Randomized, Double-Blind Clinical Trial of Topical Imiquimod 5% with Parenteral Meglumine Antimoniate in the Treatment of Cutaneous Leishmaniasis in Peru
- 1 Department of Microbiology and Immunology, McGill University, Montreal, Canada
- 2 McGill Centre for Tropical Diseases, McGill University Health Center, Montreal, Canada
- 3 Instituto de Medicina Tropical Alexander von Humbolt, Universidad Peruana Cayetano Heredia, Lima, Perú
- Reprints or correspondence: Dr. Brian Ward, McGill Centre for Tropical Diseases, McGill University Health Centre, Montreal, Canada 42810 (brian.ward{at}mcgill.ca).
Abstract
Background Current treatments for cutaneous leishmaniasis are limited by their toxicity, high cost, and discomfort and the emergence of drug resistance. New approaches, including combination therapies, are urgently needed. We performed a double-blind, randomized trial of therapy with parenteral antimony plus topical imiquimod, an innate immune-response modulator, versus therapy with antimony alone, in subjects with cutaneous leishmaniasis for whom an initial course of antimony therapy had failed.
Methods Forty subjects with clinical resistance to antimony were recruited in Lima, Peru, between February 2001 and December 2002. All subjects received meglumine antimoniate (20 mg/kg/day im or iv) and were randomized to receive either topical imiquimod 5% cream (Aldara; 3M Pharmaceuticals) or vehicle control every other day for 20 days. Lesions and adverse events were evaluated during treatment and at 1, 2, 3, 6, and 12 months after the treatment period.
Results The mean number of lesions was 1.2 per person; 71% of the lesions were facial and 76% were ulcerative. There were no major differences between the groups, and all but 2 subjects completed therapy. Mild adverse events were reported by 73% of the subjects, but only erythema occurred more commonly in the imiquimod group (P ⩽ .02). Lesions resolved more rapidly in the imiquimod group: 50% of the imiquimod group achieved cure at 1 month after the treatment period versus 15% of the vehicle cream group (P ⩽ .02); 61% of the imiquimod group at 2 months versus 25% of the vehicle cream group (P ⩽ .03); and 72% of the imiquimod group at 3 months versus 35% of the vehicle cream group (P ⩽ .02). Residual scarring in the imiquimod group was less prominent than in the vehicle cream group.
Leishmaniasis is a serious public health problem throughout the developing world. At least 350 million people are at risk of infection in 88 countries [1, 2]. Disease is caused by the Leishmania family of protozoan parasites, which are transmitted by Phlebotomine sand flies. In cutaneous leishmaniasis, nodules or ulcerative lesions can persist from months to years and lead to disfiguring scarring (reviewed in [3, 4]). Some species also cause mucocutaneous and visceral disease, which can result in significant morbidity and mortality. The spectrum of pathologies in any given geographic area depends on the diversity of Leishmania species in that region [3, 4]. In Peru, anthropomorphic ceramics of the Mochica civilization clearly demonstrate the presence of leishmaniasis during the pre-Columbian period [5]. The major subspecies of Leishmania in Peru are Leishmania (Viannia) braziliensis and Leishmania (Viannia) peruviana, both of which are associated with cutaneous leishmaniasis, or “Uta,” the Quechua term for the disfiguring lesions that occur on the face and other exposed parts of the body [6].
Pentavalent antimony compounds, which were developed over 50 years ago, remain the main therapeutic agents for leishmaniasis [3, 4, 7]. However, treatment with pentavalent antimony is expensive ($300–$400 in Peru), lengthy (daily injections for up to 3 weeks), and is frequently complicated by toxicity (i.e., fatigue, cardiopathy, and pancreatitis). Furthermore, resistance to pentavalent antimony is increasing in some regions of the world where Leishmania is endemic [8], including Peru (A.L.-C., unpublished observations). Amphotericin B and pentamidine are considered second-line treatments because they can cause irreversible toxic effects (e.g., renal failure). More recently, miltefosine has been shown to be effective against Old World and New World leishmaniasis [9, 10]. However this agent is teratogenic and cannot be given to women of child-bearing age [7]. Alternative or more effective combination therapies are therefore urgently required to better manage leishmaniasis.
We have explored the potential for treating leishmaniasis with an immunomodulator, imiquimod. Imiquimod was originally developed to treat viral infections, and imiquimod 5% cream (Aldara; 3M Pharmaceuticals) has received regulatory approval in the United States (US Food and Drug Administration new drug application 020723), Canada (Health Canada, DIN 02239505), and the European Union (European Agency for the Evaluation of Medicinal Products, EC18SET1998) for the treatment of human papillomavirus [11]. We first demonstrated that imiquimod could activate macrophages to kill Leishmania amastigotes in vitro and could reduce disease severity in a mouse model of cutaneous leishmaniasis [12, 13]. In a small, open-label clinical trial in Peru [14], we observed that clinically refractory cutaneous lesions could be cured using combination therapy of standard pentavalent antimony compounds plus topical imiquimod. Subsequently to these initial findings, imiquimod was demonstrated to be an agonist for the toll-like receptor 7 (TLR7) on macrophages and dendritic cells [15]. TLR7 can direct the development of a Th1-type immune response [16]. Such immune responses are required for resolution of leishmaniasis [17].
To confirm that the combination of imiquimod and pentavalent antimony compounds was superior to pentavalent antimony compounds alone in the treatment of cutaneous leishmaniasis, we performed a randomized, double-blind, placebo-controlled trial using pentavalent antimony compounds in combination with topical imiquimod 5% or a vehicle control cream for the treatment of cutaneous leishmaniasis in Peru. We show that, when used in combination with pentavalent antimony compounds, imiquimod significantly shortens the time required for complete cure of cutaneous leishmaniasis and may reduce the severity of scarring.
Patients, Materials, and Methods
Study subjects
Subjects with cutaneous leishmaniasis were identified in rural health centers in regions of the Peruvian Andes (1500–2500 m above sea level) and the Peruvian jungle where cutaneous leishmaniasis is endemic, including the provinces of Churin, Barranca, Yauyos, Satipo, Chachapoyas, Cusco, and Madre de Dios. We considered that patients from the Andean valley regions were infected with L. (V.) peruviana and that patients from the lower Amazonian jungle regions were infected with L. (V.) braziliensis, as has been previously described [18–20]. Subjects were referred for evaluation and treatment at the Instituto de Medicina Tropical Alexander von Humbolt, Universidad Peruana Cayetano Heredia, in Lima. One objective of this study was to determine whether combined imiquimod therapy plus treatment with meglumine antimoniate could increase the cure rate among patients for whom prior therapy with meglumine antimoniate had failed.
The inclusion criteria were as follows: each subject must have had parasitologically confirmed cutaneous leishmaniasis and a history of⩾1 failed course of treatment with meglumine antimoniate. Exclusion criteria were mucosal involvement, pregnancy, breast-feeding,⩾1 lesion with an area >25 cm2, a history of liver or renal disease, allergy to antimony or imiquimod, or the presence of another significant medical condition (e.g., liver failure, renal failure, AIDS, or tuberculosis).
Between February 2001 and August 2002, a total of 220 patients with suspected cutaneous leishmaniasis were evaluated. Cutaneous leishmaniasis was confirmed in 139 patients by aspiration smear, biopsy, culture, and/or PCR. Most of the other patients had lesions caused by fungi or bacteria. Of the 139 patients with confirmed cutaneous leishmaniasis, 22 had failed⩾1 complete course of meglumine antimoniate, and 5 of those met the inclusion criteria and were enrolled. The remaining 117 patients were treated with a standard course of meglumine antimoniate; of these patients, 35 had treatment failure and met the criteria for entry into the study. Subjects also had to agree to remain in Lima for the duration of active treatment (20 days).
All patients had a full medical evaluation on enrollment. If the clinical presentation raised the possibility of a bacterial superinfection (e.g. surrounding cellulitis, actively weeping lesions, tissue maceration, and foul odor), treatment with local or systemic antibiotic therapy and daily cleaning of the lesion was initiated to resolve the infection prior to entry into the study. Lesion numbers were recorded and a plastic sheet was used to trace the perimeter of each lesion to help assess changes in lesion size at follow-up visits. Parasitologic confirmation was accomplished by Giemsa staining of either lesion scrapings or biopsy specimens, by culture in Novy-Macneal Nicolle media, and/or by PCR. The study protocol and consent form were approved by the Research Ethics Committees of McGill University and Universidad Peruana Cayetano Heredia (Lima, Peru) and followed the Helsinki Declaration of 2000. All of the subjects or their guardians provided written, informed consent.
Study design
All 40 subjects enrolled in the study received standard therapy (20 mg/kg per day) with meglumine antimoniate (Glucantime; Aventis Pharma) for 20 days. This drug was administered intramuscularly to 9 children ⩽5 years of age (5 of whom were from the imiquimod group and 4 of whom were from the vehicle control group) or by slow intravenous infusion (over 15 min) in older subjects. Subjects were also randomized to receive either imiquimod 5% cream or placebo vehicle cream (the vehicle used for imiquimod but with inactive ingredients: isostearic acid, cetyl alcohol, steryl alcohol, white petroleum, polisorbate 60, sorbitan mosnosterate, glycerin, xanthan gum, purified water, benzyl alcohol, methylparaben, and propylparaben; 3M Pharmaceuticals). A thin layer of cream was applied to each lesion every other day for 20 days (i.e., 10 applications). Each numbered treatment package had 10 sachets that were identical in appearance and contained either the imiquimod cream or the vehicle cream. Each lesion typically received 125–250 mg of cream every other day. The entire area of each lesion (including a 0.5 cm margin of normal skin) was treated. Cream was applied in the morning by the physician and was rubbed into the lesion(s) until no longer visible. Occlusive dressings were not used, but a sterile plastic “cap” was used for the lesions not on the face, to prevent cream removal by clothing. Subjects were instructed to remove the cap after ∼1 h and to wash the lesions with soap and water after ∼8 h.
After the 20-day period of treatment, follow-up evaluations were scheduled to occur in either in Lima or provincial health centers at months 1, 2, 3, 6, and 12 months after the end of treatment. On each day of active treatment and at follow-up visits, characteristics of adverse effects were solicited from patients by means of a structured questionnaire. A semi-quantitative grading scale for evaluating adverse effects was used: grade 1 was defined as no significant interference with daily activities; grade 2 was defined as mild interference with daily activities, but no treatment required; and grade 3 was defined as severe interference with daily activities and treatment or intervention required. Only adverse effects categorized as grades 1 or 2 were observed; note that many symptoms are typically reported in subjects with leishmaniasis, even when they do not receive treatment). Photographs of each lesion were obtained, and clinical outcome was formally estimated on days 0, 10, and 20 of active treatment and at each follow-up visit. When multiple lesions were present, the clinical outcome was defined by the status of the least well healed lesion. Clinical outcomes were defined as follows: “clinical cure” was defined as complete reepithelialization without signs of inflammation; “clinical improvement” was defined as reduction in lesion size and inflammation but without full reepithelialization; and “treatment failure” was defined as no improvement, with the lesion unchanged or worse compared with its status at the start of treatment. In patients with >1 lesion, the final response score was based on the lesion with the least improvement. At termination of the study (12 months), patients for whom therapy had failed were offered outpatient treatment with intravenous amphotericin B (0.5 mg/kg in 500 mL of 5% dextrose every other day, for a total cumulative dose of 7.5–15 mg/kg or a maximum of 1 g).
Statistical analysis
All statistical analyses were performed using SigmaStat software, version 9.0 (SPSS). Student's 2-tailed t test was used to compare the means of continuous variables (i.e., age, duration of disease, and number of lesions). Median values were compared using the Wilcoxon rank-sum test. Differences in clinical status were compared using the Fisher's exact test. Differences were considered significant when P values were <.05.
Results
A total of 40 patients for whom⩾1 course of standard therapy with pentavalent antinomy compounds had failed were enrolled. The demographic characteristics of the patients are presented in table 1. There were no significant differences between the imiquimod group and vehicle control group with respect to any parameter. Most of the subjects were either domestic workers or farmers, and there was an even distribution of geographical origins between mountain and jungle regions for the 2 treatment groups.
View this table:
Table 1
Demographic characteristics of subjects with leishmaniasis treated with imiquimod 5% cream or placebo vehicle cream.
The principal characteristics of the lesions are outlined in table 2. A total of 75 lesions were evaluated: 35 lesions in the imiquimod group and 40 in the vehicle control group. There were no significant differences between lesion type (e.g., nodular or ulcerative), lesion size (median, 0.5 cm2 for the imiquimod group vs. 0.5 cm2 for the vehicle control group), or lesion duration (median, 8.8 months for the imiquimod group vs. 5.2 months for the vehicle control group). Nine of the subjects had swollen lymph nodes, and 20 of the lesions initially had evidence of bacterial superinfection, but differences between the groups were not significant.
View this table:
Table 2
Characteristics of lesions in subjects with leishmaniasis treated with imiquimod 5% cream or placebo vehicle cream.
One patient who received treatment with imiquimod developed ataxia. Neurotoxic effects of antimony therapy have been reported [21], and the patient's ataxia began before the patient had received the first application of imiquimod. He recovered uneventfully after therapy with antimony was stopped and was treated and cured with amphotericin B. No other serious adverse events were reported. A second patient randomized to the imiquimod group was discovered to have a mucosal lesion at the time active treatment was initiated and was excluded from the study. This patient was also treated successfully with amphotericin B. Local adverse effects are summarized in table 3. Edema, itching, burning, and/or local pain occurred in many subjects but were reported with equal frequency by subjects treated with imiquimod and those treated with the placebo cream. Only mild erythema was more common among subjects in the imiquimod group and was evident during most of the 20-day treatment period (11 of 20 subjects for the imiquimod group vs. 4 of 20 subjects for the vehicle cream group; P = .02). Local symptoms were not sufficiently uncomfortable for any subject to abandon therapy.
View this table:
Table 3
Adverse effects experienced by subjects with leishmaniasis treated with imiquimod 5% cream or placebo vehicle cream.
The evolution of lesions among subjects in the imiquimod and vehicle cream groups is shown in table 4 and figures 1, 2, and 3. There were striking differences in the proportion of subjects who had achieved clinical cure at months 1, 2, and 3 after the end of treatment (P < .02, P < .03, and P < .02, respectively); imiquimod-treated subjects healed more rapidly than did those treated with the placebo vehicle. At follow-up visits at 6 and 12 months after the treatment period, differences between groups were not statistically significant.
Figure 1
The percentage of subjects who were completely cured at various times after the completion of treatment. Cure was defined as complete reepithelialization without signs of inflammation. The first time point (Day 21) represents the end of the 20-day course of treatment, and the others represent months after the completion of treatment.
Figure 2
The evolution of single lesions in representative subjects with leishmaniasis presumed to be due to Leishmania (Viannia) peruviana (mountain isolate) who were treated with meglumine antimoniate and imiquimod 5% cream (upper panels) or with meglumine antimoniate and vehicle cream (lower panels). These individuals originated from mountain locations where L. (V.) peruviana is endemic. Both the speed of lesion healing and scarring are improved in the subjects treated with combined meglumine antimoniate and imiquimod. Day 0 represents baseline.
Figure 3
The evolution of single lesions in representative subjects with leishmaniasis presumed to be due to Leishmania (Viannia) brazilensis (jungle isolate) who were treated with meglumine antimoniate and imiquimod 5% cream (upper panels) or with meglumine antimoniate and vehicle cream (lower panels). These individuals originated from jungle locations where L. (V.) brazilensis is endemic. Both the speed of lesion healing and scarring are improved in the subjects treated with combined meglumine antimoniate and imiquimod. Day 0 represents baseline.
View this table:
Table 4
Response of lesions to treatment among subjects with leishmaniasis treated with imiquimod 5% cream or placebo vehicle cream.
Subjects whose condition had not responded to therapy at month 12 after the end of treatment were offered therapy with amphotericin B, and all lesions eventually resolved. These subjects were not distinguishable from subjects who responded to treatment, according to any of the study parameters (e.g., age; duration, size, or location of the lesion[s]; the presence of single vs. multiple lesions; or geographic origin). We found no significant difference in the cure rate according to the geographic origin of the patients: 77.3% of subjects from mountain regions were cured (figure 2), and 61.1% of subjects from jungle regions were cured (figure 3) (P = .56).
Scar quality was not an original outcome of our study design and there is no well standardized scale for the assessment of scar quality in cases of leishmaniasis. Nevertheless, the differences we observed in wound healing between the subjects treated with imiquimod and those treated with the vehicle cream were striking. Although all personnel remained blinded to group assignment until termination of the study, the speed with which some lesions healed and the superior quality of the residual scars made several of the investigators certain that they could predict who had received imiquimod and who had received vehicle cream prior to unblinding. Representative photos of lesion evolution are presented in figures 2 and 3.
Discussion
The treatment of cutaneous leishmaniasis in 2005 is a challenge for many reasons, including migration of infected individuals, changed expectations of therapy, drug resistance, and the AIDS pandemic. New treatment paradigms that exploit our growing understanding of the immunology and immunopathology of leishmaniasis are needed. This study represents a significant advance for the therapy of parasitic diseases because the rationale for combined therapy was based on advances in basic immunological concepts.
Imiquimod is a nonnucleoside imidazoquinolinamine. Our initial interest in this molecule arose from a report that imiquimod could stimulate the release of cytokines from human peripheral blood mononuclear cells, including macrophages [22]. Subsequently, we demonstrated that imiquimod could activate murine macrophages to synthesize nitric oxide and kill intracellular Leishmania donovani amastigotes both in vitro and in vivo [12]. We further established that an imiquimod analogue, S28463, was able to activate murine macrophages to express a variety of genes associated with inflammation [13]. The recent observation that imiquimod is a TLR7 agonist helps to explain both its antiviral and antileishmanial activities [15]. TLR7-mediated activation of dendritic cells and macrophages results in the release of IL-12, which would be expected to polarize towards a Th1-type cellular immune response [16]. It is now generally accepted that the Th1 response is critical for successful resolution of Leishmania infections [17]. Competent T cell responses may be required for the activity of antimonials [23], and recent evidence has confirmed that imiquimod can indeed enhance Th1 responses [24]. The increased incidence of erythema (inflammation) among subjects that we observed in the imiquimod group is certainly consistent with TLR7-mediated induction of a local, innate immune response.
Our first attempt to use imiquimod to treat cutaneous leishmaniasis in the absence of pentavalent antimony was not successful. Although some subjects initially experienced improvement, most did not complete the trial because of the inflammatory response and the absence of obvious benefit [14]. This is consistent with a recent report that imiquimod alone had only a transient beneficial effect for Syrian patients with cutaneous leishmaniasis [25]. Together, these observations suggest that imiquimod-mediated immunomodulation alone is insufficient to resolve an established Leishmania infection in humans, and that it should only be used in combination with leishmanicidal compounds. Further studies are needed to determine whether imiquimod can synergize with other leishmanicidal agents or if treatment with it would permit lower doses or shorter courses of antimonial drugs to be used. A lower requirement for antimony therapy could significantly reduce both toxicity and cost, enhancing the feasibility of using such combination therapies in developing world populations. Moreover, the adjuvant effects of imiquimod may permit the development of completely topical therapeutic approaches for cutaneous leishmaniasis. For example, it has recently been reported that paromomycin ointment has a cure rate of 66% for cases of cutaneous leishmaniasis [26]. It is certainly plausible that response rates could be increased with combination therapy with paromomycin plus imiquimod. Even if no alternate or antimony-sparing therapy combinations can be identified, the acceleration of lesion resolution with combination antimony-imiquimod therapy that we observed is a significant benefit for those afflicted by cutaneous leishmaniasis.
In regions where leishmaniasis is endemic, the disease primarily occurs in children, who were underrepresented in the current study because of the requirement for treatment in Lima. The social and psychological costs for a child living with an open ulcer on the face for 9–12 months are likely to be quite large. Furthermore, the risk of bacterial superinfections and scarring are probably substantially reduced by an accelerated healing process. The rapidity of lesion resolution may therefore contribute indirectly to the improved scar quality observed. However, imiquimod may also have direct effects on scar formation. It has recently been reported that topical application of imiquimod 5% cream can reduce keloid formation and scarring after surgical excisions [27, 28].
Finally, 25% of subjects in each treatment arm had infections that were ultimately refractory to treatment. According to the study's parameters, these subjects were not distinguishable from those whose condition responded to treatment. In Peru, the rate of successful isolation of L eishmania from patients with prior antimony treatment failure is only 10%–20% (A.L.-C., unpublished data), making identification of parasite species difficult in this study. Our presumptive species-specific diagnoses are therefore based largely on the geographic site of acquisition of infection and the epidemiology of such infections in Peru [18–20]. As a result, we do not know whether different response rates for different Leishmania species contributed to the treatment failures we observed. In future trials, a modified PCR protocol that can distinguish between the different Leishmania species (L. [V] peruviana and L. [V] braziliensis) endemic to Peru will be used [29].
At the start of the 21st century, many events in Peru and the rest of the world have increased the number of people exposed to Leishmania-infected sand-fly bites (e.g., migration, environmental encroachment, adventure tourism, and military conflicts) [1]. The growing recognition of leishmaniasis as a global public health problem has added a sense of urgency to the search for new therapeutic approaches. The current study suggests that combination therapy with imiquimod and antimony has advantages (i.e., accelerated healing and improved scar quality) over monotherapy with antimony in the treatment of refractory cutaneous leishmaniasis.
Acknowledgments
We thank the participating patients, without whom this study could not have been accomplished. We appreciate the skilled help of our assistants (nurses and technicians), and we thank our colleagues in the Peruvian Health Centers. Drs. Marcela Lazo, Nancy Mugruza, and Rocio Espinoza were responsible for patient recruitment, selection, and follow-up.
We wish to express our gratitude to Drs. Miller, Meng, and Tomai of 3M Pharmaceuticals for their valuable input and also to Ms. Mary Jane Maser and Kathleen Laska of 3M Pharmaceuticals for their patience and support.
Financial support. We wish to acknowledge the financial support of the World Health Organization/Tropical Disease Research and 3M Pharmaceuticals. G.M. and B.W. also acknowledge support from Canadian Institutes of Health Research for the research that provided the foundation for this study.
Conflicts of interest. All authors: no conflicts.
Footnotes
-
Presented in part: 52nd Annual Meeting of the American Society of Tropical Medicine and Hygiene, Philadelphia, Pennsylvania, December 2003.
- Received September 10, 2004.
- Accepted December 10, 2004.
- © 2005 by the Infectious Diseases Society of America
References
This Article
- AbstractFree
- » Full Text (HTML)Free
- Full Text (PDF)Free
Classifications
Services
Citing Articles
Google Scholar
PubMed
Related Content
Share
- Email this article
-
CiteULike
Connotea
Delicious
Facebook
Google+
Mendeley
Twitter
Published on behalf of
IDSA Members: For your free access to this journal, log in via the IDSA members area.
The Journal
Impact Factor: 8.186
5-Yr impact factor: 7.898
For The Media
For Authors
- Services for authors
- Self-archiving Policy
- IDSA Conflict of Interest policy
- Instructions for Authors
- ICMJE Form
- Submit Now!
Open access options for authors visit Oxford Open
This journal enables compliance with the NIH Public Access Policy
Corporate Services
Alerting Services
Related IDSA Publications
