Max Grogl

Topical Paromomycin with or without Gentamicin for Cutaneous Leishmaniasis

BACKGROUND There is a need for a simple and efficacious treatment for cutaneous leishmaniasis with an acceptable side-effect profile. METHODS We conducted a randomized, vehicle-controlled phase 3 trial of topical treatments containing 15% paromomycin, with and without 0.5% gentamicin, for cutaneous leishmaniasis caused by Leishmania major in Tunisia. We randomly assigned 375 patients with one to five ulcerative lesions from cutaneous leishmaniasis to receive a cream containing 15% paromomycin-0.5% gentamicin (called WR 279,396), 15% paromomycin alone, or vehicle control (with the same base as the other two creams but containing neither paromomycin nor gentamicin). Each lesion was treated once daily for 20 days. The primary end point was the cure of the index lesion. Cure was defined as at least 50% reduction in the size of the index lesion by 42 days, complete reepithelialization by 98 days, and absence of relapse by the end of the trial (168 days). Any withdrawal from the trial was considered a treatment failure. RESULTS The rate of cure of the index lesion was 81% (95% confidence interval [CI], 73 to 87) for paromomycin-gentamicin, 82% (95% CI, 74 to 87) for paromomycin alone, and 58% (95% CI, 50 to 67) for vehicle control (P<0.001 for each treatment group vs. the vehicle-control group). Cure of the index lesion was accompanied by cure of all other lesions except in five patients, one in each of the paromomycin groups and three in the vehicle-control group. Mild-to-moderate application-site reactions were more frequent in the paromomycin groups than in the vehicle-control group. CONCLUSIONS This trial provides evidence of the efficacy of paromomycin-gentamicin and paromomycin alone for ulcerative L. major disease. (Funded by the Department of the Army; ClinicalTrials.gov number, NCT00606580.).

Safety and Efficacy of Intravenous Sodium Stibogluconate in the Treatment of Leishmaniasis: Recent U.S. Military Experience

The efficacy and toxicity of sodium stibogluconate (SSG) at a dosage of 20 mg/(kg.d) for either 20 days (for cutaneous disease) or 28 days (for visceral, mucosal, or viscerotropic disease) in the treatment of leishmaniasis is reported. Ninety-six U.S. Department of Defense health care beneficiaries with parasitologically confirmed leishmaniasis were prospectively followed for 1 year. One patient was infected with human immunodeficiency virus; otherwise, comorbidity was absent. Clinical cure occurred in 91% of 83 cases of cutaneous disease and 93% of 13 cases of visceral/viscerotropic disease. Adverse effects were common and necessitated interruption of treatment in 28% of cases, but they were generally reversible. These included arthralgias and myalgias (58%), pancreatitis (97%), transaminitis (67%), headache (22%), hematologic suppression (44%), and rash (9%). No subsequent mucosal leishmaniasis was identified, and there were no deaths attributable to SSG or leishmaniasis.

Inefficacy of Allopurinol as Monotherapy for Colombian Cutaneous Leishmaniasis: A Randomized, Controlled Trial

New World cutaneous leishmaniasis classically presents as an ulcerating papule or nodule. The ulcer then reepithelializes during a period of a few to many months. Infection frequently spreads to draining lymph nodes; less frequently, it spreads to the mucous membranes of the nose and mouth. The disease is treated to relieve the discomfort of a weeping ulcer and to prevent mucosal disease. Treatment currently requires large daily doses of pentavalent antimony (20 mg/kg of body weight per day) by injection for 20 days [1]. This regimen creates considerable morbidity: Severe arthralgias or myalgias occur in at least 50% of patients, gastrointestinal discomfort develops in 30% of patients, hepatocellular enzyme levels are elevated in 30% of patients, diminution of T-wave height as seen on electrocardiography occurs in at least 10% of patients, and-rarely-thrombocytopenia and neutropenia occur [1]. Antimony treatment also causes chemical pancreatitis in almost all patients; gastrointestinal discomfort may be a symptom of clinical pancreatitis [2]. The treatment of cutaneous leishmaniasis, therefore, leads to morbidity similar to that of the disease itself. Such considerations have led to a 20-year search for an oral, well-tolerated therapeutic agent. The hypoxanthine analogue allopurinol, an inhibitor of uric acid formation in mammalian cells, was shown by LaFon and colleagues [3] to inhibit purine anabolism in Leishmania. In a randomized study done in Colombia, Martinez and Marr [4] found that a daily regimen of allopurinol (20 mg/kg per day) and antimony (20 mg/kg per day) for 15 days was much more effective than therapy with antimony alone (20 of 25 patients receiving allopurinol plus antimony [80%] recovered completely compared with 12 of 33 patients [36%] receiving antimony alone). However, the rate of cure for antimony alone in this study was surprisingly low [5]. Because of the controversy about the Colombian study and the lack of a well-controlled study on the efficacy of allopurinol in treating clinical leishmaniasis, we planned a randomized, blinded, controlled, large-scale clinical trial of the efficacy of allopurinol. To improve our ability to evaluate the efficacy of therapy, we chose the same endemic area (Colombia) and parasite (Leishmania panamensis) on which the major 1992 report focused. Methods Study Design In this randomized, controlled, partially double-blinded phase III study, we compared allopurinol with placebo and Glucantime (Rhone Poulenc, Paris, France). One hundred eighty-seven patients with cutaneous leishmaniasis were randomly assigned to one of three treatment groups. Patients in the first group received allopurinol, 300 mg (three 100-mg tablets) four times daily for 28 days, so that the dosage given was approximately 5 mg/kg four times daily or 20 mg/kg daily for 28 days. Patients in the second group received placebo, three tablets four times daily for 28 days. Patients in the third group received Glucantime, 20 mg of antimony/kg daily (no maximum daily dose) intramuscularly for 20 days. Patients in the allopurinol and placebo groups were assigned to treatment in a double-blinded manner. Study Sample, Inclusion Criteria, and Exclusion Criteria The study sample was composed of patients who were clinically suspected of having cutaneous leishmaniasis and who were from the following regions of Colombia: Arma, Dabeiba, Herveo, La Mesa, Marquetalia, Medellin, San Carlos, San Luis, Taraza, Valdivia, and Victoria. Patients were eligible for the study if they were 6 to 60 years of age, had cutaneous leishmaniasis as confirmed by the presence of parasites, had not received treatment for leishmaniasis with recognized agents during the previous 6 months, did not have lesions close to the eyes or on the mucosa, had body weight that was appropriate for height, and were amenable to prolonged follow-up. Exclusion criteria were the presence of concomitant diseases that required medical intervention, abnormalities in the complete blood count, abnormal glutamate oxaloacetate aminotransferase levels, abnormal creatinine levels, abnormal uric acid levels, and pregnancy. The ethical review committee of the Antioquia University School of Medicine and Hospital San Vicente de Paul, Medellin, Colombia, approved the study, and all patients gave written, informed consent. Parasitologic Diagnosis Leishmaniasis was diagnosed by visualizing amastigotes in lesion material or culturing promastigotes from lesion material. Two scrapings from one lesion on each patient (one scraping taken from the border of the lesion and one taken from the center) were examined with Giemsa stain for amastigotes; needle aspirates of the same lesion were added to culture medium to permit promastigotes to grow. Cultures were maintained at 26 C for 4 weeks before they were reported as negative. In each isolate that was cultured, species were identified by the use of monoclonal antibodies [6]. Conduct of Clinical Trial The first patient was entered into the study in April 1992; the last follow-up examination was completed in November 1995. Before the start of therapy, a complete history, a physical examination, and laboratory testing were done for each patient as described above. A photograph of each patients lesions was also taken. Patients were monitored every 10 days during the treatment phase of the study. Oral therapy was self-administered, and compliance was self-recorded. At each monitoring session, a 10-day supply of pills was dispensed and the patients compliance record was checked and verified by counting the number of pills. Glucantime was administered by medical support personnel. In addition, the occurrence and severity of anticipated adverse effects were recorded at each monitoring session. Lesions were examined before the start of therapy; at the end of therapy; and 1.5, 3, 6, 9, and 12 months after the end of therapy. Attendance at monitoring sessions and follow-up appointments was aided by telephone calls and home visits by study staff. At each evaluation, the induration (measured by using the ballpoint-pen technique) and the area of ulceration were both measured in two directions, which we designated R1 and R2. The areas of the indurated and ulcerated regions were calculated using the formula R1 x R2. Definition of Responses Response of lesions to therapy was determined clinically. Lesions treated with standard Glucantime regimens may increase in size or may not completely heal by the end of therapy; however, they generally heal by 1.5 months after therapy. We therefore used the response pattern of lesions treated with Glucantime as our standard in developing our definitions. For each lesion, we used the following definitions and dispositions: Complete clinical response: Complete reepithelialization of the ulcer and disappearance of all induration. Lesions that showed a complete clinical response were followed for as long as 12 months to verify lack of relapse. Clinical improvement: Fifty percent to 99% reepithelialization of the ulcer area and diminution of induration relative to the previous examination. Lesions that showed clinical improvement at the end of therapy or 1.5 months after the end of therapy were followed until either a complete clinical response or no clinical response was seen at subsequent follow-up sessions. No clinical response: Less than 50% enlargement or diminution of the ulcer area and of induration. If no clinical response was seen at the end of therapy, the lesion was monitored further. If no clinical response was seen at a later follow-up, therapy was considered to have failed. Failure to respond: 1) Greater than 50% enlargement of lesion size at the end of therapy or at subsequent follow-up or 2) no clinical response at an examination done 1.5 months or more after the end of therapy. Relapse: The reappearance of the lesion at the original site after a complete clinical response or the appearance of lesions involving the mucosa. A patient was considered cured if all of the patients lesions had a complete clinical response by the third month of therapy and no relapse had occurred by the 12-month follow-up appointment. Therapy was considered to have failed if any of the patients lesions did not respond to therapy or relapsed. Evaluation of Responses Patients, study investigators, and monitors were blinded to therapy with allopurinol compared with placebo. After the end of follow-up for the last patient, three independent, blinded evaluators determined efficacy and reached a consensus for each patient. The randomization code was then broken. Toxicity was determined by one evaluator before the code was broken. Results Patient Characteristics Five of the original 187 randomly assigned patients were excluded from the study: Two patients violated the study protocol, 1 had an uncertain parasitologic diagnosis, 1 had a clinical course that could not be interpreted, and 1 had co-infection with Sporothrix schenckii. The characteristics of the 182 patients studied are shown in Table 1. The groups were well matched for age, sex, number of lesions per patient, location and characteristics of lesions, and duration of the presence of lesions before treatment. Table 1. Patient Characteristics* Study Site Characteristics and Parasitology Patients from Arma and La Mesa had disease caused by L. braziliensis. Patients from the other nine regions had disease caused by L. panamensis. Ninety-seven percent to 100% of lesions were smear positive and 61% to 75% were culture positive for Leishmania organisms. Because our study was designed to investigate the efficacy of treatment for infection with L. panamensis, we stopped entering patients into the study at Arma and La Mesa as soon as the strain present at those sites was recognized. For the 182 analyzable patients, 153 patients (84%) had documented infection with L. panamensis or were from regions in which L. panamensis was endemic; 29 patients (16%) had infection

Leishmania spp.: Development of pentostam-resistant clones in vitro by discontinuous drug exposure

Antimony unresponsiveness in mucocutaneous and visceral leishmaniasis is a serious clinical problem. Information on the mechanisms and characteristics of drug resistance in parasites that suggest chemotherapeutic strategies to overcome resistance is of practical importance. We developed nine lines of Leishmania resistant to drugs, the major emphasis being on pentavalent antimony (Sb) complexed to carbohydrate in the form of sodium stibogluconate (Pentostam), one of the only two antileishmanial agents with a clearly favorable therapeutic index. Resistance to Pentostam (33- to 212-fold increase) was obtained in promastigotes of Leishmania in vitro by exposure to gradually increasing concentrations of drug over several passages. Resistance to Sb was found to be either stable or unstable. Stable resistance to Sb required (greater than 3) exposures of the initial sensitive clones to Pentostam and tended to stabilize with increased time under pressure. In general, resistance obtained in a clone after only a few (less than or equal to 3) step treatments was low and unstable. Differences in the susceptibility to Pentostam were found between strains isolated from patients with American cutaneous leishmaniasis. In addition, natural isolates of Leishmania from patients represented a heterogeneous population of parasites as demonstrated by a biphasic concentration response to Sb (typical of mixed population dynamics) and by marked differences in susceptibility to Pentostam among clones prepared from single isolates. These results suggest that the emergence of parasite resistance to antimonial treatment is a potential risk of inadequate dose therapy.

WR279,396, a Third Generation Aminoglycoside Ointment for the Treatment of Leishmania major Cutaneous Leishmaniasis: A Phase 2, Randomized, Double Blind, Placebo Controlled Study

Background Cutaneous leishmaniasis (CL) is a disfiguring disease that confronts clinicians with a quandary: leave patients untreated or engage in a complex or toxic treatment. Topical treatment of CL offers a practical and safe option. Accordingly, the treatment of CL with WR279,396, a formulation of paromomycin and gentamicin in a hydrophilic base, was investigated in a phase 2 clinical study in Tunisia and France. Methods A phase 2, randomized, double blind, vehicle-controlled study was conducted to assess the safety and efficacy of topical WR279,396 when applied twice a day for 20 days as treatment for parasitologically confirmed CL. The study protocol established the primary efficacy end point as complete clinical response (CCR) defined as 50% or greater reduction in the ulceration size of an index lesion by day 50 (D50) followed by complete re-epithelialization by D100, and no relapse through D180. Results Ninety-two subjects were randomized. Leishmania major was identified in 66 of 68 isolates typed (97%). In the intent-to-treat population, 47 of 50 WR279,396 treated participants (94%) met the definition of CCR, compared with 30 of 42 vehicle-placebo participants (71%) [p = 0.0045]. Erythema occurred in 30% and 24% of participants receiving WR279,396 and placebo, respectively [p = 0.64]. There was no clinical or laboratory evidence of systemic toxicity. Conclusion Application of WR279,396 for 20 days was found to be safe and effective in treating L. major CL, and offers great potential as a new, simple, easily applicable, and inexpensive topical therapy for this neglected disease. Trial Registration ClinicalTrials.gov NCT00703924

Methodology of Clinical Trials Aimed at Assessing Interventions for Cutaneous Leishmaniasis

The current evidence-base for recommendations on the treatment of cutaneous leishmaniasis (CL) is generally weak. Systematic reviews have pointed to a general lack of standardization of methods for the conduct and analysis of clinical trials of CL, compounded with poor overall quality of several trials. For CL, there is a specific need for methodologies which can be applied generally, while allowing the flexibility needed to cover the diverse forms of the disease. This paper intends to provide clinical investigators with guidance for the design, conduct, analysis and report of clinical trials of treatments for CL, including the definition of measurable, reproducible and clinically-meaningful outcomes. Having unified criteria will help strengthen evidence, optimize investments, and enhance the capacity for high-quality trials. The limited resources available for CL have to be concentrated in clinical studies of excellence that meet international quality standards.

Successful topical treatment of murine cutaneous leishmaniasis with a combination of paromomycin (aminosidine) and gentamicin

Cutaneous leishmaniasis is presently treated with 20 days of parenteral therapy with a frequently toxic drug (antimony). Topical formulations of paromomycin (15%) plus methylbenzethonium chloride (MBCL, 12%) or plus urea (10%) in soft white paraffin have been tested for Old and New World disease in humans. We compared the efficacy of a new topical formulation, WR 279,396 (paromomycin [15%] plus gentamicin [0.5%]) to the clinical formulations in the treatment of cutaneous disease in BALB/c mice. Sixty-day-old lesions were treated twice a day for 10 days, and the response to therapy was determined over a further 70 days. For ulcers due to Leishmania major or to Leishmania mexicana, 100% of lesions in the WR 279,396 group healed by day 20 after therapy and did not relapse by day 70; 83% of lesions healed without relapse in the paromomycin-MBCL group. In the paromomycin-urea group, 100% of L. major lesions healed by day 30 but 30% relapsed. For ulcers due to Leishmania panamensis or Leishmania amazonensis, all lesions treated with WR 279,396 healed and did not relapse; < 50% of lesions treated with paromomycin-MBCL healed by day 30, and all lesions relapsed by day 70. In addition to being active, WR 279,396 was not toxic in this model and appears to have a cosmetic effect (promoting hair growth, healing, and limiting the size of the scar).

Identification of potent chemotypes targeting Leishmania major using a high-throughput, low-stringency, computationally enhanced, small molecule screen.

Patients with clinical manifestations of leishmaniasis, including cutaneous leishmaniasis, have limited treatment options, and existing therapies frequently have significant untoward liabilities. Rapid expansion in the diversity of available cutaneous leishmanicidal chemotypes is the initial step in finding alternative efficacious treatments. To this end, we combined a low-stringency Leishmania major promastigote growth inhibition assay with a structural computational filtering algorithm. After a rigorous assay validation process, we interrogated ∼200,000 unique compounds for L. major promastigote growth inhibition. Using iterative computational filtering of the compounds exhibiting >50% inhibition, we identified 553 structural clusters and 640 compound singletons. Secondary confirmation assays yielded 93 compounds with EC50s ≤ 1 µM, with none of the identified chemotypes being structurally similar to known leishmanicidals and most having favorable in silico predicted bioavailability characteristics. The leishmanicidal activity of a representative subset of 15 chemotypes was confirmed in two independent assay formats, and L. major parasite specificity was demonstrated by assaying against a panel of human cell lines. Thirteen chemotypes inhibited the growth of a L. major axenic amastigote-like population. Murine in vivo efficacy studies using one of the new chemotypes document inhibition of footpad lesion development. These results authenticate that low stringency, large-scale compound screening combined with computational structure filtering can rapidly expand the chemotypes targeting in vitro and in vivo Leishmania growth and viability.

Limited efficacy of injectable aminosidine as single-agent therapy for Colombian cutaneous leishmaniasis

Ninety military patients with cutaneous leishmaniasis in Colombia were randomly allocated to 3 treatment regimens of parenteral aminosidine sulphate: (i) 12 mg aminosidine base/kg/d for 7 d, (ii) 12 mg/kg/d for 14 d, and (iii) 18 mg/kg/d for 14 d. With the 89 evaluable patients, the cure rates 12 months after the end of treatment were 10%, 45%, and 50%, respectively. Fifty-eight of the 66 patients who were not cured had lesions that enlarged or were unchanged by 1.5 months after treatment follow up. The other 8 patients had lesions that relapsed between 3 and 12 months after therapy. Even in group (iii) the cure rate was inferior to that (> 90%) with antimony or pentamidine previously reported in this patient population. This study indicates that parenteral aminosidine alone is less likely to be successful in the treatment of cutaneous lesihmaniasis than visceral leishmaniasis, for which a 74% cure rate has been reported. Further trials might consider the combination of aminosidine with other antileishmanial drugs.

Leishmania mexicana: Chemistry and biochemistry of sodium stibogluconate (Pentostam)

The chemical properties of the primary antileishmanial agent sodium stibogluconate (Pentostam), and the interaction of Pentostam with Leishmania mexicana amastigotes, have been investigated with the aid of [125Sb]Pentostam. The molecular weight by P2 chromatography showed [125Sb]Pentostam to be of multiple species of MW = 100-4000 Da, rather than the one species of 746 Da predicted by the commonly hypothesized structural formula. Nonradioactive Pentostam had a lower osmolarity (789 mOsm for a 100 mg Sb/ml solution) than predicted (1644 mOsm), which indicates that the multiple components of Pentostam (Sb and derivatives of gluconic acid) are more closely complexed with each other than previously thought. When incubated with L. mexicana amastigotes, labeled drug was bound to at least six polypeptides of molecular weights ranging from 14,000 to 68,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Interaction with the polypeptides is presumed to contribute to the antileishmanial action of Pentostam.