Aminosidine and its combination with sodium stibogluconate in the treatment of diffuse cutaneous leishmaniasis caused by Leishmania aethiopica

TRANSACTIONS OF THE

ROYAL SOCIETY OF TROPICAL MEDICINE

AND HYGIENE (1994) 88, 334-339

Aminosidine and its combination with sodium stibogluconate in the treatment of diffuse cutaneous leishmaniasis caused by Leishmania aethiopica Sabawork Teklemariam’a’, Amha Gebre Hiwotl, Dominique Frommell, Tivadar L. Mike’>*, Gunilla Ganlov2 and Anthony Bryceson3 ‘At-maw Hansen Research Institute (AHRI), P.O. Box 1005, Addis Ababa; Ethiopia; 2All Africa Leprosy Rehabilitation and Training Centre (ALERT), P.O. Box 165, Addis Ababa, Ethiopia; 3110spital for Tropical Diseases, London, NW1 OPE, UK

Abstract Treatment of diffuse cutaneous leishmaniasis (DCL) causedby Leishmania aethiopica remains unsatisfactory as the parasite is relatively insensitive to antimonial compounds. Reports of the clinical effectiveness of aminosidine sulphate, especially in combination with sodium stibogluconate, in visceral leishmaniasis and the finding that this antibiotic is potent against L. aethiopicain vitro, prompted us to evaluate its usefulness in DCL. Two patients with long-standing, active DCL were treated for 60 d with aminosidine sulphate, 14 mgikgid parenterally. The skin lesions resolved completely in both patients although they relapsed subsequently. Synergism between aminosidine and stibogluconate was demonstrated in vitro against parasites isolated from the patients. This led us to administer combined therapy, aminosidine sulphate 14 mgikgid and sodium stibogluconate 10 mgikgid, to the 2 patients in relapse and to another, third patient. Treatment was continued for 2 months beyond parasitological cure. Side effects were minimal. Following treatment, a return of specific cell-mediated immunity occurred, as expressedby a moderate infiltration of lymphocytes into the lesions and by lymphocyte proliferation in vitro in the presenceof live Leishmania antigen, with synthesis of interleukin-2 and interferon y with one patient and interleukin 4 with the other. During follow-up periods of 2 to 21 months after treatment, no sign of relapsewas seen. Introduction In the Ethiopian highlands cutaneous leishmaniasis, caused by Leishmania aethiopica, occurs in 2 distinct clinical forms, localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). In most casesthere is a single lesion which heals spontaneously within 6 months to 3 years, leaving a flat atrophic scar. DCL is rare and is characterized by disseminated nodular lesions, rich in organisms, which may involve large areas of the skin. The diseaseruns a chronic and nonhealing course due to a specific defect in the patients’ immune response(BRYCESON,1969,197O). Treatment of DCL remains unsatisfactory becauseL. aethiopica is poorly sensitive to the commonly used drugs, the pentavalent antimonials sodium stibogluconate and meglumine antimonate. The second line drugs, which have been available for the past 25 years (pentamidine and amphotericin B), are difficult to administer and toxic (BRYCESON, 1970, 1987). Thus there is still an urgent need for the introduction of effective and less toxic drugs. The anti-leishmanial activity of aminosidine sulphate, an antibiotic derived from Streptomyces chrestomyceticus, has been known for many years from studies in vitro and in vivo (MATTOCK & PETERS, 1975; EL ON & GREENBLATT, 1983). The chemical structure of aminosidine sulphate is identical with that of paromomycin, which is nroduced from a different strain of Stre~tomyces. Aminosidine sulphate has been found active & vi&o against L. aethiopica, with a median effective dose (ED50) of 4.0 ug/mL, lower than that of sodium stibogluconate, 25.3 ug SbimL (GEBRE-HIWOTet al., 1992). Recently, aminosidine sulphate, administered in combination with sodium stibogluconate in the treatment of visceral leishmaniasis, was reported to be more effective than either drug given alone (CHUNGE et al., 1990;THAKUR et al., 1992). The present study was initially designed to assessthe effectiveness of parenteral aminosidine sulphate in DCL caused by L. aethiopica. It soon became apparent that a 60 d course of 14 mgikgid of aminosidine sulphate as monotherapy was effective in clearing parasites from slit skin smearsand in permitting resolution of skin lesions, but the disease relapsed. In an attempt to prevent relapse, combined chemotherapy with aminosidine sulphate and sodium stibogluconate was tried. Addressfor correspondence:Dr S. Teklemariam,AHRI, Box 1005, Addis Ababa, Ethiopia.

P.O.

Patients and Methods The study was conducted between December 1990 and November 1992 at the All Africa Leprosy Rehabilitation and Training Centre (ALERT)Hospital and the Armauer Hansen Research Institute (AHRI) in Addis Ababa, Ethiopia, and received the approval of the Institutions’ joint ethics committee. Patients

Two male and one female patients with active DCL, but who were otherwise healthy, were selected for the study. All 3 patients had mistakenly been treated elsewhere for leprosy. The first patient had also been treated since 1976 for DCL with several drugs (pentamidine, rifampicin, ketaconozole, itraconozole, intra-lesional interlet&in 2) and with local heat (NEVA et al., 1984), all of which had proved unsuccessful (AKUFFO et al., 1990a, 1990b). However, he had not received any treatment since February 1988. The other 2 patients had not received any anti-leishmanial drug previously. Each patient presented with classical DCL: slowly enlarging,, widespread, non-ulcerative nodules and plaques contammg numerous amastigotes. Standard blood and urine laboratory tests, chest radiographs, and electrocardiograms (ECGs) were unremarkable, and audiograms normal. On histopathological examination the lesions were seen to be covered by thin epidermis with a flattened undersurface. There was a narrow, clear subepidermal zone. The dermis showed confluent sheets of macrophages containing large numbers of amastigotes, with a slight admixture of lymphocytes and plasma cells. Aminosidine sulphate (Carlo Erba Farmitalia, Milan, Italy) was given by intramuscular injection in a dose of 14 mgikgibody weight daily for 60 consecutive days. Patients were assessedand interviewed for side effects. Skin smears and cultures, plasma urea and creatinine levels, urine analysis and audiograms were performed weekly. Aminosidine alone was used to treat patients 1 and 2. Later, it was administered in combination with sodium stibogluconate (Pentostam@,Wellcome Foundation) at a dose of 10 mg Sbikg body weight, once daily by intramuscular injection to treat patient 3, and patients 1 and 2 in relapseafter their treatment with aminosidine alone. Parasite monitoringand isolation

Slit skin sampleswere taken weekly from each of 4 active lesions from each patient. Smears were made and stained with Leishman’s stain. Parasites were counted

335

Fig. 1. Appearance of patients 1 and 2 before (A, C) and after CB,D) treatment rvith aminosidine alone.

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Fig. 2. Parasitological results from slit skin smears taken during treatment with aminosidine alone (A, C) and with aminosidine plus sodium stibogluconate (B, D, E) in patients 1 (A, B), 2 (C, D) and 3 (E). Closed symbols indicate positive cultures, open symbols indicate negative cultures. Mean smear grade (vertical axis)=loglo no. of parasites per 1000 high power microscope fields: mean of smears from 4 sites. Horizontal axis shows weeks of treatment.

337 (1988), using as antigen live promastigotes derived from patients’ parasites. Supernatants from the lymphocyte transformation test were stored at - 70°C. Levels of interleukin (IL) 2, IL-4, interferon (1FN)y and tumor necrosis factor (TNF) a, were measured using solid-phase enzyme immunoassays according to the instructions of the manufacturer (Genzyme Corporation, Boston, Massachusetts,USA).

and graded on a logarithmic scale(WHO, 1990). The absence of parasites in 100 high power microscope fields was considered negative. The weekly slit skin samples were also introduced into NNN medium overlaid with RPMI-1640 (Sigma, St Louis, Missouri, USA) supplemented with 200 units/mL of penicillin and 200 ug/mL of streptomycin. Cultures were kept at 26°C with biweekly examination for the presence of promastigotes under an inverted microscope. Cultures were recorded as negative if no parasite growth was observed within 3 weeks.

Results Tolerance of drug regimens

No clinical. biochemical or renal toxicitv was observed during 60 d’of aminosidine sulphate monotherapy in either patient. During combined therapy, urea and creatinine levels rose to 60 mg/dL and l-7 -mg/dL respectivelv in natient 1 (according to the Merckotestm kit used at AVERS the normal level gf urea is 2&40 mg/dL while that of creatinine is 0.7-1.1 mg/dL). Therefore, the aminosidine dose was reduced to 12 mgikg and the frequency to 3 times per week, while sodium stibogluconate was continued at the same dosage. Subsequently urea and creatinine levels decreasedto 25 mg/dL and 0.8 mg/dL respectively at the end of treatment. Urine analysis revealed red blood cells and granular casts occasionally. Liver enzymes showed inconsistent trends. The ECGs remained normal. No abnormality developed in the other 2 patients. During monotherapy audiograms were obtained weekly and no disturbance was observed. During combined therapy audiograms were continued until the 10th week. A final audiogram at the end of the treatment was examined from all 3 patients and no hearing disturbance was detected. No patient complained of vestibular symptoms.

Drug sensitivity assay in vitro

Promastigotes were cultured in RPMI-1640 medium supplemen;d with 10% foetal calf serum (FCS), 200 units/mL of penicillin and 200 PgimL of streptomycin and kept frozen at -70°C in 7.5% dimethyl sulphoxide until required. Drug sensitivity of the parasites was tested using the human THP-1 monocyte cell line model (GEBRE-HIWOT et al., 1992). In brief, THP-1 cells maintained in RPMI-1640 supplemented with 10% FCS and antibiotics as described above at 37°C were stimulated to differentiate with 1 pM retinoic acid for 3 d, transferred into medium supplemented with 5% heat inactivated normal human serum, and then infected with promastigotes of the isolate(s) of interest. Infected cells were exposed to serial dilutions of aminosidine sulphate in flat bottomed microtitre plates for 6 d. Cells from each well were harvested on to slides by cytocentrifugation. The percentage of infected cells was counted under an oil immersion microscope lens and the ED 50 was determined from dose-responsesigmoidal regression plots. To study the combined effect of aminosidine sulphate and sodium stibogluconate on the isolates, the method described by ELION et al. (1954) was followed. In brief, infected THP- 1 cells were exposed for 6 d to dose ranges with various combinations of a fixed concentration of one drug and variable concentrations of the other. A seriesof dose-response curves was constructed and the ED50 values at different drug ratios were used to calculate the fractional inhibitory concentrations (FIC). The combined effect of the 2 drugs was evaluated from isobolograms constructed from FIC values.

Efficacy of aminosidinesulphate monotherapy

During treatment skin lesions shrunk gradually and became soft. By the end of the treatment period all lesions had resolved comuletelv, asillustrated in Fig. 1. Parasitological data obtained from slit skin sme’;rsduring and after treatment of patients 1 and 2 are shown in Fig. 2. By 9 weeks, slit skin smears and cultures had become negative. Skin biopsies taken 5 weeks after the onset of therapy revealed no amastigote. The inflammatory infiltrate was dominated by macrophages showing a slight foamy change in the cytoplasm. Several lymphocytes and occasional Langhans’ type giant cells were also present. Between the 5th and 10th weeks after the onset of therapy, the number of inflammatory cells gradually decreased.By the 10th week the dermis revealed nodular fibrosis with slight lymphohistiocytic infiltration.

Immunological tests

The leishmanin used for intradermal testing was derived from L. maior (Pasteur Institute. Teheran. Iran. lot 109). Blood mononuclear cell proliferation assays were performed in vitro according to AKUFFO et al.

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Fig. 3. Isobolograms showing the combined action of aminosidine sulphate and sodium stibogluconate on parasites isolated from patients no. 1 (i) and no. 2 [ii). The fractional inhibitory concentration (FIC)of each drug in combination with the other was calculated from dose response curves: see text for details.

338 The first patient showed signs of clinical relapse 3 months after the end of treatment, while the second relapsed after one month. Relapseswere confirmed by skin smear, culture, and histology. Combined therapy

Patients 1 and 2 in relapse, and patient 3, were treated with aminosidine and sodium stibogluconate for 2 months beyond parasitological cure: in-total, 6 months for oatients 1 and 2 and 9 months for the female uatient 3. kesults of slit skin smearsare shown in Fig. 2. Disappearanceof parasites occurred between the 12th and 21st weeks; in patient 1, parasitescould still be isolated in culture at 10 weeks. In natient 3 an incisional bioosv from a poorly resolved lesioi on her left leg, at the 24;h’week of treatment, showed a few amastigotesthough skin smears and cultures were negative. A positive skin test with leishmanin developed in patients 2 and 3 at the end of combined therapy, but not in patient 1. Lymphocyte proliferation responsesin vitro to L. aethiopica antigen were depressed before treatment, with stimulation indices of 0.8 and 1.9 respectively; one month after the completion of the combined treatment, stimulation indices increased by a factor of 10 and 6.8 for patients 1 and 2 respectively. In patient 2, levels of IFNy (4800 pg/mL), TNF-(w (1125 pg/mL), and IL-2 (1000 pg/mL), in culture supernatants were significantly higher than in unstimulated cell cultures; in patient 1, an increaseof only IL-4 (to 3000 pg/mL) was observed. Twenty-one, 2 and 17 months following releasefrom treatment, respectively, patients 1, 2 and 3 remained symptomless and repeated slit skin smearswere negative. Drug sensitivities of isolates of Leishmania in vitro

Parasites isolated from patients 1 and 2 before treatment were found to be sensitive in vitro to aminosidine sulphate with ED50 values of 4.0 and 7.1 PgimL. At the time of relapse after 60 d of aminosidine sulphate monotherapy, freshly isolated parasites showed a marked decreasein their sensitivity in vitro, with ED50 values of 21.9 PgimL and 21.3 PgimL respectively. Parasitesisolated from patient 3 before treatment were sensitive to aminosidine sulnhate with an ED50 of 15.0 ue/mL. The combinaiion of aminosidine sulphati withsodium stibogluconate showed synergistic activity on the 2 isolates with decreased aminosidine sulphate sensitivity (Fig. 3). Sodium stibogluconate alone was poorly active, with ED50 values of 78.2 pg SbimL and 55.0 pg SbimL for the isolates from patients 1 and 2. In the presenceof sodium stibogluconate at 9 pg SbimL, the-activity of aminosidine sulnhate increased bv 1.5 and 2.5 times in patients 1 and 2,’ respectively, and-the ED50 was lowered to the serum level attainable in vivo (DONNO & RIMOLDI, 1974). Similarly, the activity of sodium stibogluconate increased 3.8 and 7.2 times respectively, and the EDSOsfell to attainable serum levels (REES et al., 1980). Discussion

Aminosidine is not absorbed from the gut. Following intramuscular injection, oeak blood levels of 20 mg/L and 40 ma/L are reached one hour after administratioi of 0.5 g and 1 g, respectively, to adults (DONNO & RIMOLDI. 1974). It is excreted unchanged through the kidney (F~LLA~H et al., 1980). Like alraminogl;cosides, it may induce nephrotoxic and ototoxic side-effects

Aminosidine sulphate is clearly effective against L. aethiopica, as shown by good clinical responses and studies in vitro. Synergy between aminosidine and sodium stibogluconate, first noted in vitro, was suggestedin vivo by eventual parasite clearance from the sites of lesions. As the mode of action of neither drug is fully known, it is impossible to elucidate the mechanism of synergy. Nor is it clear whether this synergy was, or may be, expressed in patients. Clinically, synergy might be expressed by more rapid elimination of parasites, or by more prolonged remission. In our patients the rate of elimination of parasites was the same with each regimen. The fact that the relapse parasite isolates from patients 1 and 2 were only one-fifth and one-third as sensitive to aminosidine as the original isolate suggeststhe possibility of an added effect of the antimonial, which would have to be synergistic as the parasite is unresponsive to 10 mg Sbikg daily when given on its own (BRYCESON, 1970). Investigations have also shown that promastigotes of L. major developed resistance to paromomycin after long term exposure in vitro (EL-ON et al., 1991). The prolonged remission in our casesmight be attributed to synergy or to prolonged treatment. CHUNGE et al. (1990) and THAKUR et al. (1992) also concluded that the 2 drugs had an additive effect in patients with visceral leishmaniasis. Further studies on synergy are needed urgently as they could permit lower, less toxic doses of each drug and reduce the risk of drug. resistance. RelapseGf DCL is a well recognized eventuality. Its likelihood is significantly lower in those patients who regained Leishmania-specific cell mediated immunity after treatment (BRYCESON, 1970). After cessation of therapy, skin test conversion occurred in patients 2 and 3, a return of T cell function that was corroborated by lymphocyte proliferation assaysin vitro that resulted in measurable levels of IFNy in the culture supernatants. These results suggest that immunotherapy merits investigation as an adjunct, or a sequel, to chemotherapy of DCL \;;wT

et al., 1987; HARMS et al., 1989; BADARO et al.,

Acknowledgements

Carlo Erba Farmitalia kindly supplied aminosidine sulphate and the Wellcome Foundation freely provided the sodium stibogluconate. We are grateful to Dr P. Olliaro and Dr D. B. A. Hutchinson for their co-operation. The Pharmaceutical Division of the Ministry of Health of Ethiopia approved the trial. We thank Dr Hailu Kefene, former Head of the Armed Forces Hdspital, Addis Ababa, for allowing us to use audiographic facilities, and Dr Alehegne Mekonnen for performing the audiograms. ALERT and AHRI provided all the necessarysupport for patient care and research. Mr Kassa Beimnet took the clinical photographs, Mr Alemayehu Kifle was responsible for the Leishmania cultures and Mrs Mulunesh Negash provided secretarial assistance. We are grateful to them and to the nursing staff of ALERT who conscientiously cared for the patients. AHRI is supported by the Norwegian and Swedish Development Agencies (NORAD and SIDA), and ALERT by the Ministry of Health, Ethiopia, and the International Federation of Anti-Leprosy Associations (ILEP). Dominique Frommel is a staff member of the French National Institute of Health (INSERM), Paris, secondedto AHRI. Last but not least we are grateful to the 3 patients who volunteered to take part in this prolonged and uncomfortable study. References

a seriesof 10 casesin London, high-tone deafnessand re-

Akuffo, H. O., Fehninger, T. E. & Britton, S. (1988). Differential recognition of Leishmaniaaethiopica antigens by lymphocytes from patients with local and diffuse cutaneous leishmaniasis.Joumul of Immunology, 141,2461-2465. Akuffo, H., Dietz, M., Teklemariam, S., Tadesse, T., Amare, G. & Yemane Berhan, T. (1990a). The use of itraconazole in the treatment of leishmaniasis caused by Leishmania aethiopica. Transactions of the Royal Society of Tropical Medicine

versible nephrotoxicity were noted in 2 and one case respectively (SCOTT et al., 1992). It is thus advisable to monitor for toxicity, especially as there is no simple means of measuring serum levels of the drug.

Akuffo, H., Kaplan, G., Kiessling, R., Teklemariam? S., Dietz, M., McElrath, J. & Cohn, Z. A. (1990b). Admimstration of recombinant interleukin-2 reduces the local parasite load of patients with disseminated cutaneous leishmaniasis.

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Received 4 June 1993; revised 20 September 1993; accepted for publication 22 September 1993

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