Drug resistance in leishmaniasis: Newer developments.

Leishmaniasis is a vector borne protozoan disease and it remains a major public health problem world-wide. Lack of an effective vaccine and vector control program makes the chemotherapy as the primary tool for leishmaniasis. Antimonials were used as the first line of treatment for many years. Emergence of resistance against this drug has become a major concern. Literatures and studies published on anti-leishmanial drug resistance, newer drug discovery for leishmanial resistance etc., in PubMed, Medline and Google search and reviewed thoroughly. Various newer drugs have been identified but, are in limited use because of high cost, toxicity, resistance etc., Recently, many newer mechanisms of drug resistance have been identified which may boost in future designing and development of drugs.

INTRODUCTION

Leishmaniasis is an important parasitic disease, caused by protozoa Leishmania. There are 21 Leishmania spp., prevalent world-wide with the potential to cause human infection.[1] The disease presents with four major clinical manifestations (visceral leishmaniasis [VL], cutaneous leishmaniasis [CL], mucocutaneous leishmaniasis and post-kalaazar dermal leishmaniasis [PKDL]). VL is the most serious presentation amongst all and may lead to death with lack of treatment. Leishmania donovani causes VL or kalaazar. It is transmitted by sandfly Plebotomus argentipes. The parasite exists in two stages: Amastigote or intracellular forms in man and promastigote or extracellular form in sandfly. It is endemic in many parts of the world such as Africa, Latin America and Central Asia. The prevalence of leishmaniasis has been reported approximately 12 million cases with an annual mortality rate of 60,000.[2] Around 2 million new cases of leishmaniasis are detected every year out of which 50,000 cases were diagnosed as VL.[2] More than 90% of these cases were reported from the five developing countries, i.e., Sudan, Brazil, Bangladesh, India (Bihar) and Nepal.[3] The disease is endemic in several other poor and developing countries; hence, now it is included in the list of the neglected tropical diseases. Around 200 million people from India are at risk estimating approximately 67% of the world's risk population.[4] it is especially prevalent in Bihar (>90%), Uttar Pradesh, West Bengal and Jharkhand. Free availability of anti-leishmanial drug in India increases the chances of misuse; thereby increases the emergence of drug resistance.[2] It was observed that 73% of total VL patients first consult to an unqualified medical practitioner, who may not have the exact knowledge about drug dosage, duration of intake etc., Irregular and incomplete intake of drug leads to progressive tolerance and significant contributor in development of drug resistance in India. Cost of the drug is also one of the major contributing factors in development of resistance. Among all the drugs, antimonials are the only one which can be affordable. Majority of patients unable to complete the full course of the other drugs leads to the development of resistance. In the other hand, >90% of CL cases are reported from Afghanistan, Iran, Brazil, Peru and Saudi Arabia. Increased incidence of human immunodeficiency virus (HIV) co-infection, human migration and resettlement (especially areas where leishmaniasis is zoonotic) in recent years may cause a resurgence of the number of cases. Since there is no effective vaccine available, control of leishmaniasis is primarily depends on chemotherapy. Pentavalent antimonial remains the key drug for last several decades. During last 10-15 years, resistance to antimonials has been reported from all over the world; particularly from Bihar (India).[5] The second line drugs for VL includes amphotericin B (Amp B) and pentamidine. Various oral medications such as miltefosine and fluconazole are also got approval for VL and CL patients, respectively.[2] These second line anti-leshmanials are less popular in the developing countries because of high cost, side effects and the requirement of hospitalization. It suggests the need of newer drugs for better management of this condition. This review highlights the mechanism of resistance to anti-leishmanial drugs, newer anti-leishmanial drugs under development and strategies to preserve the efficacy of currently used drugs.

The classification of various anti-leishmanial drugs and their mechanism of action have been described below [Table 1].

Table 1

Classification of anti-leishmanial drugs

ANTIMONIALS

Antimony remains the mainstay of treatment for leishmaniasis since last 60 years. The mechanism of action is not very clear. It is known that the pentavalent form is inactive against Leishmania spp.[6] It utilizes thiols from the parasite and host cell surface and gets reduced to the trivalent form inside the macrophage, thereby act on the amastigote forms of the parasite.[3] In 1920, Professor Bramhachari first synthesized the pentavalent antimony and used as a chemotherapeutic agent in Indian VL patients.[7] Initially, a dosage of 10 mg/kg for 6-10 days showed clinical cure rate of 90% in VL patients.[3] Subsequently, higher dosage with prolong duration (20 mg/kg for 30 days) was suggested owing to failure of the treatment.[3] Unfortunately, it could not prevent the emergence of resistance of antimonials. Rather, more resistant strains were selected out and reached to an epidemic dimension in Bihar (India). Rapid spread of resistant strains in India occurred because of the anthroponotic transmission. It was observed only 64% of new VL cases from Bihar were sensitive to antimonials in additional to 100% resistant cases from Sitamari and Darbhanga district.[8] Although it is banned in India, antimonials are used as the first-line treatment in the endemic areas world-wide. Sensitivity of antimonials toward different Leishmania spp. varies differently. It is observed that, Leishmania brasilensis is more sensitive in comparison Leishmania mexicana.[9] Host factors such as decreased drug uptake, increased efflux mechanism, reduced concentration inside the parasite, inhibition of drug activation, inactivation of active drug and gene amplification are some important mechanisms responsible for the development of resistance in this group. Thiol metabolism also plays an important role in developing resistance.[310] Thiol molecule increases the oxidative stress inside the macrophage preventing glutathione formation and reduction of pentavalent antimonials to trivalent form. Therefore, increased intracellular thiol levels are associated with high antimonial resistance. The role of ABC transporters such as P-glycoprotein and multidrug resistance (MDR)-related protein in the drug resistance is proven in laboratory isolates of Leishmania spp.[10] However, its role in the field isolates is still not clear.[10]

AMP B

Amp B is a polyene antifungal agent extracted from the filamentous bacteria called Streptomyces nodusus. It is used as the second line drug in patients with leishmaniasis. It is available in two forms, i.e., plain Amp B, liposomal Amp B. The latter one is better tolerated and less toxic.[11] Its mechanism of action is multifaceted.[12] Amp B is a sterol loving agent. It binds with cholesterol and forms pores in the host cell membrane causing leakage of cellular content followed by cell death. It helps in generation of reactive oxygen free radical. These radicals subsequently cause damage to cell leading to cell death. Although, Amp B has been proven as a successful chemotherapeutic agent in the leishmania patients, there are few reports indicating its resistance. Emergence of resistance could be expected because of high frequency of use of Amp B in India. The mechanism of resistance is not very clear. Recently, an article from India has proven the development of Amp B resistance due to the molecular mechanism.[12] It was observed that, the sterols in the cell wall of the amastigote forms of the resistant strains were replaced by a precursor, cholesta 5,724-trien-3 β-ol.[12] It might have resulted due to defective trans-methylation of C-24 molecule by S-adenosyl-L-methioninie: C-24-Δ-sterol methyltransferase (SCMT) enzyme. SCMT enzyme is classified as SCMT A and SCMT B. In resistant strains, transcripts of SCMT-A is found to be absent with over expression of SCMT-B. Absence of SCMT-A is possibly responsible for the defective trans-methylation. This alteration in the membrane composition leads to decreased binding and reduced uptake of Amp B into the cell. MDR1 gene in the ABC transporters of promastigotes is responsible for drug efflux mechanism and subsequently leads to resistance.[12] There is three-fold increased expression of MDR1 genes observed in resistant strains in comparison to sensitive strains.[12] The resistant strains also showed up regulation reactive oxygen species (ROS) scavenging machinery inside the cell. The cumulative effect of all mechanisms leads decreased concentration of Amp B inside the cell conferring to its resistance.

MILTEFOSINE

Miltefosine (hexadecylphosphocholine) is the first oral drug exhibiting 94% cure rate in VL patients.[513] It had shown good results against antimony resistant VL and PKDL cases.[1415] Hence, in India, it is proposed to be the first-line drug in the kalaazar elimination program.[1617] The mechanism of action of this drug is unclear. It binds with the cell membrane, gets internalized by the help of two membrane proteins, i.e., L. donovani miltefosine transporter (LdMT) and L. donovani Ros3 (LdRos3).[18] It deranges the alkyl-lipid metabolism of the parasite and cause apoptotic cell death. However, because of anthroponotic transmission in Indian subcontinent; misuse of the drug and its longer half-life are the two factors that alarms acquiring of quick resistance to this drug.[19] Recently, there are reports of miltefosine resistant cases from Nepal.[20] The proposed mechanism of resistance was inactivation of genes responsible for drug uptake, i.e., LdMT and LdRos3.[21] In an Indian study, four-point mutation was noticed showing variable drug response to miltefosine.[19] However, no significant genomic difference was observed between the genes of LdMT and LdRos3 between the sensitive and resistant strains. Hence, it is likely that, several other factors are also responsible for the mechanism of resistance of miltefosine apart from point mutation.

PAROMOMYCIN

PM is a broad spectrum antibiotic extracted from the bacterial spp. Streptomyces rimosus var. paromomycinus. It is active against several parasites. In 2002, it was first introduced as anti-leishmanial drug in the form of PM sulphate.[22] Low cost, fewer side-effects, better efficacy and shorter duration of administration make this drug popular and thought to be a candidate for the first line therapy for VL patients.[23242526] It belongs to the aminoglycoside group of antibiotic; hence, the mechanism of action is by inhibition of protein synthesis. There are several clinical trials undergoing to know the efficacy results of this drug by different routes. However, there is a likely chance of acquiring resistance with PM monotherapy. To combat these problems, newer formulations like PM loaded with albumin microsphere and PM with liposome have come up exhibiting better results.[27]

NEWER DEVELOPMENTS

The following newer compounds are in different stages of drug development and preclinical trials. The in vitro efficacy of these compounds has been shown in various animal studies.

2-substituted quinoline

2-substituted quinoline is a medicinal plant derivative, shows excellent activity against the new world CL.[28] Significant action against L. donovani has been observed in BALB/c mouse.[29] It is one of the most active quinoline against promastigote and amastigote forms of L. infantum and L. amazonensis.[29] In vitro activity of this compound against L. donovani found to be more efficacious in comparison to miltefosin and sitamaquine.[30] It does not possess in vitro antiplasmocidal or antitoxoplasmic activity.

8-aminoquinolines

It is an antiprotozoal drug originally introduced as an antimalarial drug. Recently, phase II trial in India and Kenya showed its potency against L. donovani.[31] It causes apoptosis such as death of the parasite by chromatin fragmentation, enhanced reactive oxygen production, elevation of intracellular Ca+2 ion.[32] Recently, it is suggested to be considered as a candidate for combination therapy causing less toxicity and therapeutic failure.

DRUGS TARGETING METABOLIC PATHWAY

New anti-leishmanial drug can be designed and developed targeting the metabolic pathways and biochemical structures of the parasite.

Protein kinases are the key regulatory proteins and major target of metabolic pathway of Leishmania spp.[29] Among the various kinases, mitogen-activated protein kinase, L. mexicana mitogen activated protein kinases-1 (LmxMPK-1), LmxMPK-2 are found to be essential for the survival of amastigotes inside the host and pathogenesis.[33] There are different libraries available, which one can screen and differentiate the parasite specific kinase from the human kinases. The above information can be utilized for the better optimization and designing of new drug. Among different cyclin dependent kinases, Cdc2-related kinase-3 (CRK-3) observed to be the most potent and present in the amastigote form of L. donovani.[3435] Another new inhibitor named as indirubin has been proven to be highly potent and active against L. donovani.[35] Its mechanism of action is linked with the CRK-3 and other protein kinases.

ROLE OF INTERFERON GAMMA

In VL cases, the amastigote forms quench cholesterol from the cell membrane of macrophage during their intracellular stay; thereby making defective antigen presentation function.[36] This also interferes in the signaling of the IFNγ receptor oligomerization. Exogenous supply of cholesterol not only reinstates the receptor but also restores the IFNγ level; which is more host related than the parasite.[36] Hence, cholesterol liposomal formulation of IFNγ is a newly emerging therapeutic option in both drug sensitive and resistant cases, which enhances the macrophage mediated killing.

COMBINATION OF DRUGS

Combination drug regimen for the treatment of VL is come into knowledge to combat the drug resistance developed by monotherapy. It can potentially broaden the spectrum, increase the activity of the drug by additive or synergistic action, decrease the duration and dosage, reduces the side effects and thereby reduces the cost of treatment and the emergence of drug resistance. Among the anti-leishmanials, miltefosine and paramomycin were found appropriate for combination therapy because of less interference with other drugs. Recently, a randomized control trial was carried out in East Africa for the treatment of VL comparing PM 20 mg/kg/day for 21 days monotherapy with PM and sodium stibogluconate combination (PM 15 mg/kg/day and SSG 20 mg/kg/day for 17 day) and SSG (30 mg/kg/day for 30 days) alone. The 17 day combination regimen found equally efficacious and safer in comparison to 30 days regimen.[37] A phase III randomized control trial has been planned to observe the safety and efficacy of miltefosine alone, miltefosine with liposomal Amp B versus SSG with liposomal Amp B in primary VL patients in East Africa.[38] Recently, techniques are available to screen the action of the drugs; thereby detecting the drug resistance.[39] These methods also screen the natural compounds in the endemic region with the available libraries and would be able to discover new drugs.

HIV and kalaazar co-infection

HIV co-infection in VL patients remains a major challenge for therapeutic management. These groups of patients develop early resistance and may show treatment failure or relapse of the disease with a rate of 0-85%.[40] Treatment of these co-infections is not very clear and varies from country to country depending upon the policy. Some follow treatment of antiretroviral therapy followed by anti-leishmanial therapy, whereas few follow maintenance therapy with Amp B.[40] However, many trials on protease inhibitor for the drug dosage, efficacy and drug concentration are going on currently to evaluate the action against Leishmania species.[41]

STRATEGIES TO COMBAT THE DRUG RESISTANCE AND PRESERVE THE EFFICACY OF AVAILABLE DRUGS

In addition to the pharmacologic factors, a number of host factors are responsible for the emergence and spread of drug resistance. These include improper monitoring of drug resistance, diagnostic methods, lack of compliance, drug availability and affordability, drug quality, limited drug distribution, limited access to peripheral health care facility for early treatment etc., Therefore, to prevent further emergence and spread of drug resistance and to preserve the efficacy of currently available drugs, the following strategies should be followed:

To determine drug resistance improved phenotypic and genotypic methods with good sensitivity should be developed and used

There is a need of development of good diagnostic tests, which can monitor the drug response as well as therapeutic outcome of the patient

Guideline based management should be introduced and hence that proper selection of anti-leishmanials will be done

Directly observed therapies should be implicated to increase compliance

Drug affordability should be improved by joining the pharmaceutical companies with World Health Organization or Non-Governmental Organizations

Continuous drug availability to the different health care facilities should be done to ensure the successful control of the disease

Strict rules for avoidance of suboptimal use or misuse of anti-leishmanial drugs should be implemented and maintained at the national and peripheral level

Large scale control trials should be conducted to evaluate the safety and efficacy of combined regimens.

CONCLUSION

The treatment of leishmaniasis still relies on very few drugs which are toxic, expensive and difficult to administer. Emergence of drug resistance along with lack of standard molecular markers for its detection makes the situation worse. Care must be taken to prevent the emergence and spread of drug resistance by proper understanding the mechanism of resistance, monitoring the drug use and its response. By understanding the resistance mechanism, many intracellular targets can be exploited, which will provide clues for designing newer drugs. Several large scale trials based on combination therapy going on, which may be extrapolated into successful management of leishmaniasis. Despite good knowledge on the epidemiology leishmaniasis, it still remains uncontrollable. Hence, efforts should be made to develop newer drugs which should be cheap, less toxic and easy to administer.