was observed in treatment efficacy with regard to duration of infection. Mice with advanced infection responded to treatment identically to those with early infection. Of the 3 American strains used in this study, L. m. mexicana was the most susceptible to topical treatment. These results are similar to those obtained with the Israeli (EL-ON et al., 1984) and the Saudi strains of L. major. L. b. panamensis showed only moderate susceptibility to topical treatment. In man, infection with L. b. panamensis may develop into the mucocutaneous form, involving mucosal junctions. In these cases, local treatment is not recommended since it is most unlikely to affect organisms already disseminated (JOLLIFFE & BRYCESON, 1983). However, if reliable sensitive diagnostic techniques for disseminated parasites can be developed, such treatment might be considered as a treatment of choice, in addition to parenteral treatment, particularly with drugs that produce low skin concentration when given parenterally. The high percentage of survival of the infected treated mice indicates that the parasite in the dermal lesion might be responsible for the dissemination of the disease by serving as a continuous supplier of invading parasites. This hypothesis is supported by an additional study, not presented in this paper, in which mice infected with L. major LRC-L137, treated locally for a period of 10 days starting 60 days after infection, developed 2.37 times smaller spleen weight 50 d afterwards, compared with the untreated control. Balb/c mice infected with L. major suffer from chronic topical and systemic disease which, if not treated, may causedeath within 4-5 months (DJOKOTAMNOU et al., 1981). After topical treatment of the dermal lesions relapse of the diseaseoccurred in all treated animals. Lesions generally were found at the site of first infection asweil as at the other sites on the skin (EL-ON et al.. 1984). Relanseof treated mucocutaneous and visceral leishman~sis has been renorted in humans (FILSER et al., 1982; ZAAR ec al., 1982). Relanse of the disease in tonicallv treated infected Balbjc mice conceivably occu&ed becauseparasitesin the internal organs were not killed, and reinfected the skin. This hypothesis is supported by a previous study (El-On, unpublished) indicating that infections of L. major LRCL137, relapsing after topical treatment, responded to second topical treatment as well as the original infection. The demonstration bv DJOKO-TAMNOU et al. (1981), of the similarity between the development of L. major in mice and visceral leishmaniasis in man, is relevant; partially cured kala-azar in humans is often followed by so-called post kala-azar dermal leishmaniasis in which, as in Balbic mice infected with L. major, parasites from the internal organs attack the skin. Paromomycin was found also to be highly effective against L. major and L. mexicanaamastigotesin vitro (MATTOCK&PETERS, 1975; EL-ON &GREENBLATT, 1983). L. m. mexicana and L. major in man are parasites of the skin; they are not found in the

J.

EL-ON

AND

A.

peripheral blood and rarely metastasizeto other sites or invade the reticuloendothelial cells of the viscera (MARCIAL-ROJAS,1975). This study has demonstrated that a mixture of paromomycin and methylbenzethonium chloride can kill L. m. mexicanu as well as L. major from Saudi Arabia when applied directly to the infected lesion; a further controlled study should be considered.

References

Crofts, M. A. J. (1976). Use of amphotericin B in mucocutaneous leishmaniasis.Journal of Tropical Medicine and Hygiene, 79, 111-113. Djoko-Tamnou, J., Leclerc, C., Modabber, R. & Chedid, L. (1981). Studies on visceral Leishmania tropica infection in BALBIC mice. Clinical and Experimental Immunology, 46, 491-498. El-On, J. & Greenblatt, C. L. (1983). An in vitro model for testing the effect of anti-leishmanial drugs of possible use in topical treatment. Current Therapeutic Research, Clinic33, 660-669.

El-On, J., Jacobs, G. I’., Witztum, E. & Greenblatt, C. L. (1984). The development of topical treatment for cutaneous leishmaniasis due to Leishmania major in experimental animals. Antimicrobial Agents and Chemotherapy, 26, 745-751.

El-On, J., Weinrauch, L., Livshin, R., Even-Paz, Z. & Jacobs, G. P. (1985). Topical treatment of recurrent cutaneous leishmaniasis with ointment containing paromomycin and methylbenzethonium chloride. British Medical Journal,

737

HAMBURGER

Weinrauch, L. (1986). Topical treatment of cutaneous leishmaniasis. 3ournal of Investigative Dermatologv, 87, 287-288. Filser. T.. Heim. M. E.. Kollmeir. W. & Heene. D. L. (1982): Visceial leishmaniasis iA Europe. Prakitioner, 226, 1559-1564. Jolliffe, D. S. & Bryceson, A. D. M. (1983). Cryosurgery in cutaneous leishmaniasls. British Yournal of Dermatolom, 109, 489-490.

Kern, P. (1981). Leishmaniasis. Antibiotics and Chemotherapy, 30, 203-233.

Acknowledgements

This work was supported by Teva Pharmaceutical Industries, Jerusalem, Israel and by the WHOiUNDP~orld Bank Special Programme for Research and Training in Tropical Diseases.

al and Experimental,

D.

Martial-Rojas, R. A. (1975). Pathology of protozoa1 and helminthic diseases. Huntington, New York: Robert E. Krieger Publishing Co., 850 pp. Mattock, N. M. & Peters, W. (1975). The experimental chemotherapy of leishmaniasis. III. Detection of antileishmanial activity in somenew synthetic compounds in tissue culture model. Annals of Tropical Medicine and Parasitology, 69, 449-462. Neal, R. A. & Hale, C. (1984). A comparative study of susceptibility of inbred and outbred mouse strains compared with hamsters to infection with New World cutaneous leishmaniasis. Parasitology, 87, 7-13. Schewach-Mille!, M., Fisher, B. K. & Semah, D. (1981). Leishmaniasls recidivans treated with sodium stibogluconate. Curis, 28, 67-68. Sebai, Z. A., Morag, T. A. & Suroor, F. J. (1975). Treatment of cutaneous leishmaniases with sodium stibogluconate (Pentostam) in SaudiArabia.Journal of the Egyptian Public Health Association, 50, 59-62.

Steck, E. A. (1974). The leishmaniases. Progressin Drug Research, 18, 289-351.

Zaar, K., Wunderlich,, F. & Belehu, A. (1982). Electron microscopial stuches on cutaneous leishmaniasis in Ethiopia. I. The diffuse form and its treatment with pentamidine. Annals of Tropical Medicine and Parasitology, 76, 595-605.

291, 704-705.

El-On, J., Livshin? R., Even-Paz, Z. Hamburger, D. &

Accepted for publication

14 June

1986

Corrigendum Jha, T. K. (1983). Evaluation of diamidine compounds (pentamidine isethionate) in the treatment of resistant casesof kala-azar occurring in North Bihar, India. Trans. R. Sot. crop. Med. Hyg., 77 (2), 167-170. Page 167, column 1, (Materials for

and Methods),

para.

1, line

5:

“doses of 0.4 mgikg bodyweight” read “doses of 4 mglkg bodyweight”.