Combination Therapy for the Treatment of Cutaneous Leishmaniasis Compared with Standard Drugs, In Vitro or In Vivo: A Systematic Review

Abstract

The obligate intracellular parasite, Leishmania, causes leishmaniasis as an infectious disease. The parasite is transmitted through female sandfly bites and infects the host mononuclear phagocytes, leading to severe skin lesions and fatal systemic infection. The quick diagnosis and effective treatment can prevent the development scars and chronic or uncontrollable disease forms. The current chemical and physical treatments have limitations; hence, the use of plant compounds and their derivatives is a new proposed treatment method. Therefore, in this review, the recent advances in combination therapy of cutaneous leishmaniasis were investigated. The information was obtained from all articles published in PubMed, SciELO, ScienceDirect, Scopus, Google Scholar, and Web of Science databases (1998–2022). Search terms used were “Combination Therapy” AND “Cutaneous Leishmaniasis.” The data showed increased synergistic efficacy or shortening the course of treatment and few adverse effects in the combined treatment of (CL), in vitro and in vivo. Expanding the combination therapy in clinical trials could open a new insight in treatment strategies of CL.

INTRODUCTION

Leishmaniasis is one of the common diseases between humans and animals that mankind has faced since 650 BC and even more.[1] This disease is caused by different protozoan species of Leishmania genus and causes different clinical manifestations in humans and some animals. The most common forms of the disease are cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL), which are transmitted by female Phlebotomus mosquitoes.[2] Currently, more than 12 million people in the world suffer from this disease, and the annual prevalence of the disease in the world is estimated at 1–1.5 million people.[3] Based on incidence rate and disease causing capacity, leishmaniasis is considered as one of the six important infectious diseases by the World Health Organization (WHO).[4] Although CL that causes by Leishmania major or Leishmania tropica has less mortality compared to other diseases, it causes many problems due to the long duration of the wound, the creation of unpleasant scars on the face, the possibility of secondary infections, the heavy treatment burden for the society, and the secondary drug complications. Studies showed that quick diagnosis and effective treatment not only prevent the development of unpleasant scars but also reduce the chronic and uncontrollable forms.[1] Clinical diagnosis is based on histopathology of lesions, isolation of the parasite from the lesion, parasite culture, PCR, and microscopic methods.[5] CL treatments include chemical (local and systemic) and physical treatments (cryotherapy). The current treatment of CL is pentavalent antimoan chemical compounds (such as glucantim and pentostam), with limitations of high costs, toxicity, and low effectiveness. There are many reports of parasite resistance to the mentioned drugs, as well as an increase in disease relapse in different parts of the world, that reveal the need to new drugs.[6] One of the solutions proposed by WHO for eliminating pathogenic microorganisms is the use of plant compounds and their derivatives. As medicinal plants with many secondary metabolites have the primary active ingredients with new antibacterial and antifungal effects,[7,8] the discovery of antimicrobial properties of natural compounds has been considered. According to the conducted studies, some medicinal plants due to their antimicrobial properties have a significant effect on the speed of the wound healing.[9]

Another proposed strategy is the use of two or more medicinal compounds with synergistic properties to increase effectiveness and reduce side effects so that the synergistic effect of two compounds can reduce the possible side effects of common drugs and increase the efficiency of the treatment. Hence, in recent years, the different effects of combined treatments using the examining their interaction effects in different concentrations have been reported in different diseases.[10,11,12] In addition, combination therapy can be effective in the treatment of patients with coinfections. The study of examined medicinal combination therapies in different regions of the world can detect the basis for appropriate strategies of better treatment and less economic and psychological burden in societies. Therefore, considering the importance of combined treatments in CL, the present study reviewed the studies relative to the recent advances in combined treatments of CL.

MATERIALS AND METHODS

Information sources and search strategies

This systematic review was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.[13] The process of identifying articles was developed using the following electronic databases in which the effect of combination therapy was assayed on treatment of CL: PubMed, SciELO, ScienceDirect, Scopus, Google Scholar, and Web of Science databases.

The PICO criteria in this review were applied: population: in vitro/in vivo samples; intervention: combination therapy; comparator: placebo or standard treatment; and outcome: healing and improvement of lesions. A comprehensive search was performed using keywords and subject medical headings including combination therapy, cutaneous leishmaniasis, combined with, pulse, combined with Boolean operator “OR” and “AND.”

Study selection and data extraction

Inclusion criteria included all original in vitro/in vivo studies (met the PICO criteria) with full-text written in English language without geographical restriction between 1997 and 2022 reporting the role of combination therapy in the treatment of cutaneous leishmaniasis. Articles without any of aforementioned criteria, including unpublished abstracts/conference proceedings, case reports, retrospective or prospective case series, letters to the editor, brief communications, duplicates, and studies on human, were excluded. Additional relevant articles were manually obtained through citation tracking, and the reference lists of included articles and finally appropriate articles were included [Figure 1].

Figure 1.

Study flow diagram

Afterward, the studies were screened carefully by two independent investigators (PM and MA) to eliminate irrelevant articles. As the titles and abstracts of each paper were read in its entirety, data elements were extracted. The information including first author’s name, publication year, sample characteristics, types of leishmaniasis, leishmania species, and treatment specification extracted from studies and entered into a Microsoft Excel sheet. Then, the requisite data were rechecked for accuracy before data report and interpretation. When there was any discrepancy in report, the third reviewer was invited to resolve the issue. Agreement between reviewers was obtained through discussion and consensus. The results were described on a study-by-study narrative base.

RESULTS

A total of 91 records (in vitro, in vivo, clinical trials) were found following the initial search of databases and after removing duplicates and/or noneligible articles, as shown in Figure 1. Twenty-five articles (in vitro, in vivo) against L. major or L. tropica had eligibility to be included in this systematic review.

In vitro: There were 19 combination therapy articles against various Leishmania species such as L. braziliensis, L. tropica, L. major, L.infantum, and L.mexicana based on the inclusion criteria defined by consensus. Fourteen articles from Iran,[9] Egypt (1), United Kingdom (1), France (1), Kenya (1), and Pakistan (1) were selected. Seven articles studied combination therapy against CL on L. major and sevens article on L. tropica (see Table 1). In some selected articles, pentavalent antimonials (Glucantime or Pentostam) were administered in combination with different drugs such as amphotericin B, Methotrexate, paromomycin, miltefosine or allopurinol), Nicotinamide, Verapamil, Green Synthesized Copper nanoparticles, Albumin nanoparticles, and Biogenic Selenium Nanoparticles. Other studies used combinations of other drugs or treatments which were Amphotericin B (AmB) and chitosan platelets, Allium sativum and Aloe secundiflora, 10-Hydroxy Chondrofoline and Tafenoquine, Doxorubicin, and its pegylated liposomal formulation (Doxil, Caelyx), Chloroquine with paromomycin.

Table 1.

The inhibitory effects of combination therapy on leishmania parasites in vitro

n	Year	Leishmaniasis	Leishmania species	Combination therapy	Results	Ref.
1	2019	ACL	L. major	Levamisole with Glucantime	Levamisole increased immunity	Bamorovat M[21]
2	2021	CL	L. major	Green Synthesized Copper Nanoparticles Combined with Glucantime	Combination therapy prohibited the growth of L. major amastigotes and caused the NO production in a dose-dependent manner.	Albalawi AE[19]
3	2017	CL	L. major	Paromomycin puls Chloroquine	Combined treatment decreased EC50s by over 5-fold against L. major.	Wijnant G-J[26]
4	2020	ACL	L. tropica	Nicotinamide plus Glucantime	Inhibiting effects on L. tropica compared to each drug only.	Oliaee RT[17]
5	2019	CL	L. major	Amphotericin B with chitosan platelets	-Platelets showed antiparasitic activity, whereas native chitosan was inactive. -Chitosan plus AmB-DOC platelets had an additive effect on L.major amastigote	Malli S[20]
6	2019	CL	L. tropica	Amphotericin B with glucantime	The niosomal combination increase the apoptotic values and expression levels of IL-12 and metacaspases genes, and also reduced the levels of IL-10 with a dose-response effect.	Mostafavi M[14]
7	2018	CL	L. major	Meglumine antimonate loaded albumin nanoparticles	This nanodrug had the highest anti-leishmanial activity and the lowest cytotoxicity against macrophages.	Barazesh A[23]
8	2017	CL	L. major	Doxorubicin and its pegylated liposomal formulation (Doxil, Caelyx)	This combination, at low concentrations, had the anti-leishmanial effect on L. major.	Shokri A[22]
9	2016	CL	L. major	Allium sativum and Aloe secundiflora	This combination was less toxic and more effective against L. major.	Nasimiyu KC[34]
10	2022	CL	L.tropica	10-Hydroxy Chondrofoline and Tafenoquine	10-hydroxy chondrofoline was the most potent in vitro with an LD50 value of 43.80 μΜ after 48 h incubation, while tafenoquine LD50 value was 53.57 μΜ.	Shah SI[16]
11	2014	CL	L.tropica	Biogenic Selenium Nanoparticles and glucantime	Combined treatment decreased the mean number of amastigotes in macrophages	Mahmoudvand H[24]
12	2017	CL	L.tropica	Methotrexate with Glucantime	Combination therapy reduced the mean of infection and the mean of number of amastigotes in macrophage cells compared to positive control	Mahmoudvand H[25]
13	2013	CL	L.tropica	Glucantime with Paromomycin, Miltefosine or Allopurinol	Various concentrations of MA plus allopurinol, miltefosine or paromomycin significantly prohibited the growth of L. tropica amastigote in the macrophage compared to MA alone or positive control.	Riabi TR[27]
14	2012	CL	L.tropica	Verapamil with Glucantime	-In combined treatment, IC50=116.03 μg/ml was less than MA alone (IC50=225.14 μg/ml) for promastigote stage. -The amastigote was more sensitive to MA with verapamil compared to MA alone.	Shokri A[15]

Nine articles showed that the growth rate of amastigotes or promastigotes of L. major or L. tropica is suppressed, based on a dose-dependent mode. For instance, the simple and niosomal combination of AmB and glucantime against the L. tropica promastigote and amastigote demonstrated a simultaneous increase in the percentage of apoptotic cells from 10.97 and 8.45 (at 12.5 + 12.5 µg/mL) to 67.32 and 61.71 (at 200 + 200 µg/mL), respectively.[14] Furthermore, in assaying the antiparasitic effect of meglumine antimoniate (MA, 100 or 50 µg/ml) combined with paromomycin (100 µg/ml), miltefosine (100 µg/ml), or allopurinol (100 µg/ml), in vitro was isolated resistant L. tropica (dose-dependent).[15] Moreover, meglumine antimonate in an amalgamation with verapamil against L. tropica showed IC₅₀ values of 116 µg/mL compared to meglumine antimonate only 225.14 µg/mL.[15] The combination of 10-hydroxy chondrofoline and tafenoquine also had an LD₅₀ value of 43.80 µM and 53.57 µM against L. tropica, respectively.[16] Moreover, the higher effectiveness of nicotinamide plus glucantime (dose-dependent) was detected in reducing L. tropica growth, promoting immune response and inhibiting the ARG level.[17] In addition, the IC50 for the combination of Allium sativum and Aloe secundiflora at ratio (1:1) against L. major was 391.79 µg/ml and infection rate of 17% and multiplication index of 48.65% was reported. In contrast, pentostam alone was able to inhibit the growth of L. major promastigotes in vitro at a concentration of 12.5 µg/ml.[18] Moreover, IC50 value of Green Synthesized Copper Nanoparticles combined with Glucantime was 21.3 ± 0.42 µg/mL that this combination significantly caused 93.1% infectivity reduction in promastigotes and suppressed the growth of L. major amastigotes, based on a dose-dependent mode.[19] In vitro evaluations of antileishmanial activity of AmB and chitosan platelets also showed moderate intrinsic activity on L. major axenic amastigote, as the IC50 was 60.24 µg/mL.[20] Furthermore, levamisole in combination with glucantime (at 200 µg/ml concentration) caused the highest apoptotic values (32.23%) against L. major.[21]

Five articles also demonstrated the efficacy of combination therapy on L. major or L. tropica at low concentrations. For example, combination of Doxorubicin and Doxil significantly inhibited growth of L. major promastigotes and amastigote by at least 1 µg/mL.[22] Furthermore, meglumine antimonate-loaded albumin nanoparticles (10 µg/ml) had the highest effect (IC50 = 8.35) against L. major and the lowest toxicity (CC50 = 309.7) on J774 macrophages.[23] In addition, the IC₅₀ values of MA plus Biogenic Selenium Nanoparticles for sensitive and glucantime-resistant strains of L. tropica were 1.5 and 2.8 µg/mL, respectively. In contrast, the IC₅₀ values of MA for glucantime-sensitive and resistant strains were 11.6 and 43.6 µg/mL, respectively.[24] In addition, the findings of OD and IC50 showed that Methotrexate plus MA (SS: 16.1 µg/ml, RS: 39.8 µg/ml) had a higher antileishmanial effect than MA (SS: 52.2 µg/ml, RS: 170 µg/ml) or Methotrexate alone (SS: 22.2 µg/ml, RS: 51.4 µg/ml) on promastigotes and amastigote of sensitive and resistant L. tropica to MA.[25] Furthermore, the combination of 10 µM Chloroquine to Paromomycin reduced 50% effective concentrations against L. major (EC₅₀s) by over 5-fold (33.9 ± 5.9 to 10.7 ± 1.8 µM).[26]

In vivo: Considering the inclusion criteria, eleven combination therapy articles against L. tropica and L. major were selected, from Iran (1), Egypt (1), Brazil (1), Saudi Arabia (1), United Kingdom (1), United State (3), France (1), Kenya (1), and Pakistan (1). Ten articles studied combination therapy against CL on L. major and one article on L. tropica (see Table 2). Nine articles showed that the combination therapy as effective as standard drugs caused significant reduction in lesions size and parasitic load in spleen of hamster and BALB/c mice. The results of one article showed the combination therapy for short duration induced dramatic clinical improvement but relapse rapidly occurred after cessation of therapy. One article on the combination therapy resulted in a significant reduction in lesion size but not in parasite load. Furthermore, one article revealed the combination therapy reduced lesion size and spleen parasitic load significantly but did not prevent visceralization.

Table 2.

The effect of combination therapy on the treatment of cutaneous leshmaniasis in vivo

n	Case	Year	Leishmania species	Combination therapy	Results	Ref
1	Swiss strain albino mice	2016	L. major	Oral azithromycin plus miltefosine	Although, the combination therapy caused significant clinical improvement for short duration, relapse developed after therapy cessation.	Amer EI[35]
2	BALB/c mice	2009	L. major	Oral Miltefosine with paromomycin gel	The combined treatment was as effective as topical paromomycin only to decrease the parasite load and lesion size.	Aguiar MG[36]
3	BALB/c mice	2021	L. major	Green Synthesized Copper Nanoparticles Combined with Glucantime	Combination therapy prohibited the proliferation of L. major amastigote, and led to recovery and improved CL.	Albalawi AE[19]
4	BALB/c mice	2017	L. major	Paromomycin-Chloroquine	The combination therapy significantly decreased lesion size but not parasitic load.	Wijnant G-J[26]
5	BALB/c mice	1995	L. major	Interleukin 12 and Pentostam	The combined therapy induced improvement related to a switch from a Th2 to a Th1 response.	Nabors GS[29]
6	BALB/c mice	2002	L. major	Interleukin-12 and Indomethacin	Mice treated with indomethacin improved with exogenous IL-12 during the first 2 weeks of infection and unlike control mice, developed an enhanced Th1-type response related to increased resistance to infection.	Li J[30]
7	BALB/c mice	1997	L. major	Sodium stibogluconate and gamma interferon	This compound healed the infection and induced Th1-type responses.	Li J[31]
8	BALB/c mice	2019	L. major	Amphotericin B and chitosan platelets	Significant reduction of the inflammatory granuloma and a decrease of the parasitic load compared to AmB-DOC alone.	Malli S[20]
9	BALB/c mice	2019	L. majlue or	Amphotericin B with glucantime	The niosomal combination was efficient in decreasing the lesion size and spleen parasitic load.	Mostafavi M[14]
10	BALB/c mice	2016	L. major	Allium sativum and Aloe secundiflora	Combination therapy significantly decreased lesion size and splenic parasite burden, but did not prevent visceralization.	Nasimiyu KC[34]
11	BALB/c mice	2022	L.tropica	10-Hydroxy Chondrofoline and Tafenoquine	The combination therapy significantly decreased lesion size (0.70±0.03 mm) compared to negative control (1.2±0.3 mm).	Shah SI[17]

DISCUSSION

In the present review, we found the combination therapy with different drugs was more efficacious to decrease the course of treatment and cytotoxic effects of chemical drugs in the CL treatment, in vitro and in vivo. The standard treatment for CL is pentavalet antimony compounds such as meglumine antimoniate (MA) (Glucantime) and sodium stibogluconate (Pentostam) with low efficacy.[27] Previous studies have showed that glucantime in combination with other drugs is more efficacious against L. tropica, or L. major compared to glucantime alone. For example, the synergistic effect of methotrexate with meglumine antimoniate led to inhibit the growth of promastigotes and amastigotes of MA-sensitive ad resistant L. tropica.[28] Findings of the study by Mostafavi et al.[14] also showed that there is a synergistic effect between niosomal formulation of AmB and glucantime in inhibition of extracellular and intracellular growth of L. tropica and also L. major in vivo that seems to be an appropriate formulation in leishmaniasis treatment due to low cost and easy to use. Furthermore, glucantime along with different concentrations of miltefosine, paromomycin, or allopurinol showed a synergistic effect on L. tropica amastigotea in comparison with each drug alone. Hence, synergistically, this combination reduced the number of Leishmania parasite.[27] Shokri and et al.[15] also used MA alone and in combination with verapamil (as a calcium channel blocker) against L. tropica at less concentration. In addition, Mahmoudvand and et al.[25] investigated the effects of Methotrexate (MTX) (as an antimetabolite and antifolate drug) alone and in combination with MA on the promastigote and amastigote of MA-susceptible and resistant L. tropica strains. The OD and IC50 results showed that MTX with MA (at the low doses and short times) had a higher antileishmanial effect than MA or MTX alone on promastigotes and amastigote of both strains of L. tropica. Furthermore, the results showed that the combination of MA and MTX effectively reduced the number of amastigotes in each macrophage at all concentrations compared to MA or MTX alone.

Moreover, the immunomodulatory potential of combination therapy in developing immune system was obtained through in vitro experiments. For example, the study of Oliaee RT and et al.[17] showed higher efficacy of the Nicotinamide/MA combination in preventing the growth of L. tropica in a macrophage model through rising the immune response. They found the Nicotinamide/MA combination increased levels of IFN-γ, IL-12p40, and TNF-α and reduced IL-10 with a dose-response effect. Furthermore, Nabors GS et al.[29] reported that treatment with IL-12 or anti–IL-4 antibody, in combination with Pentostam, induces healing in L. major-infected mice with a switch from Th2 to Th1 response. In addition, Li J and et al.[30] reported that L. major-infected BALB/c mice and treated with indomethacin and IL-12 combination controlled their infections and developed Th1-type response (high levels of IFN and NO2 (indicative of enhanced NO production) and low levels of IL-4), while treatment with either indomethacin or IL-12 alone failed to stop disease progression. In addition, Li J and et al.[31] showed that treatment with IFN-g plus Pentostam can decrease the treatment duration and dose of antimony required to cure. Mice receiving combined therapy had small lesions with fewer parasites than control mice or mice which were treated with either the Pentostam or IFN-g alone. The results also indicated that immunotherapy may enhance the development of a protective response of Th1 in sensitive mice through a mechanism involving suppression of regulatory cytokines and increased production of IL-12 and NO. In another study, Li J and et al.[32] reported the decreased levels of IL-4 and TGF-b mRNAs in lesions of L. major-infected CB6F1 mice treated with IL-12 plus antibody against IL-4.

Recently, nanoparticles (NPs) such as silver, gold, copper, and selenium (as drug carriers) have been used to treat CL due to their nanoscale sizes and high surface-to-volume ratios that allow more active sites for interacting with biologic molecules like microorganisms.[33] For example, Barazesh and et al.[23] used encapsulation of glucantime with albumin (as a nontoxic polymeric carrier that is removed by inflammatory tissues). They found that this nanodrug had the highest effect and the lowest cytotoxicity against L. major and J774 macrophages, respectively. Furthermore, Mahmoudvand and et al.[24] evaluated the antileishmanial effects of Se-NPs in combination with MA and alone on promastigotes and amastigotes of L. tropica sensitive and resistant to glucantim and found the combined treatment prohibited the growth of promastigotes of both strains in a dose-dependent manner in compared to MA alone, but promastigotes were more sensitive to Se-NPs (alone or in combination with MA) than amastigote forms. The difference in the sensitivity of the promastigote and amastigote stages to different concentrations of Se-NPs may be related to the structural, biochemical, and morphological characteristics, as well as other factors such as the phago-lysosomal membrane of the macrophage, which decreases the entry of Se-NPs into the macrophages and reduces the concentration of Se-NPs around amastigotes inside the macrophage. Furthermore, results revealed that the level of macrophages infection reduced significantly when promastigotes of both strains of L. tropica were pretreated with Se-NPs. Similarly, Se-NPs in combination with MA significantly decreased the mean number of amastigotes of both strains compared to each drug alone. In addition, Albalawi and et al.[19] showed that preincubation with low concentrations of Cu-NPs + MA reduced the mean number of L. major promastigotes compared to the nontreated promastigotes or promastigotes that were preincubated in Cu-NPs or MA, separately. As well as, they resulted the main antileishmanial mechanisms of Cu-NPs is triggering NO for recovering CL in BALB/c mice.

Moreover, other studies suggested combination therapy as a reliable and an alternative approach. For example, oleoyl chitosan/α-CD platelets exhibited high antileishmanial activity and exerted an additive effect when accompanied with AmB-DOC against L. major axenic amastigotes in vitro. In vivo experiments also highlighted the histological and immunohistochemical (IHC) evidences that combination between AmB-DOC and chitosan platelets could be an interesting strategy for the treatment of CL. As, histological and IHC tests of dermal lesions of L. major -infected mice and treated with the combination showed a significant reduction of the inflammatory granuloma and the parasitic load compared to AmB-DOC alone.[20] Furthermore, in vitro results of Shah and et al.[16] study detected the combination of Tafenoquine (as an 8-amminoquinolone that is typically used for the treatment of Malaria) and 10-hydroxy chondrofoline could be the most potent drug against CL as compared to MA. In vivo activity also was conducted using injecting 10-hydroxy chondrofoline in the foot of L. tropica -infected BALB/c mice, where it caused a statistically significant reduction in lesion size when compared with negative control.

Some studies also have shown that plant-derived products with synergistic effects could be considered as a new approach in the treatment of leishmaniasis. For instance, Nasimiyu KC and et al.[34] analyzed the combining effect of aqueous extracts of A. sativum and A. secundiflora on L. major, in vitro and in vivo. The combination therapy with mentioned extracts resulted to high anti promastigote, anti-amastigote activities, less toxic to Vero cells as compared to pentostam and this drug reduced lesion sizes in L. major-infected BALB/c mice significantly, but did not prevent visceralization. In addition, Chloroquine (CQ) improves the effects of specific antibiotics against intracellular pathogens. For instance, the high-dose combination of CQ and PM provides only limited improved efficacy (mild effect on the evolution of lesion sizes without an additional reduction in parasite burdens) in L. major-infected mice compared to PM monotherapy.[26] Moreover, Amer and et al.[35] tested the combination of miltefosine with azitheromycin (a semi-synthetic macrolide antibiotic) against L. major in their effective doses only for 10 days, and obtained dramatic clinical improvement of lesions with significant reduction of parasite burden, however relapse rapidly developed after cessation of therapy. In contrast, Aguiar and et al.[36] showed the combination of topical paromomycin and oral miltefosine was effective in the treatment of L.major- infected mice, and led to parasite load reduction in the skin and spleen, and healing the lesions. Consequently, results of mentioned studies showed that combination therapy is efficient and has a major role to control leishmaniasis in vitro and in vivo.

CONCLUSION

The combination of strong and weak antileishmanials drugs in compared to monotherapeutic regimens showed increased synergistic efficacy, reduced resistance, the short course of treatment, and less side effects in the treatment of CL, in vitro and in vivo. Hence, this study suggests combination therapy to be surveyed in clinical trials with more population.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.