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Leishmaniasis caused by protozoan parasites Leishmania, is a disease of poverty as its victims are among the poorest. According to ranking after malaria it is a second most prevalent parasitic disease. Leishmaniasis has been considered as a tropical affliction that constitutes one of the six entities on the list of most important diseases of World Health Organization/Tropical Disease Research (WHO/TDR) viz. Malaria, Schistosomiasis, Filariasis, Chagas disease, African Trypanosomiasis, Leishmaniasis, Leprosy, Tuberculosis.

1. History of Leishmaniasis:

Representations of skin lesions and facial deformities have been found on pre-Inca pottery from Peru and Ecuador dating back to the first century AD. There are evidences that some forms of leishmaniasis prevailed as early as this period (WHO website). There are detailed descriptions of oriental sore by Arab physicians including Avicenna in the 10th century, who described it as Balkh sore from northern Afghanistan, and there are later records from various places in the Middle East including Baghdad and Jericho; many of the conditions were given local names by which they are still known. In the Old World, Indian physicians applied the Sanskrit term kala-azar (KA) (meaning ‘black fever’) to an ancient disease later defined as visceral leishmaniasis (VL). KA was first noticed in Jessore in India in 1824, when patients suffering from fevers that were thought to be due to malaria failed to respond to quinine. By 1862 the disease had spread to Burdwan, where it reached epidemic proportions. In 1901, William Leishman identified certain organisms in smears taken from the spleen of a patient who had died from ‘dum-dum fever’. Initially, these organisms were considered to be trypanosomes, but in 1903 Captain Donovan described them as being new. The link between these organisms and KA was eventually discovered by Major Ross, who named them Leishmania donovani. The search for a vector was a long one, and it was not until 1921 that the experimental proof of transmission to humans by sand flies belonging to the genus Phlebotomus was demonstrated by Edouard and Etienne.

2. Risk Factors and Definition of the Problem

In India, a country with a high leishmaniasis burden, 88% of leishmaniasis patients have a daily income of less than USD 2 and poor economic level. The number of cases of leishmaniasis is increasing, mainly because of man-made environmental changes that increase human exposure to the sand fly vector. Extracting timber, mining, building dams, widening areas under cultivation, creating new irrigation schemes, expanding road construction in primary forests such as the Amazon, continuing widespread migration from rural to urban areas, and continuing fast urbanization worldwide are among the primary causes for increased exposure to the sand fly.

3. Types of Leishmaniasis

The leishmaniases which causes considerable morbidity and mortality is the collective name for a number of diseases which have diverse clinical manifestations. Leishmaniases has traditionally been classified in three major forms on the basis of clinical symptoms. The most deadly form is visceral leishmaniasis (VL), which if left untreated, leads to full-blown disease and invariably leads to death. A number of other species of Leishmania cause cutaneous (CL) and mucocutaneous (MCL) leishmaniasis, which, if not fatal, are still responsible for considerable morbidity of a vast number of people in endemic foci. Leishmaniasis is spreading in several areas of the world as a result of epidemiological changes which sharply increase the overlapping of AIDS and VL.

Cutaneous Leishmaniasis (CL)

This is the most common form of Leishmaniasis, also known as ‘Oriental sore’ which first appears as a persistent insect bite. Simple skin lesions appear at the site of sand fly bite (fig 1) which self-heal within few months but leaves scars. The incubation period can last from few days to months. Gradually, the lesion enlarges, remaining red, but without noticeable heat or pain. Resolution of the lesion involves immigration of leucocytes, which isolate the infected area leading to necrosis of the infected tissues, and formation of a healing granuloma in the floor of the lesion.

The disease is mostly prevalent in Mediterranean region, Central Asia and many places of Central Africa. Man is the definitive host whereas gerbils, cats, dogs, and rodent act as the natural reservoir of CL. Sand flies of genus Phlebotomus serve as transmitter for this disease. CL is usually caused by L. major, L. tropica, L. aethiopica, in old world and by L. mexicana, L. venezuelensis, L. amazonensis, L. braziliensis, L. panamensis, L. guyanensis and L. peruviana in new world.

Fig1. Severe skin ulcer development due to sand fly bite, a specific and primary symptom of cutaneous Leishmaniasis followed by lesion enlargement, redness, but without noticeable heat or pain.

Variations of CL: Diffuse cutaneous leishmaniasis (DCL)

This is a chronic, progressive, polyparasitic variant that develops in context of leishmanial-specific anergy and is manifested by disseminated non-ulcerative skin lesions, which can resemble lesions of lepromatous leprosy. DCL is restricted to Venezuela and Dominican Republic in the western hemisphere, and to Ethiopia and Kenya in Africa. Its main causative organisms are L. aethiopica (old world) and L mexicana species complex (new world).

Mucocutaneous Leishmaniasis (MCL)

This form of disease, also known as ‘‘espundia’’, causes extensive destruction of naso-oral and pharyngeal cavities with hideous disfiguring lesions, mutilation of the face and great suffering for life. MCL is occasionally reported from Sudan and other Old World foci. Classical MCL is, however, restricted to L. braziliensis infections in which, following the apparently complete resolution of the initial oriental sore, sometimes many years later, metastatic lesions appear on the buccal or nasal mucosa. MCL usually exists as an azoonotic infection in which lifecycle is being transmitted from rodent to rodent and mammal by the forest sand fly Lutzomyia spp. The reservoir hosts include rodents, opossums, anteaters, sloths and dogs etc. Human infection occurs when human invade the forest habitats.

The causative agents of MCL in old world are L. aethiopica (rare), and in new world are L. braziliensis, L. guyanensis, L. mexicana, L. amazonensis and L. panamensis.

Visceral Leishmaniasis (VL)

VL is the most dreaded and devastating amongst the various forms of leishmaniasis. VL is also known as Kala-Azar, Black Sickness, Black Fever, Burdwan fever, Dumdum fever or Sarkari Bimari etc. It is the most severe form of disease and if left untreated, is usually fatal. The parasite is responsible for a spectrum of clinical syndromes, which can, in most extreme cases, move from an asymptomatic infection to a fatal form of VL. It is characterized by prolonged fever, splenomegaly, hepatomegaly, substantial weight loss, progressive anemia, pancytopenia, and hypergammaglobulinemia (mainly IgG from polyclonal B cell activation) and is complicated by secondary opportunistic infections (Fig.2). The parasite invades and multiplies within macrophages (free mononuclear phagocytic cells) and affects the reticuloendothelial system including spleen, liver, bone marrow, and lymphoid tissue. The outcome of fully developed VL is death, usually said to be due to concomitant infection resulting from the weakened immunological state of the patient.

$D:\Mukesh Samant 2006-07\Mukesh thesis\Thesis figures\Final VL.tif$

Fig.2. Clinical symptoms of visceral leishmaniasis. Hepato-splenomegaly and substantial weight loss are main features.

VL is typically caused by L. donovani complex, which includes three species: L. donovani donovani, L d. infantum, and L. d. chagasi. L. donovani is the causative in the Indian subcontinent and East Africa. L. infantum causes VL in the Mediterranean basin and L. chagasi is responsible for the disease in Central and South America . VL (occasionally other syndromes) is emerging as an important opportunistic infection among people with HIV-1 infection.In fact, the parasite may be a cofactor in the pathogenesis of HIV infection.

There are more than 21 morphologically indistinguishable species of Leishmania that infect humans. Conventionally, they are classified and named mainly according to their geographical distribution and clinical characteristics of the disease they afflict.

The Post Kala-azar Dermal Leishmaniasis (PKDL) is a type of non ulcerative cutaneous lesion. After recovery from infection, VL patients may develop a chronic form of CL i.e., PKDL which is developed in about 10% of kala-azar patients generally one or two years after completion of sodium stibogluconate (SSG) treatment and requires a long and expensive treatment.

4. Geographical Distribution of Leishmaniasis

Leishmaniasis occurs in 88 countries in tropical and temperate regions, of which 72 are either developing or least developed. Approximately 1,98,000 people are affected with these diseases worldwide with 5,00,000 million new cases occurring each year, but the true picture remains largely hidden since a substantial number of cases are never recorded. The disability-adjusted life years (DALY) burden was 2,357,000 and total deaths were 59,000 in 2001. It has been estimated that 90% of CL cases occur in 7 countries: Afghanistan, Algeria, Brazil, Iran, Peru, Saudi Arabia and Syria whereas MCL is endemic in Mexico and Central and South America (Fig 3).The annual estimate for the incidence and prevalence of kala-azar cases worldwide is 0.5 million and 2.5 million, respectively and of these, 90% cases occur in India, Nepal, Bangladesh and Sudan. PKDL is prevalent in India, Sudan and Kenya.

Global Status of Visceral Leishmaniasis

VL is endemic in 62 countries, with 200 million people at risk, an estimated 500,000 new cases each year worldwide and 41,000 recorded deaths in the year 2000. The disease burden associated with VL, measured in DALYs was estimated to be 1,980,000 (1,067,000 for male and 744,000 for female in year 2000. VL is caused by L. donovani in the Indian subcontinent, Asia, and Africa (in adults and children), and by L. infantum in the Mediterranean region, southwest and central Asia and by L. chagasi in South America. In Sudan, for example, a major decade-long epidemic of VL occurred from 1984 to 1994. As this was the first epidemic in the area, populations were highly susceptible. Some studies estimate that the disease caused 100,000 deaths in a population of around 300,000 in the western upper Nile area of the country (http:// www.who.int/mc/ diseases/leish/ diseaseinfo.htm). The health ministers of India, Nepal and Bangladesh signed a memorandum of understanding on 18 May 2005, pledging to eliminate KA from their countries. The five elements of the elimination strategy are access to early diagnosis and treatment (particularly by the most vulnerable groups), strengthening disease and vector surveillance, integrated vector management, social mobilization, networking and research.

National Status Visceral Leishmaniasis

KA is present in India for more than 100 years. The incidence of KA in India is among the highest in the world. KA is a disease of poor people whose daily income is less than 1 dollar a day. Epidemics of KA occurred in Bengal in the years 1832, 1857, 1871, 1877, and 1899. In India about 100,000 cases of VL are estimated to occur annually. It has recently posed a serious threat in India involving 38 out of 42 districts of Bihar state, 8 districts of West Bengal and 2 districts of Eastern Uttar Pradesh. In 1977, a sample survey in Bihar estimates the number of cases to be about 1, 00,000 with 4,500 deaths whereas in 1991 infected cases reached to 2, 50,000 with 75,000 deaths. The state of Bihar now accounts for more than 90 percent of the cases. Because of the rapid manner in which the disease was spreading, an alarming situation existed.

$D:\Mukesh Samant 2006-07\Mukesh thesis\Thesis figures\VL in Word and India.tif$

Fig.3. The worldwide distribution of visceral leishmaniasis (A), VL affected states of India (B) and VL affected districts of Bihar (C).

5. Vectors of the Disease

Leishmaniasis is transmitted by the Phlebotomus spp. in the Old World and Lutzomyia spp. in the New World. P. argentipes is the proved vector of KA in India. Of the 500 known Phlebotomine species, only some 30 of them have been positively identified as vectors of the disease. Sand flies are very small in size (< 3.5 mm) (Fig. 4) and may be hard to see. These are usually most active in twilight, evening and night hours (from dusk to dawn) and less active during the hottest time of the day. Female sand fly lays its eggs in the burrows of certain rodents, in the bark of old trees, in ruined buildings, in cracks in house walls, in animal shelters and in household rubbish (WHO website). High incidence of VL is reported during pre-monsoon season that coincides with vector abundance and increased man-vector contact due to sleeping habits of children in open space in summer.

$D:\Mukesh Samant 2006-07\Mukesh thesis\Thesis figures\Sand fly.TIF$

Fig.4. Sand fly, the vector host of Leishmania parasite

6. Morphology and Digenetic Life cycle of Leishmania donovani

In India, VL is caused by L. donovani. Indian VL is anthroponotic and is transmitted chiefly through the bites of the female sand fly, P. argentipes. Leishmania exists in two forms (i) promastigotes: these are extracellular, elongated, flagellated, motile and ranges in size from 2 µm ´ 2-20 mm. This form exists in sand fly and in in vitro cultures (ii) amastigotes: these are intracellular, round to oval, aflagellated, non- motile and ranges in size from 2-5 mm. This form resides and multiplies within the phagolysosomes of macrophages of reticuloendothelial system of the vertebrate host. Following the sand fly bite, some of the flagellates entering the circulation are destroyed while others enter the cells of the reticuloendothelial system. Here they undergo change into amastigote form which multiply by binary fission, with the multiplication continuing until the host cell is packed with the parasites and ruptures, liberating the amastigotes into circulation (Fig.5).

$D:\Mukesh Samant 2006-07\Mukesh thesis\Thesis figures\Life cycle of Leishmania.jpg$

Fig.5. The life cycle of Leishmania. Parasite shuttles between vector host, sand fly and human host. [Adapted from Kumari et al, (2008)]

7. Factors Responsible for Virulence and Survival of Parasite:

Cell surface glycoconjugates play a pivotal role in parasite virulence and infectivity. Expression of complex and unique glycoconjugates at the parasite cell surface appears to be crucial for their survival and development in the sand fly vector and the mammalian host macrophage. Sialoglycans as well as lipid-bound (LPG) and protein-bound (sAPs) and (PPGs) phosphoglycan-containing glycoconjugates are the predominant molecules on the cell surface and in the secretory products of the parasites and are the target of intense research efforts.

8. Leishmania/HIV Co-infections

Epidemiology of VL is further changing due to widespread migration of population and emerging HIV/VL co-infection which is emerging as an extremely serious problem. The risk of VL among AIDS patient’s increases by 100 to 1000 times in endemic areas as well as VL accelerates the onset of AIDS in HIV infected people. To date, it has been reported from 31 countries, with most of the cases from Southern Europe, where 25–70% of adult patients with VL are co-infected with HIV. The first case of VL/HIV co-infection in India was identified from the State of Bihar in the year 2000. Since AIDS epidemic is looming large on the horizon of new millennium in India, the State of Bihar needs to be looked seriously for VL/HIV co-infections. These co-infections impose specific difficulties in terms of diagnosis and treatment (often results in frequent failure and relapses due to drug resistance).

9. Control Strategies of the Disease:

Efficient case management based on early diagnosis and treatment is the key to limit morbidity and prevent mortality. Effective treatment of patients is also a measure to control reservoir and transmission in anthroponotic foci, particularly for cases of PKDL, which are thought to act as a long term reservoir of the disease. In addition, vector control should be implemented wherever feasible. Spraying of houses with residual insecticides has been an important measure in the past in India but is not much used now. Insecticides used in malaria control programme are effective on sand fly. In foci where sand flies bite at night, impregnated bed nets have decreased the incidence of leishmaniasis. Indian government started Leishmaniasis Elimination Programme in 2001 with the targets of prevention of death by 2004, zero level incidence by 2007, zero level prevalence by 2010, and elimination by 2012.

Based on

1. Handman, E., 2001. Leishmaniasis: current status of vaccine development. Clin Microbiol Rev 14, 229-243.

2. Palatnik-de-Sousa, C.B., 2008. Vaccines for leishmaniasis in the fore coming 25 years. Vaccine 26, 1709-1724.

Post-kala-azar dermal leishmaniasis (PKDL), an aftermath of kala-azar (KA) or visceral leishmaniasis (VL) is an unusual dermatosis with gross cutaneous lesions mainly comprising of hypopigmented macules, erythema, and nodules. The lesions persist for long periods, and complications arise when mucous membranes are affected, the most serious being blindness due to ocular involvement. The disease is relatively common in the Indian subcontinent (India, Nepal, and Bangladesh), East Africa (Sudan, Ethiopia, and Kenya), and China, where the causative agent for VL is L. donovani. However, the disease is exceptional in the American and European continents, where VL is caused by L. chagasi/infantum. This entity was first described by Brahmchari in 1922 in cured VL, confirmed by demonstration of Leishman-Donovan (LD) bodies in slit skin smear and termed as dermal leishmanoid. Later it was renamed PKDL since eruptions follow the visceral disease, commonly called KA. There are distinct features in PKDL in Sudan, and in the Indian subcontinent. In India, PKDL develops as a dermatosis in 5%–15% of treated VL patients with a usual interval of 2-3 yr but it may occur much earlier (i.e., after 6 months) or much later (up to 32 yr). In 15-20 per cent of PKDL cases no preceding history of VL is available, suggestive of subclinical infection. On the contrary, in Sudan the disease manifests in 56%–62% of VL patients, usually soon after, or sometimes even during the treatment. The interval between VL and PKDL is short, with all cases occurring 0-13 months after treatment, usually within first 6 months. About 8 per cent of cases in Sudan have no previous VL history while parallel VL and PKDL is reported in 18 % cases.

The incidence of PKDL has important implications in transmission of VL, as PKDL provides the only known reservoir of the parasite in India. Therefore, it is necessary to identify and cure PKDL for control of KA. However, the diagnosis of PKDL remains a challenge and the disease is often confused with leprosy. Diagnosis of PKDL is often based on previous history of kala-azar, origin from an endemic area, histopathology and finally response to anti-leishmanial therapy. The histopathology of PKDL varies according to the type of lesion taken for study. It is a diffuse infiltrate composed of lymphocytes, macrophages and plasma cells in the entire dermis in biopsies from nodules and around the pervascular area in biopsies from macules. LD bodies are demonstrable in around 50%, at times numerous, failing which a patient search is required. Slit-skin smears from indurated lesions are better for demonstration of LD bodies than histopathology. Other better methods for demonstration of LD bodies include immuno histochemical stains and electron microscopy which may not be accessible to all. PCR is the most sensitive and specific diagnostic method for PKDL.

So far, little is known about the factors of parasite/host origin that drive the parasite to cause a shift in the site of infection from viscera to dermis and thereby the clinical manifestation of the disease. It is not known whether the parasite in PKDL lesions is the residual parasite after VL infection or is introduced upon re-infection by sand fly vector. Reactivation of the persistent infection is considered to underlie PKDL pathology. Genes showing up regulation in PKDL like major surface proteins gp63, PSA-2 and Amastin, may be the factors that contribute to persistence after VL and play a role in altered clinical manifestation in PKDL. A role of cytokines IFN-g, TNF-a and IL-6 is implicated in distinct PKDL pathogenesis. Interference with type 1 effector activity in PKDL may be due to minimal expression of IFN-gReceptor1 or simultaneous presences of elevated levels of IL-10, IL-6 and TGF-b that have counter acting activities. Upon treatment, the restoration of IFN-gR1, coupled with down regulation of counter acting cytokines, facilitates the action of signals associated with IFN-g, yielding parasite clearance. Higher expression of chemokines (MCP-1, MIP-1a, MIP-1b) in PKDL indicates a role of these molecules in parasite perseverance by host cell recruitment at the site of infection. Distinct Immune profile is observed in Sudanese PKDL patients where IL-10, IFNg and IL-4 present in majority of tissue lesions, while in Indian PKDL here is preponderance of IFNg, TNFa, IL-10, TGFb, IL-6 and IL-4 in lesion tissues. In Sudan VL cases with high level of IL-10 are prone to develop PKDL, while in India there is negligible IL-10 at post treatment stage in VL cases. Further, Sudanese VL patients with high C-Reactive protein levels (>40µg/ml) are prone to PKDL development but no such association is observed in Indian cases.

Intriguingly, treatment of PKDL requires sodium antimony gluconate (SAG) therapy for a duration exceeding 4 times that required for treatment of KA, at the same dose. The chequered role of antimony in medicine has reached a climax in the leishmaniases, lending credence to the pithy remark made almost 2 decades ago that apart from antimonials no other heavy metal treatment in any disorder has enjoyed such a reputation and remained unchanged over decades. KA is the severest form in which pentavalent antimonials commendably scaled down the mortality to as low as 5% from that of 95% in the pre-antimony era. Being poorly absorbed orally, the mode of administration of antimony is parenteral. In contrast to the trivalent form, pentavalent compounds are less toxic, high doses can be given as they are quickly excreted, and the sodium salt is better tolerated than the potassium one. Acquired resistance and relapse led experts to recommend 20mg/kg/day to a maximum of 850mg , 8.5 ml (10mg/ml) of sodium stibogluconate (SSG) or 10 ml (85 mg/ml) of meglumine antimoniate, in KA for a minimum of 20 days, which could extend up to 40 days if required. Common side-effects were stated to be anorexia, vomiting, nausea, malaise, myalgia, headache and lethargy, followed uncommonly by electrocardiographic changes, and rarely renal damage. A subsequent review into toxicity and efficacy of SSG in various studies recommended that the dose of 20mg/kg/day for 28 days could be routinely used without an upper limit, with a provision to extend if required.

Summing up the use of SSG during latter half of previous century in highly endemic areas of India, it was seen that efficacy had diminished over years as treatment in some had to be prolonged to achieve cure, while some did not respond indicating that resistance had developed probably due to exposure to suboptimal doses in the past. More importantly, ECG changes were seen in a significant number of patients, some of whom died of cardiac causes. Reviewing current situation and regional variations in the responsiveness of KA in India and endemic areas of Africa it was concluded that antimonials could be continued parenterally in a dose of 20mg/kg daily for 30 days without an upper limit with a provision to use amphotericin B in areas showing refractoriness. The generic formulation like the one widely used in India (Albert David, Kolkata) is comparable in efficacy to the costlier brand (Pentostam, Glaxo-Wellcome, UK), but one had to be wary of other companies owing to high incidence of cardiotoxicity. Cardiac side effects can well be mitigated by excluding preexisting systemic disease and avoiding doses higher than 20mg/kg/day.

This regimen of SSG for 4 months administered intramuscularly is tolerable in PKDL, with mild ECG changes and raised plasma urate with arthralgia ocassionaly, none of which precluded completion of therapy. In some cases the duration needs to be extended to 200 days to combat refractoriness and relapse, and also to ensure subsidence in those with extensive lesions. ECG abnormalities revert to normal on stopping the drug, and arthralgia, pain and swelling at site of injection are managed with analgesics and brief discontinuation of therapy; neuritic symptoms and change in taste were occasionally encountered. Between 1997 till 2007 a total of 126 cases of PKDL, 112 adults and 14 children have been treated at our centre in Delhi, mainly as outpatients with this regimen. The intravenous route was preferred to the less tolerated intramuscular one that prevented them from work. Twenty of these did not return after the first visit and six had been given ketoconazole as SSG was not available at that time. Of the remaining 100 all of whom showed signs of regression by the third week, only 20 managed to complete the recommended 120 days’ therapy within 5 months. Some took between 40 to 60 injections and remained irregular or hesitant to continue. Most were unable to take beyond 4 to 6 weeks and nearly all children refused to go any further. Compromises had to be devised to facilitate treatment. The injections were made biweekly, at times 10mg/kg/day (5ml) to ensure compliance, and daily allopurinol or rifampicin was added to the regimen. It became evident however that reducing the frequency of SSG markedly delayed the clinical response, even when used with other drugs. When given daily intravenously mainly as inpatients in the dose of 10mg/kg/day along with rifampicin, results have been good. The irregularity in therapy resulted from body aches, giddiness, metallic taste, loss of appetite, thrombosed veins and severe joint pains. Few of them experienced vomiting, febrile episodes and one had postural hypotension. None had cardiac abnormalities and 2 of those who completed therapy had mildly elevated liver enzymes. All these patients were engaged in physical work for livelihood and found treatment worse than disease. In addition they had to buy disposable syringes, the drug too when we were unable to provide, face considerable difficulty in accessing new veins when previous sites became thrombosed and also a suitable medical person to administer the injection. Cyclical intramuscular SSG for 20 days alternating with 20-day drug-free intervals ranging from 6 to 10 cycles has shown poor response indicating the pressing need for a good alternative. This has led some to prefer the much longer oral regimen with allopurinol instead of SSG in those with less extensive lesions and children. Experience with African PKDL which requires 2 to 4 months of treatment with SSG has been similar and shorter but expensive regimens have been explored.

When treatment rallies around a month like in KA it is tolerable, but if prolonged, rheumatic side-effects like myalgia and joint pains dominate, which make irregular therapy inevitable. Studies have surprisingly commented little on the relative lack of cardiac side-effects in PKDL, despite the long duration of therapy with SSG as compared to KA. It may relate to systemic disease and general condition of the patient in KA. Pancreatitis, mainly subclinical and unusually rare in those with KA or PKDL, has occurred during SSG therapy for cutaneous leishmaniasis as evidenced by increased serum amylase or lipase levels associated with signs or symptoms of abdominal discomfort but patients could complete treatment. A recent study inferred that both hyperamylasemia and raised liver transaminases were clinically insignificant and not deterrents to treatment. Fatalities due to pancreatitis have occurred in those co-infected with HIV. Apart from drug resistance, the varying clinical response and toxicity of SSG may be related to a bimodal type of drug clearance in the patient accounting for rapid and slow eliminators. Higher amount of antimony than that specified by the manufacturers or traces of the more toxic trivalent compound in the preparation could also account for toxicity.

Reducing the volume injected by increasing concentration of antimony could minimize local side-effects. Unfortunately at levels above 100mg/ml SSG tends to supersaturate forming crystals and precipitates. Alternatively, duration of therapy could be shortened by immunotherapy in addition to the recommended dose of SSG and more studies are required in this direction. Liposome entrapped Pentostam achieved better results with low doses in experimentally infected mice with L donovani indicating enhanced drug delivery, but this was not further perfected and popularized for wider use. Pending a good multi-drug formulation, an acceptable therapy for PKDL is required. Since the duration is likely to be longer than in KA and the patients are in otherwise good health without the need for hospitalization, oral therapy is preferable to ensure compliance. Currently miltefosine, the only drug to fulfill this requirement, is beset with high cost of therapy and is not freely available as yet. Cure of antimony unresponsive Indian PKDL has been obtained with oral miltefosine at a dose of 50mg twice daily for 3 months or at 50mg thrice daily for 2 months. l miltefosine treatment is also successful in PKDL with HIV co-infection. However results of Miltefosine trials in PKDL are awaited to find out the optimum dose and duration. Amphotericin B, both in the liposomal and non-liposomal forms has been well tried in KA, but in PKDL more studies are required to recommend the adequate dose and duration of therapy. Till then antimonial appears to be the Hobson’s choice with amphotericin B in reserve for refractory or antimony-resistant patients of PKDL.

1. Introduction

Leishmania are protozoan parasite(s) that causes a wide spectrum of clinical manisfestation in human(s) collectively referred to as leishmaniasis ranging from self healing cutaneous (CL), mucocutaneous (MCL) skin ulcers to life threatening visceral diseases. (VL or kala azar)(Murray et al., 2005¹). Leishmaniasis is a devastating disease that affects about two million people each year and threatens one fifth of the world’s population, new treatments are desperately needed (Murray et al., 2005).

The Leishmania parasites are transmitted by an invertebrate sandfly vector, Phlebotomus and exist in two major developmental stages. Infected sandflies introduces the metacyclic forms of the promastigote stage into the bloodstream of the vertebrate host. The promastigote differentiates into amastigote stage that is adapted for survival in the phagolysosome of the macrophages, the cell responsible for pathogen elimination. During promastigote to amastigote differentiation, the parasites are subjected to drastic environmental changes, including sharp rise in temperature, a drop in extracellular pH, an increased exposure to oxygen and nitrogen reactive species, an extracellular proteolytic activity and nutritional starvation (Barak et al.,2005). The association of Leishmaniasis with HIV has also been reported from 33 countries, where up to 70% of potentially fatal visceral leishmaniasis (VL) cases are

associated with HIV infection, and up to 9% of AIDS cases suffer from newly acquired or reactivated VL. No effective vaccines are available against Leishmania infections as yet (Handman, 2001) and treatment relies solely on chemotherapy with pentavalent antimonials as first-line drugs and amphotericin B and pentamidine as second-line drug (Murray, 2000). Resistance to antimony is now observed in several parts of the world most notably in the state of Bihar, India, where more than 50% of the patients are unresponsive to treatment, or relapse after conventional chemotherapy (Sundar et al., 2000). Ketoconazole, allopurinol, and miltefosine are been used as drug against Leishmania parasite as the biochemical pathways such as the ergosterol biosynthetic pathway, the exquisite requirements for purine salvage and high levels of ether lipids differs from its mammalian host. With the exception of miltefosine, these drugs are not effective against visceral leishmaniasis. There are reports of development of resistance against miltefosine in Leishmania (Seifert et al., 2003).

The route to drug target identification has been through comparative biochemistry of host and parasite enzymes, metabolites or protein identified in parasite. Biochemical analysis, genome sequencing of three Leishmania species (L. major, L. braziliensis, L. infantum) have identified potentially useful target enzymes, transporters, metabolites, hypothetical proteins that are distinct to parasite and their mammalian host.

2. Comparision of three Leishmania genome

Genome sequences of three species of Leishmania (L. major, L. infantum and L. braziliensis) are now available at gene database (http://www.genedb.org). The chromosomes of Leishmania differ from those of the trypanosome species in not having extended telomeric regions containing species specific genes (Peacock et al., 2007). The complete genome of L. major, L. infantum and L. braziliensis has provided many new potential targets to be used in conjunction with comparison and functional genomic studies. Such comparative genomic study will allow us to identify the molecules or biochemical pathways that have been successfully targeted in other pathogens. The genome mining will also aid in large scale proteomics studies generating expression profiles of Leishmania parasites and gene targets for treatment development.

Comparison of L. braziliensis and L. infantum revealed marked conservation of synteny and identified only ~200 genes with a differential distribution between the three species. L. braziliensis, contrary to Leishmania species possesses components of putative RNA mediated interference (RNAi) pathway, telomere associated transposable elements and spliced leader associated (SLACS) retrotransposons. Differentially distributed genes between the species encodes protein for parasite survival in the macrophages and pertaining to host parasite interaction (Peacock et al., 2007). The genome sequence have given us a shortcut to a small number of largely novel genes ‘‘Given their lack of similarity to human genes, they present a limited repertoire of potential targets for drugs and vaccine development allowing researchers to optimize the use of limited resources”. Cyclopropane fatty acid synthase (CFAS) an enzyme that may be involved in producing components of the cell membrane, is present in the genome of L. braziliensis and L. infantum, but is absent in the human genome. CFAS is also involved in virulence and persistence in Mycobacterium, causative agent of tuberculosis, so the identification of this potential target CFAS gene in Leishmania raises the exciting possibility that some virulence factor are conserved between bacterial and eukaryotic intracellular pathogens (Peacock et al., 2007). Biological studies for the function of 50% of Leishmania genes are lacking. The comparitive genome study would provide a route to find those that might be essential to each species (Rochette et al., 2008)

3. Thiol Metabolism

The enzymes of thiol metabolism and in some cases the thiols themselves, of parasitic protozoa differ in many interesting ways from those of mammals. Trypanosoma and Leishmania are most remarkable in that they have trypanothione reductase (TR) instead of glutathione reductase (GR). This enzyme is responsible for maintaining the parasites, reducing intracellular milieu by keeping trypanothione [N¹, N8-bis-(glutathionyl) spermidine] in the dithiol state. The crucial role of TR for thiol homeostasis and its absence from mammalian cells suggest that it might be well suited as a target molecule for rational drug development. The trypanothione system, which replaces the nearly ubiquitous glutathione/glutathione reductase (GR) system, protects the parasites from oxidative damage and toxic heavy metals, and delivers the reducing equivalents for DNA synthesis. The parasite system is far less efficient than mammalian glutathione peroxidases in detoxifying hydroperoxide, but has the advantage of much broader substrate specificity, with lipid hydroperoxides also being reduced. The relatively low activity of the tryparedoxin peroxidase system is in accordance with the high sensitivity of the parasites to oxidative stress. Trypanosomes and Leishmania have superoxide dismutase (SOD), but lack catalase and glutathione peroxidase. Thus, the trypanothione system seems to be the only mechanism to detoxify hydrogen peroxides.

3a.Trypanothione Reductase

Trypanothione is kept reduced by the flavoenzyme TR. Several reverse genetic approaches have undoubtedly shown that TR is essential in different Leishmania species as well as in bloodstream of T. brucei (Krieger, 2000) and is thus an attractive target molecule for structure-based drug design. Within the past 15 years, numerous compounds have been elucidated that inhibit TR, but not human GR, which is the closest related host enzyme. Despite knowledge of the three-dimensional structure of the protein and of complexes with its substrates and an inhibitor, as well as several high-throughput and virtual screening approaches, inhibitors of TR that are suitable to enter the clinical phase are still elusive. This lack of success might be attributable to several factors. The extremely wide active site of the parasite enzyme represents an obstacle for a structure-based drug design. In addition, the pharmacokinetic properties of the potential inhibitors are crucial because of insufficient uptake, rapid extrusion or metabolism play significant roles in determining the in vivo efficacy of a drug. Another important point is the in vivo half-life of a target enzyme, and this has not been determined for TR in any of the trypanosomatid species. Thus, it is still not clear if reversible, irreversible or turncoat inhibitors are likely to be the most promising candidates.

3b.Thioredoxin reductase

Thioredoxin reductase (TrxR) is a pyridine nucleotide-disulphide oxidoreductase as are GR, TR, and lipoamide dehydrogenase. TrxR maintains the levels of reduced thioredoxin, a protein involved in the activity of ribonucleotide reductase, transcription factors and cell signaling, and the detoxification of reactive oxygen species. Most studies to date on TrxR of parasitic protozoa have concerned the enzyme of P. falciparum. Current evidence suggests that it is a promising drug target, although validation is awaited. Interestingly, P. falciparum TrxR differs from the human enzyme in not only containing selenocysteine, but having a C-terminal cysteine pair separated by four amino acids. The unusual nature of the C-terminal domain prompts thoughts on whether it has a distinct role in the parasite e.g., as a thiol that acts as a general reductive reagent in the cell and so a special adaptation of the parasite for counteracting oxidative stress) and how it interacts with the parasite’s thioredoxin (which is presumed, but yet to be discovered (Krauth-Siegel and Coombs, 1999).

4. Folate Metabolism

A biochemical pathway that has been exploited in the past for the treatment of infectious disease is the folic acid biosynthetic pathway. Inhibitors of folate metabolism are known to be important for malaria, bacteria and cancer chemotherapy. Perturbation in the folate pathway analog which inhibits the mTHF recycling pathway rapidly leads to nucleotide imbalance and thus causing cell death. Leishmania cannot synthesize folate de novo (e.g. - folate and pterin) and must import these metabolites from an exogenous source (Ouellette et al., 2002). A novel class of transport membrane proteins is responsible for their uptake (Richard et al., 2002; Richard et al., 2004; Cunningham et al 2001). Enzymes of the total Leishmania folate pathway have been studied and these include the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) and the folyl polyglutamate synthetase (El Fadili et al., 2002; El fadili et al., 2003). The genome sequencing of Leishmania species (Ivens et al., 2005), http:/www.genedb.org and their analysis have highlighted the presence of several proteins implicated in folate metabolism (Ouellette et al., 2002). L.major genome sequence showed the presence of two isoforms of SHMT gene cytosolic and mitochondrial suggesting the folate metabolism of Leishmania to be compartmentalized (Gagnon et al., 2006). For better understanding of the properties of SHMT, and complexities of folate metabolic pathway of Leishmania the enzyme has been overexpressed and characterized in L. donovani (Vatsyayan and Roy, 2007). The folate pathway provided a valuable target for microorganisms such as bacteria and plasmodium for drug intervention. However till date no drug targetting the folate pathway have been found to be effective against Leishmania infection. There seems to be two reasons for this, first Leishmania cannot synthesize folate (biopterin and folate) de novo and must import these metabolites from exogenous source (Ouellette et al., 2002). Secondly the enzymatic reduction of folate to become active as tetrahydrofolate (THF), a coenzyme required for one carbon (C1)transfer reactions, can be catalyzed by both DHFR-TS and pteridine reductase1(PTR1). When DHFR-TS is inhibited PTR1 is overexpressed, hence it is necessary to block both DHFR-TS and PTR1 for effective interference with folate metabolism (Opperdoes and Coombs, 2007). Several genes that encode enzymes of folate biosynthesis are not in the Leishmania genome but twelve genes that encode a novel class of membrane transport protein responsible for folate transport has been reported. Putative SHMT inhibitors, including thiosemi-carbazide, have poor activity against Leishmania but further work may lead to more potent inhibitors. The development of antifolate drugs for leishmaniasis treatment requires further studies of key enzymes of this pathway

5. Glycolytic Pathway

In all kinetoplastida studied so far the majority of the glycolytic enzymes are localized in organelles called glycosomes, whereas in other organisms these are cytosolic. In blood stream form of T. brucei glycolysis is the main source of energy as they lack functional Krebs cycle while in Leishmania and T. cruzi the glycolytic pathway is less important but because their glycosome contain important anabolic and anapleuratic pathways that are interdependently linked to glycolysis, as any compound designed to act against African trypanosomes may also be effective against these parasites. Due to this compartmentation, many regulatory mechanisms operating in other cell types cannot work in trypanosomes. This is reflected by the insensitivity of the glycosomal hexokinase (HK) and phosphofructokinase (PFK) to compounds that act as activity regulators in other cell types (Bakker et al., 2000; Michels et al., 2000). Blocking of Parasite enzyme without producing damage to glycolysis in host remains challenging. Several approaches can be considered- 1) Exploitation of metabolic differences 2) Exploitation of differences in 3 D structure 3) Exploitation of unique reactive residue in or near the active site of the parasite enzyme

5a.Hexokinase

Hexokinase (HK) catalyzes the conversion of glucose to glucose 6-phosphate. The sequence of hexokinase from L. major was found to encode an enzyme with a molecular mass of 51.74 kDa. The L. major genome was found to have two copies of hexokinase coding sequences in tandem with an intergenic spacer of 2.58 kb. The HK gene was transcribed in large amounts in the promastigote stage, whereas there is only weak expression in the amastigote stage as determined by RT-PCR analysis (Umashanker et al., 2005). HK was also purified from L. mexicana from glycosome of promastigotes. The specific activity increased with the ageing of promastigote culture (Pabon et al., 2007)

5b.Glucose 6 phosphate isomerase

Glucose-6-phosphate isomerase (PGI) is an intracellular enzyme that catalyzes the reversible conversion of D-glucose 6-phosphate (G6P) to D-fructose 6-phosphate (F6P). The native Leishmania PGI is a homodimeric molecule of 60 kDa per monomer with 47% sequence identity to human PGI (Cordeiro et al., 2004). Crystal structure has been reported for L. mexicana and a significant difference was detected in the position of the small domain that presents a more open conformation in the enzyme of the parasite. As a result, a larger active-site cavity is created, providing a possible explanation for the different values of kinetic parameters, as measured for the human and parasite enzymes. This larger cavity may be used in future rational drug design strategies, to develop selective inhibitors of the parasite enzyme which are too large to be accommodated by the catalytic site of the mammalian PGI ( Cordeiro et al., 2004).

5c.Phosphofructokinase

Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1, 6-bisphosphate (FBP) in an essentially irreversible reaction. The gene of PFK has been cloned and characterized from L. donovani and T. brucei. L. donovani has a single PFK gene copy per haploid genome that encodes a polypeptide with a deduced molecular mass of 53.988 kDa while human enzyme has subunit of 85 kDa .The predicted amino acid sequence contains a C-terminal tripeptide that confirms to an established signal for glycosome targeting. L. donovani PFK showed most sequence similarity to inorganic pyrophosphate (PP_i)-dependent PFKs, despite being ATP-dependent. It thereby resembles PFKs from other Kinetoplastida such as T.brucei, T.borreli (Lopez et al., 2002). Furanose sugar amino amides as a novel class of inhibitors for both enzymes with IC₅₀ values of 23 μM against T. brucei PFK. The residue Lys 277 involved in specific inactivation of parasite PFK was identified by site directed mutagenesis. Based on promising results, other compounds could be developed with more reactive electrophilic centre directed towards this residue.

5d.Triose Phosphate Isomerase

Triose Phosphate Isomerase (TIM) is an important enzyme of glycolytic pathway which interconverts glyceraldehyde 3- phosphate to dihydroxyacetone phosphate. The TIM gene from L. donovani was cloned, over expressed, analyzed and submitted to data bank (Kumar and Roy, 2006 Accession no- DQ649411). Homology search showed 88.10, 66.67, and 49.2 % identity with L. mexicana, T.cruzi, and Human respectively. In L. donovani Glutamate was found at position 66 instead of Glutamine. The dimer of TIM is quite stable and is the active form of the protein (Knowles and Albery, 1977). In fact, there are several reports that suggest that mutations at the subunit interface of the protein destabilize the dimer leading to either complete inactivation or drastic decrease in the activity of the enzyme. The knock out of this enzyme in T. brucei established that TIM is also a vital enzyme as it would lead to complete suppression of growth arrest (Sandra Helfert and Christine Clayton, University of Heidelberg). Growth inhibition has been observed after transfection of bloodstream-form T. brucei with the gene of S. cerevisiae TIM expressed as a cytosolic enzyme which indicates that mislocalization of TIM is detrimental for trypanosomes. In the case of TIMs from parasites such as T. brucei, T. cruzi, P. falciparum, and L. mexicana, a cysteine residue is present at the position 14 (Gomez-puyou et al., 1995) which has been target in various studies, interestingly mammals have methionine at this position. One major substitution was detected at the interface region of L. mexicana protein a glutamate is found at position 66 instead of glutamine in all other available species (Kohl et al., 1994). The glutamine is thought to be important for the stability of dimeric enzyme.

5e.Glyceraldehyde 3- phosphate Dehydrogenase

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde 3-phosphate to D-glycerate 1, 3-bisphosphate. This enzyme is homotetrameric and it appears that the active site of the enzyme and the neighboring nicotinamide binding site for NAD⁺ are well conserved (Verlinde et al., 2001). However, the binding site for the adenosine portion of NAD⁺ exhibits significant differences between parasite enzyme and host. This difference can be exploited for designing selective drugs.

6. Polyamine Pathway

The polyamine pathway of protozoan parasites has been successfully targeted in anti parasitic therapies. Polyamines are ubiquitous organic cations found in virtually every eukaryotic cell and plays critical role in key cellular processes such as growth, differentiation and macromolecular biosynthesis. All the gene of polyamine pathway i.e ornithine decarboxylase (ODC), S-adenosylmethionione decarboxylases (AdoMetDC) and spermidine synthase (SPDS) has been cloned and their knockouts by gene replacement has demonstrated the essential role of each of these enzymes in L. donovani (Roberts et al., 2001; Roberts et al., 2002) unless they can access sufficient amount of exogenous putrescine and spermidine but the parasites exhibit negligible uptake capacity (Hanson at al., 2005). However polyamine transport itself has been characterized at biochemical level in various protozoa (Basselin, 2000). The inhibition of any of the polyamine the parasite cannot synthesize trypanothione, a conjugate of spermidine and glutathione that is unique to Trypanosoma and Leishmania. Trypanothione is a reducing agent with many protective and regulatory functions and consequently its depletion is detrimental to the parasites. Recent studies on polyamine supplementation shows that L. donovani lacks an intact back conversion pathway thus the pathways operating in promastigotes stage of parasite differ crucially from that in the host.

Enzymes involved in polyamine pathway of Leishmania are:

6a.Arginase

Ornithine, the first amino acid from which polyamines are generated is produced from arginine by arginase enzyme. An arginase activity has been detected in L. mexicana and L. amazonansis, while T. cruzi and T. brucei lacks arginase activity. Arginase provides a building block for production of polyamines so it has been touted as a potential antileishmanial drug target, because N(omega)-hydroxyarginine, an inhibitor of arginase that is produced by the macrophages during the formation of nitric oxide, can reduce polyamine levels in Leishmania amastigotes and lowers parasitic loads (Iniesta et al., 2001). The lethal nature of arginase knockouts establishes that Leishmania promastigotes have only a single avenue for ornithine biosynthesis (Roberts et al., 2004). The arginase gene from L. donovani has been cloned, analyzed and submitted to data bank (Roy et al., 2006 Accession no-DQ649412). Complete ORF codes for 330 amino acids with GC content of 60.3%. Homology search of L. donovani with L. mexicana and Human showed 99%, and 42 % identity respectively.

6b.Ornithine decarboxylases

Ornithine decarboxylase is the first enzyme of polyamine pathway, catalyzes ornithine to putrescine. It is also validated as drug target because of major differences between parasite ODC and Human ODC. The parasite ODC is quite stable (half life > 6 hr in T. brucei and > 20 hr in L. donovani) as compared to human ODC ( half life < 1 hr). ODC knockouts were incapable of growth in polyamine deficient medium. The DFMO and 3-aminoxy-1-aminopropane (APA) are the strong inhibitors of ODC. In recent studies it was observed that L. donovani ODC overexpression exhibited significant resistant to sodium stibogluconate (Singh et al., 2007).

6c.S-adenosylmethionione decarboxylases

S-adenosylmethionione decarboxylases (AdoMetDC) generate decarboxylated S-adenosylmethionione (dcAdoMet) which serves as the aminopropyl group donor for spermidine and spermine. The latter is absent from Leishmania. A very potent and selective irreversible inhibitor of AdoMetDC is 5{[(2)4amino-2-butenyl] methylamine}5’ deoxyadenosine (MDC 73811;AbeAdo). AbeAdo was shown to inhibit L. donovani promastigotes with an EC₅₀ value of 40 µM (Roberts et al., 2002). Depletion of putrescine causes marked accumulation of AdoMet and DCAdoMet that may lead to abberant methylation in parasites.

6d.Spermidine synthase

Spermidine biosynthesis is catalyzed by spermidine synthase which transfers an aminopropyl moiety from decarboxylated S-adenosylmethionione to putrescine. The L. donovani spermidine synthase is present as a single copy gene in genome exhibiting 56% identity with human. Two sequences encoding the spermidine metabolizing enzymes deoxyhypusine synthase and homospermidine synthase have been isolated from L. major and published in gene bank. The product of these two genes are excellent drug targets (Akopynts et al., 2001). S-adenosyl-1, 8-diamino-3-thiooctane (AdoDATO) is potent inhibitor of T. brucei spermidine synthase.

7. Sterol Biosynthetic Pathway

Isoprenoid compounds are ubiquitous in prokaryotic and eukaryotic cells with the sterols usually the most abundant isoprenoid group present in eukaryotes. Sterols perform a structural function as constituents of cellular membranes and this has been referred to as their ‘bulk membrane’ role. The most extensively examined parasitic protozoa as far as sterols are concerned are the trypanosomatids. It has emerged that these parasites have close similarities to fungi in relation to their sterol composition and sterol biosynthesis. This has offered an opportunity for the development of chemotherapy by targeting the sterol biosynthetic pathway using the types of drugs already successfully employed against fungal pathogens.

The enzymes of this pathway are attractive targets for the specific treatment of leishmaniasis, because the etiological agents for the disease that is the leishmanial parasites have a strict requirement for specific endogenous sterols (ergosterol & analogs) for survival and growth and cannot use the abundant supply of cholesterol present in their mammalian host. There are differences in the enzymes in the biosynthetic pathways of ergo sterol and cholesterol. A number of enzymes in the ergosterol biosynthetic pathway have been investigated as potential drug targets for these organisms and have shown great promise. Thus, C14a-demethylase,sterol 24- methyltransferase,3-hydroxy-3-methylglutaryl CoA reductase, squalene epoxide, squalene synthase and farnesyl pyrophosphate synthase have been studied both individually and in combination, with varying degrees of success (Lorente et al., 2005). Ergosterol biosynthesis inhibitors with potent in vitro activity and special pharmacokinetic properties in mammals can induce radical parasitological cure in animal models of several forms of leishmaniasis (Urbina, 2002).

7a.S-adenosyl-L-methionine: Delta (24 (25))-sterol methenyltransferase

Trypanosomatids contain predominantly ergostane-based sterols, which differ from cholesterol, the main sterol in mammalian cells, in the presence of a methyl group in the 24 position. The methylation is initiated by S-adenosyl-L-methionine: Delta (24 (25))-sterol methenyltransferase, an enzyme present in protozoa, but absent in mammals. The importance of this enzyme is underscored by its potential as a drug target in the treatment of the leishmaniasis (Carmen Jiménez-Jiménez, 2008). The C-24 transmethylation reactions involving S-adenosyl methionine as the methyl donor and a Δ²⁴⁽²⁵⁾-sterol or Δ^24(24′)-sterol substrate can be inhibited by various azasterols with a nitrogen substitution in the side chain and such compounds have been tested against trypanosomatids (Roberts, 2003).

7b.Sterol C14 alpha-demethylase

Recent work with the sterol C14 alpha-demethylase inhibitor D0870, a bis triazole derivative, showed that this compound is capable of inducing radical parasitological cure in murine models of both acute and chronic Chagas' disease. Other inhibitors of this type, such as SCH 56592, have also shown curative, rather than suppressive, activity against T. cruzi in these models. Leishmania species have different susceptibilities to sterol biosynthesis inhibitors, both in vitro and in vivo. L. braziliensis promastigotes, naturally resistant to C14 alpha-demethylase inhibitors such as ketoconazole and D0870, were susceptible to these drugs when used in combination with the squalene epoxidase inhibitor terbinafine (Urbina, 1997).

7c.3-hydroxy-3-methylglutaryl CoA Reductase

In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. This protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. In Leishmania HMG-CoA reductase is up-regulated when sterol synthesis is inhibited by drug pressure and this activation is apparently performed via post-transcriptional control (Montalvetti et al., 2000). The lack of sensitivity to mevalonate and sterols is consistent with the absence of a membrane domain and may be a consequence of unique biological properties of the isoprenoid biosynthetic pathway in protozoa. Trypanosomatids are early branching eukaryotic cells and their cell organization differs considerably from that of mammalian cells ( Peña-Díaz et al., 1997) Specific features present in trypanosomatids but absent from their hosts may be exploitable in providing targets for rational drug design .

7d.Squalene Synthase

Squalene synthase catalyzes the first committed step in sterol biosynthesis and is currently under intense study as a possible target for cholesterol-lowering agents in humans, but it has not been investigated as a target for anti-parasitic chemotherapy. Growth inhibition and cell lyses induced by Hydroxy biphenylquinuclidines (BPQ-OH) an inhibitor in both parasites (L.mexicana and T.cruzi) was associated with complete depletion of endogenous squalene and sterols, consistent with a blockade of de novo sterol synthesis at the level of squalene synthase. Ultra structural analysis of the treated parasites revealed several changes in the morphology of promastigote forms. The main ultra structural change was found in the plasma membrane, which showed signs of disorganization, with the concomitant formation of elaborated structures. Alterations in the mitochondrion-kinetoplast complex such as mitochondrial swelling, rupture of its internal membrane and an abnormal compaction of the kinetoplast were also observed. Other alterations included the appearance of multivesicular bodies, myelin-like figures, alterations of the flagellar membrane and presence of parasites with two or more nuclei and kinetoplasts ( Rodrigues et al., 2005). The squalene synthase gene from L. donovani was cloned, analyzed and submitted to data bank (Bhargava and Roy, 2006 Accession no- AM229310). The 1245 bp confirmed clone contained an open reading frame of 415 amino acids giving a predicted mass of 47.35 KDa. Comparision of the LdSSN deduced amino acid sequence with SQS from different species showed the highest identity with Leishmania major (91%), followed by T.cruzi (57%), T.bruzi (48%), Mus musculus (45%) and human (44%). The two signature sequences of squalene synthases were present at position 164-179 and at 200-227. The secondary structure prediction showed that it consists of 40.10% of alpha helix and the GC content is 59%.

7e.Farnesyl Pyrophosphate Synthase

The sensitivity of trypanosomatid protozoa to isoprenoid biosynthesis inhibitors (Docampo et al., 2001) offers a unique opportunity for drugtarget identification and the subsequent development of new anti-trypanosomatidagents. Farnesyl pyrophosphate synthase (FPPS) plays a central role in metabolism through the enzymaticgeneration of FPP, which is used for protein prenylation, for the synthesis of sterols, dolichol, heme a, and ubiquinone,and is potently inhibited by bisphosphonates. Stringentgenetic validation of putative drug targets is desirable beforethe rational design of inhibitory compounds intended for chemotherapeuticuse is undertaken. Studies validate FPPS as a drugtarget through the use of RNAi. It provides genetic evidencethat FPPS plays an essential cellular role in T. brucei and demonstratesthat the enzyme is vital for parasite survival in vitro and invivo. The finding that a similar pharmacophore can be obtainedby structure-activity investigations of in vitro growth and enzymeinhibition data further validates T.brucei FPPS as the target ofbisphosphonates ( Montalvetti et al., 2003)

8. Glyoxalase Pathway

The glyoxalase pathway is the main catabolic pathway of methylglyoxal, a toxic 2- oxoaldehyde which occurs in all living cells as a by product of glycolysis through reaction catalyzed by triose phosphate isomerase. It first reacts non enzymatically with one or both thiol of trypanothione forming hemiacetal. These hemiacetals are the substrates for glyoxylase pathway forming D- lactate as final product (Silva et al., 2005). In L. infantum it has been shown that enhancement of methylglyoxal or depletion of trypanothione leads to significant increase in the concentration of this toxic compound hence this data might be useful for research drug targeting this disease (Lages et al., 2007)

9. Topoisomerase

Topoisomerases are enzymes that use DNA strand scission, manipulation and rejoining activities to directly modulate DNA topology. These actions provide a powerful means to effect changes in DNA super coiling levels and allow some topoisomerases to both unknot and decatenate chromosomes. They are involved in replication, transcription, chromosomal condensation and segregation and many other vital cellular processes. DNA topoisomerases are the primary targets of many antitumour drugs. DNA topoisomearases are the key enzymes involved in carrying out high precision DNA transactions inside the cells. However, they are detrimental to the cell when a wide variety of topoisomerase-targeted drugs generate cytotoxic lesions by trapping the enzymes in covalent complexes on the DNA (Majumdar et al., 2006). Many antiparasitic compounds have been found to act via topoisomerases having more profound effect on the parasite protein than the host. The identification of DNA topoisomerases as a promising drug target is based on the clinical success of camptothecin derivatives as anticancer agents. The recent detection of substantial differences between trypanosome and Leishmania DNA topoisomerase IB with respect to their homologues in mammals has provided a new lead in the study of the structural determinants that can be effectively targeted (Reguera et al., 2008).

10. Protein Kinase

Protein kinases (PKs) are important regulators of many different cellular processes such as transcriptional control, cell cycle progression and differentiation, and have drawn much attention as potential drug targets to treat a wide range of diseases and syndromes, such as cancer, cardiovascular disease and Alzheimer's disease. The majority of the eukaryotic PKs reside in clusters of orthologous groups, showing synteny within the three genomes of Leishmania, however, each species also contains distinctive protein kinases. The protein kinase complement of the trypanosomatids is about 33% larger than S. cerevisiae, but twice that of the malaria parasite P. falciparum (Ward et al., 2004)

10a.Cyclin-dependent kinases

L. major has an additional unique mitotic-like cyclin, CYCA. Some cyclin-dependent kinases (CDKs) require phosphorylation on a conserved threonine residue (T loop, T160 in human CDK1) by a cdc2-activating kinase (CAK). Many trypanosomatid cdc2-related kinases (CRKs) have a conserved T-loop residue, suggesting that the CRKs might be activated in vivo by a CAK activity (Parsons et al., 2005)

10b.Map kinase

Mitogen-activated protein (MAP) kinases are important regulators of differentiation and cell proliferation in many eukaryotes. Several reports describe the identification of MAP kinases (MAPKs) and their activators, the MAPK kinases (MAPKK), from T. brucei and Leishmania (Bengs et al., 2005) of the three T. brucei MAP type kinases described, TbECK1 is the most potential drug target. These are some of the validated drug targets of Leishmania. There are more avenues to explore and avail the yet unidentified targets from the vast resource of leishmanial genome for the betterment of human life.

one single drug or drug formulation will be effective against all forms of leishmaniasis since (a) the visceral and cutaneous sites of infections impose varying pharmacokinetic requirements on the drugs to be used and (b) there is an intrinsic variation in drug sensitivity of the 20 Leishmania species known to infect humans. In addition, there are other new problems to be surmounted by novel treatments, namely: (i) the need for drugs for treatment of VL in Bihar State, India, where there is acquired resistance to the pentavalent antimonials and (ii) the need for treatment for VL and CL in immunosuppressed paitents, in particular due to HIV co - infection, where there is exacerbation of disease or emergence from latent infection due to the depleted immune response. In the latter case standard chemotherapy is frequently unsuccessful. (Alvar et al., 2006b).

Among the new drugs discovered, miltefosine, a hexadecylphosphocholine, is the first promising oral drug which can be used against leishmaniasis. Other drugs such as paromomycin, sitamaquine, azoles and azythromycin have been reported as having variable cure rates .Consequently there is still a real need for new active compounds that can provide therapeutic benefits but with fewer side effects (Pape, 2008).

2. History

In the 19th century, devastating outbreaks of a chronic progressive febrile illness with cachexia, hepatosplenomegaly, and high fatality rates were reported in Bengal and Assam, and retrospectively thought to be the first recorded VL epidemics. In 1903, Leishman and Donovan first described the organism that now bears their names in patients infected in India. Today, South Asia is estimated to account for 60% of the global VL disease burden with a sustained endemic focus stretching from Bihar and Bengal in northeastern India, across the border into southeastern Nepal, and to the east into central and western Bangladesh. The parasite in South Asia is transmitted by Phlebotomus argentipes, an endophilic vector that rests in human and animal dwellings in densely populated agricultural villages. Kala-azar incidence fell substantially during the indoor residual insecticide spray campaigns of the malaria eradication effort of the 1950s and 1960s, but the disease returned in the 1970s and transmission has been sustained since then. Superimposed on this poorly controlled endemic picture, India has experienced recurrent epidemics in the 1970s and early 1990s, and Bangladesh has seen a progressive increase in VL incidence from the mid-1990s to the present that shows no signs of abating. In most areas, there is a fairly stable incidence of two to three kala-azar cases per 1,000 populations per year, but with localized foci of intense transmission and 10-fold higher annual incidence rates. Transmission hot spots may be sustained for several years, but then appear to burn out, limited by saturation of the susceptible population (Bern et al., 2005), increases in incidence are then seen in neighboring areas. Several years after peaks in kala-azar incidence, the same communities may see large numbers of PKDL cases, in an echo of the original kala-azar outbreak. PKDL patients remain infectious for years to decades, and require prolonged antileishmanial treatment, up to 120 days, representing a significant challenge to health care systems in which kala-azar patients experience difficulty obtaining much shorter treatment courses. Facility-based studies from South Asia often report higher kala azar incidence in males than females (Thakur, 1984). Community-based data suggest that there is little difference in incidence by sex, but substantial differences in care-seeking behaviour. In South Asia, the mean duration of kala-azar illness before treatment is 3–5 months; on average, women are ill longer than men, and are more likely to die from the disease. In one highly affected village in Bangladesh, reproductive-age women were three times as likely to die from kala-azar compared to men or children; kala-azar accounted for 23% of all deaths, and 80% of those in adult women. Qualitative data from the same village suggest that women experience higher barriers to seeking care; poorer baseline iron, zinc, and vitamin A status may also play a role in higher morbidity and mortality among women. Although the morbidity and mortality caused by kala-azar and PKDL are substantial, the impact on affected individuals and their families is compounded by the expense and time involved in gaining access to appropriate diagnosis and treatment. The cost of caring for a patient with kala-azar in South Asia (US$80–US$120) approaches or surpasses the annual per capita income, and substantial additional income is lost by patients and family members unable to work. In Bihar and southern Nepal, costs have been multiplied many-fold by resistance to antimonial drugs and the imperative to use more expensive alternatives. The upsurge in PKDL cases now seen in Bangladesh will also increase difficulties for patients and their families; even if the drug is supplied gratis, the 120-day parenteral treatment course entails many other costs, such as payments for daily injections and transport to the health care facility, and is associated with much lost work time. Anecdotally, a number of PKDL patients died suddenly during treatment, consistent with antimonial cardio toxicity (Bern et al., 2008).

3. Epidemiology and Ecology

According to the World Health Organization (WHO) leishmaniasis is now endemic in 88 countries (16 developed countries, 72 developing countries) of 5 continents – Africa, Asia, Europe, North- America and South- America-affecting an estimated 12-14 million people with roughly 1.5-2 million new cases per year and a total of 350 million people at risk. It has been estimated that there are 500,000 new cases of VL and more than 50,000 deaths from the disease each year (only less than that of malaria). Both figures are approximations as VL is frequently not recognized or not reported. Majority (more than 90%) of cases occur in 6 countries- Bangladesh, India, Nepal, Sudan, Ethiopia and Brazil (Croft et al., 2006b).

In India two forms of Leishmaniasis i.e., Visceral Leishmaniasis and Cutaneous Leishmaniasis are recognized, which contributes approximately 50% of the global burden. The disease is highly endemic in Bihar (90% of cases in India), however has spread to newer areas also (Jha et al., 1998).Epidemics have been seen in Eastern Utter Pradesh and West Bengal (Gupta et al., 1997) and sporadic cases have been reported from the other part of the country too, where Kala-Azar have never seen before .The disease is prevalent in about 40 districts in Bihar, 9 districts of West Bengal and 2 districts of Eastern U.P.

Transmission of leishmaniasis to humans occurs through sylvatic, domestic, and peridomestic cycles. The distribution is dynamic: Colombia and Ethiopia have recently joined this list, and Pakistan currently faces a large epidemic of CL in Baluchistan and Sindh (WHO, unpublished data). Climate change and other environmental changes have the potential to expand the geographic range of the vectors and leishmaniasis transmission in the future. In sylvatic cycles, such as those in New World rain forests and the deserts of Central Asia, animal reservoir hosts can maintain enzootic transmission indefinitely without human disease. Sporadic or epidemic leishmaniasis occurs when humans enter the sylvatic habitat for economic or military purposes, or when human habitation encroaches on the sylvatic setting. In domestic cycles, humans or dogs form the predominant or sole infection reservoir. The foci that account for the largest number of human cases, for example, VL in South Asia and CL in Afghanistan, usually reflect anthroponotic transmission. In anthroponotic VL foci, the reservoir includes humans with untreated kala-azar but PKDL patients may maintain the infection between kala-azar epidemics. Up to half the population in highly affected foci may have asymptomatic leishmanial infection; the contribution of such individuals to transmission is presumed to be less than for active kala-azar, but has never been quantified (Costa et al., 2002; Bern et al., 2007).

4. Transmission

The transmission of leishmaniasis occurs through Female Phlebotomine sandflies (Phlebotomus genus in the old world and Lutzomyia in the new world). They seek a blood meal at or after dusk, becoming infected if they suck the blood of infected human beings (anthroponoses) or terrestrial mammals (zoonoses). The life-cycle has two distinct forms; a promastigote flagellar form found in the gut of the arthropod vector and an amastigote form, which develops intracellularly in the mammalian host. The sand fly transmits the disease by inoculation of the promastigote form into the skin. The parasites are internalized by dendritic cells and macrophages in the dermis and transform into amastigotes by losing their flagella. They multiply and survive in phagolysosomes through a complex parasite host interaction. The parasites disseminate through the lymphatic and vascular systems and infect other monocytes and the macrophages in the reticuloendothelial system, resulting in the infiltration of bone marrow, hepatosplenomegaly and sometimes in the enlarged lymph nodes (lymphadenopathy) (Chappuis et al., 2007).

About 70 of around 1000 known sandfly species transmit leishmaniasis. Vector competence in most species seems to be controlled by parasite ability to resist proteolytic enzymes during bloodmeal digestion and avoid excretion by binding to midgut epithelium. Binding is mediated by promastigote surface lipophosphoglycan and the phosphoglycan domains differ between species (Sacks, 2001). Sandfly saliva affects local host immune responses, promoting experimental cutaneous infection (Sacks & Noben-Trauth, 2002).

This present review will focus on the drugs currently available and those which are included in clinical trials, their mode of action, the experimental models and drug screening procedures with special emphasis on Visceral Leishmaniasis.

5. Conventional Therapy Against Visceral Leishmaniasis

5a. Parenterally Effective Agents

Pentavalent antimonials

N-methylglucamine antimoniate (Glucantime) and sodium stibogluconate (Pentostam) have been used as a first line of treatment for VL since the 1940s. Antimony remains the therapeutic cornerstone in all regions except two: Bihar State, India (houses around 90% of India’s and about 45% of the world’s cases) where the current approximate 35% cure response has ended the usefulness of antimony and southern Europe (Sundar et al., 2000) Relapse rates are less than 5%, but secondary resistance is likely in patients who relapse unless they are re-treated thoroughly. Effective doses of Sodium stibogluconate and meglumine antimoniate are 20 mg/kg/day up to a maximum 1275mg over 20 or 30 days given intramuscularly. The maximal tolerated dose is about 30 mg/kg/day; children tolerate these drugs better than adults. Its intracellular reduced trivalent form is the active derivative that comes about through the alteration in parasite bioenergetic pathways and trypanthione inhibition (Ephros et al., 1999; Wyllie et al., 2004).

Antimonials are toxic drugs with frequent, sometimes life threatening adverse side effects, including cardiac arrhythmia and acute pancreatitis. Patients under the age of 2 or aged 45 or over with signs of advanced disease and /or severe malnutrition are at higher risk of death during antimonial therapy owing to drug toxicity, slowness of drug action, VL complications or a combination of these factors (Chappuis et al., 2007).

Pentamidine isothionate

Pentamidine, an aromatic diamidine has been previously used as a second line of treatment for VL but its precise mode of action has yet to be elucidated. Since, it is a competitive inhibitor of arginine transport and noncompetitively inhibits putrescine and spermidine, its leishmanicidal activity is possibly mediated via its influence on polyamine biosynthesis and the mitochondrial membrane potential.Pentamidine was initially proven to be useful in Sb^v resistant kala-azar cases in India but the limiting factors were the expense and above all the unacceptable toxicity as it causes irreversible insulin dependent diabetes mellitus and death. Further, it’s declining efficacy (as only about 70% patients could be cured), has led to its being totally abandoned in India. (Sundar & Chatterjee, 2006).

Amphotericin-B & its formulations

Conventional Amphotericin B (fungizone®) is a macrolide polyene, charachterized by hydrophilic polyhydroxyl and hydrophobic polyene aspects. It is a powerful antileishmanial agent and is a first-line drug in India, where resistance to pentavalent antimonials is common. The best amphotericin B regimen is 15 doses of 1 mg/kg on alternate days. This drug is characterized by infusion related side effects and renal toxicity. Moreover this drug is costly and requires a complicated regimen. Amphotericin-B binds to membrane ergosterol leading to the formation of pores, major constituent efflux and finally parasite cell lysis (Pape, 2008).

Liposomal Amphotericin B

The liposomal amphotericin B formulation, AmBisome®, is registered treatment for visceral leishmaniasis (Meyerhoff, 1999), but use in VL endemic regions is limited by cost (US$2,800 per treatment).With recent preferential pricing offered by the manufacturer to patients in the public sector in East Africa, it is possible that AmBisome® could become economically feasible for treatment, even in resource – poor countries (DNDi Annual report 2007- 2008).

Other commercial Amphotericin B

Lipid formulations have also been manufactured, namely an amphotericin B lipid complex (Abelcet®) and an amphotericin B colloidal dispersion (Amphocil™) but their use against VL has not been as extensive as AmBisome® and they too, are costly. Other re-formulations of Amphotericin B formulations have been investigated against experimental VL but none have reached clinical development to date. Approaches to reduce cost include: (i) efficacy trials of single dose AmBisome treatment for VL, with 90 per cent cure rate reported to date, and (ii) the use of cheaper liposomal formulations, already tried for VL (Croft et al., 2006a).

Alternative amphotericin B formulations have been developed to reduce toxicity and improve drug effect. For example, arabinogalactan derivatives, nanoparticles and other lipid formulations, or chemical derivatives, have proved effective in experimental models (Croft et al., 2006a). The advantages of this association described by these authors include its physical and chemical stability when lyophilized or soluble, the easy sterilization by filtration, the drug release profile in the circulation and, consequently, good elimination by the organism, in addition to the possibility of i.v. or s.c. administration.A modified meta acrylic polymer of Amphotericin B has shown promise in experimental work carried out by the Imperial college team, London in 2007 which includes establishment of adequate efficacy in an in vivo model , size of the polymer, the ratio of the polymer to Amphotericin B and the actual dose of Amphotericin B. It is planned by DNDi to advance the most promising Amphotericin B based formulation by early 2009 (DNDi Annual report 2007- 2008).

Paromomycin

Paromomycin (formerly known as aminosidine) an aminoglycoside recently registered in India in August, 2006 for treatment of VL, is an antibiotic with good anti-leishmanial activity (den Boer & Davidson, 2006). Early results showed the initial doses of paromomycin did not work as well in Africa as it did in India. A dose escalation study was undertaken to determine if a higher dosages regimen could meet its efficacy target. It has comparatively fewer side effects like high-tone ototoxicity in 2% of patients and a significant increase in hepatic transaminases in 1.8% of patients (Sundar et al., 2007). Other advantages of paromomycin include the fact that it is active against a wide variety of pathogens, including bacteria, and its low cost (US$5–10 per treatment) (Chappuis et al., 2007). A suitable monotherapy regimen paromomycin sulphate is 15 mg/kg/day for 21 days. Paromomycin sulphate can be effectively combined with pentavalent antimonials; the combination is given for 17 days, with the two drugs injected into different buttocks (Davidson, 2005). The mechanism of its action is although unclear but in bacteria it binds to the A-site on the 16S RNA in the 30S sub units of ribosomes giving rise to non sense proteins through a misreading during protein synthesis (Kotra etal., 2000). In Leishmania, it can interfere with RNA synthesis and membrane permeability (Maarouf et al., 1997).

5b. Orally Effective Agents

Miltefosine

Miltefosine, (initially developed as anticancer drug) the first effective oral treatment for visceral leishmaniasis, including for antimony-resistant infection, opened the door to self-administered outpatient therapy. Miltefosine is licensed for visceral leishmaniasis in several regions and for cutaneous leishmaniasis in some countries. Its rapid development in India heads the preceding list of tangible treatment advances. This alkylphospholipid registered in India (Sep. 2006), Germany (2004), and Colombia (2005). Miltefosine has been tested in Nepal and in outpatients in India and testing is in progress in east Africa (Murray, 2005). It has only been available in the private market, at a retail cost of US$125–200 per treatment course (Sundar & Murray, 2005; Bhattacharya et al., 2007).It is given for 28 days at a dose of 50 mg/day (< 25 kg), 100 mg/day (> 25 kg) or 2.5 mg/kg/day (small children). Common side-effects (usually mild or moderate) are nausea, vomiting, diarrhoea and renal impairment. It is teratogenic, so cannot be given to women of child-bearing age unless contraception is guaranteed, and conception must be avoided for 2 months after treatment. It alters glycophosphatidylinositol (GPI) anchor synthesis, ether lipid metabolism, signal transduction and alkyl specific acyl coenzyme A acyl-transferase (Lux et al., 1996; 2000).

Concerns have been expressed about miltefosine’s cost as well as how to protect the high-level efficacy of this valuable agent from the effects of poor outpatient compliance and the potential development of resistance as this drug has a long half life (~150 hours) and parasite resistance is easily induced in vitro (Perez-Victoria et al., 2006). Some researchers have suggested combining miltefosine with a second agent in part to maintain its effect but also reflecting a growing interest in combination treatments for visceral leishmaniasis.

Sitamaquine

Sitamaquine, an orally active 8-aminoquinoline analog (8-aminoquinoline (8-[6-(diethylamino)hexyl]amino]-6-methoxy-4-methylquinoline), was originally developed as WR6026 by the Walter Reed Army Institute in collaboration with Glaxo Smith Kline in response to a pressing need for orally effective agents for VL, its effectiveness was validated in animal models. Several small phase I or II clinical trials have been undertaken with limited success. The cure rate for VL with sitamaquine in a Kenyan phase II study at a dose of 1 mg/kg/day for 28 days were 50 percent. Several years later, in a Brazilian phase II trial, the same dose of sitamaquine cured none of the four VL patients while a 2 mg/kg/day for 4 wk gave a maximum efficacy of 67 per cent; surprisingly, a linear correlation could not be sustained as increasing the dose to 2.5 mg/kg/day resulted in decreased efficacy concomitant with enhanced adverse effects such as nephropathy and methaemoglobinaemia. In a multicenter phase II trial in India, sitamaquine demonstrated excellent antileishmanial activity at a daily dose of 1.75 -2 mg/kg for 28 days. However, more studies are needed to evaluate some of the safety issues as this drug appears to have clinical efficacy that warrants further development. The mode of action is not known but could involved “futile redox cycling” as proposed for primaquine (Sundar & Chatterjee, 2006).

5c. Other Oral Compounds

Azoles

The last example of development in new anti-infectious drugs is therapeutic swiching also called “piggy-back therapy”. Azoles (Ketoconazole, fluconazole, itraconazole, etc.) are essentially sterol bio-synthesis inhibitors and their efficacy against L. tropica was first reported by Berman in 1981. Azoles specifically block ergosterol synthesis and as the presence of ergosterol as a membrane component is shared between fungi and Leishmania, it accounts for many antifungal sterol biosynthesis inhibitors (SBIs) to also be leishmanicidal. Most SBIs impair the biosynthesis of ergosterol by blocking14-á-demethylase, leading to the accumulation of 14-á-methylsterols. Azoles have been shown to be active against a wide range of promastigotes and amastigotes. Leishmania species differ in their sensitivity to azoles as L. donovani, L. braziliensis and L. amazonensis promastigotes are more sensitive than L. aethiopica, L. major, L. tropica and L. mexicana. However, this analogy cannot be extrapolated to clinical studies. Both ketoconazole and fluconazole have undergone evaluation in VL in India. However, despite reports of the former’s usefulness, their antileishmanial activity was not enough to induce clinical cure by themselves (Sundar & Chatterjee, 2006).

Buparvaquone

Buparvaquone (BPQ) is a hydroxynaphthoquinone and marketed as Butalex^® closely related to a well-known anti- infective drug, atovaquone. BPQ has been used as an i.m. injection for thetreatment of theileriosis in cattle. For the first time Croft et al (1992) has tested BPQ against L. donovani infected BALB/c and observed a 62% suppression of hepatic amastigote burden. Researchers are looking forward to this drug as a promising antileishmanial agent as it has several physicochemical properties suitable for topical delivery (low molecular weight, low melting point, etc.). Attempts have been made to increase aqueous solubility and absorption, and in this context two phoaphate prodrugs have been found to show potential in in vitro & in vivo antileishmanial activity against both visceral and cutaneous leishmaniasis (Garnier et al., 2007; Ma¨ntyla et al., 2004).

In 2007, DNDi- commissioned work by partners at the Universiti Sains Malaysia and at Advinus Therapeutics has shown that a new self emulsifying drug delivery system (SEDDS) could improve the oral bioavailability of BPQ to greater than 60%. In 2008, with these promising early results, DNDi has identified partners to assess BPQ as a potential optimized lead , and ongoing studies are examining toxicology , pharmacokinetics / phamacodynamics in animal models (mouse, hamsters), and reconfirmation of oral bioavailability using the SEDDS formulation. If acceptable, development will be progressed with the aim of satisfying the criteria specified for a clinical candidate (DNDi Annual report 2007- 2008).

5d. Immunomodulators

Leishmania infection progresses to kala-azar in individuals who fail to initiate Th 1 response (mediated by IL-2 and IFN-γ). Skewing of T helper cells towards a Th1 response is considered as a promising therapeutic strategy. Interferon- γ is one of the principal activators of macrophages. Clinical trials with IFN- γ alone and/or in conjunction with Sb^v were undertaken. With Sb^v it was reported to be useful in treating severe or Sb^v refractory VL in Brazil, however, in India in a large (n=156) randomized study comparing Sb^v alone with Sb^v plus IFN-γ for 15 or 30 days had disappointing results as the final cure rate with Sb^v plus IFN-γ was 42 and 49 per cent, respectively (Sundar & Chatterjee, 2006).

5e. Drug Combination Strategies

Combination therapy has more potential advantages which include delay or prevention of the development of resistance (Croft, 2004) and shorter treatment regimens that could improve compliance and reduce cost. Unrestricted use of standard antimonials have already posed potential problem of resistance (Sundar, 2001) so precautionary measures should be taken in case of monotherapy of arising drugs like miltefosine and paromomycin. Despite of remarkable work done on combination therapy for Leishmaniasis (Chunge et al., 1985; Murray & Hariprasad, 1996), it has not yet been adapted as standard treatment. Limitation is unavailability of effective antileishmanial drug. Previous studies on the interaction of miltefosine and sodium stibogluconate have shown synergism in vitro but showed no potentiation in vivo. Conversely, published reports on the combination of miltefosine with amphotericin B and miltefosine with paromomycin have shown enhanced efficacy in vivo in mice model (Seifert and Croft, 2006). Currently, clinical trials on combination therapy using paromomycin and miltefosine, AmBisome, miltefosine and paromomycin are being carried out in India by DNDi-and ICMR & RMRI and results are expected by early 2010. In African countries evaluation of shorter course combination of paromomycin + SSG as an alternative treatment for VL is also underway (DNDi Annual report 2007- 2008).

6. Experimental Models in Use in the Drug Discovery

We will focus here on the specific in vitro and in vivo assays required in the drug discovery process for Visceral Leishmaniasis.

6a. In vitro Assays

Leishmania parasite can be grown in vitro as promastigotes and amastigotes in axenic conditions. Both these stages have been exploited for development of primary drug screening procedures.

(i) Promastigotes:

Drug activity against this extracellular stage is easy to determine. However, there are significant differences between promastigotes and amastigotes in biochemistry and sensitivity to standard and experimental drugs (Croft et al., 2006a). Promastigotes assays are useful cytotoxicity indicators in bioassay-guided fractionation of plant products. A direct comparison of the drug susceptibility towards standard antileishmnial drugs, between amastigotes and axenic amastigotes, demonstrates that the latter express specific susceptibility to many if, not all the drug tested and indicates that promastigotes may not be as relevant as axenic amastigotes for drug screening purpose (Sereno et al., 2007).

(ii) Axenic Amastigotes:

Screening against axenic amastigotes presents several advantages; (1) the test is directed against the relevant stage of parasite, (2) this stage is as easy to manipulate as the promastigote model, (3) quantification of drug activity is simple and often inexpensive. This can be achieved by using a cell counter, evaluating the viability of cell population with a MTT based method, determining ornithine decarboxylase activity or using a fluorescent dye like Propidium Iodide (PI) and fluorescence-activated-cell-sorter (FACS). Since, past few years many Leishmania parasites expressing reporter genes have been selected and the capacity of some of them to be used in axenic amastigote drug screening protocol has been assessed (Sereno et al., 2005; Vergnes et al., 2005).The disadvantages are (i) assay neither test for penetration of compounds into the host cell nor for activity in the macrophage phagolysosome, (ii) not true amastigotes (metabolome etc.), (iii) have different metabolic processes than intracellular amastigotes, (iv) problem of clumping etc.

Ideally to be efficient and exhaustive, a drug screening procedure requires conditions that tightly mimic the environment encountered by the target cell. In case of Leishmania, intracellular form of the parasite (amastigotes) represent the ideal conditions since, this system involves the role played by the host cell on drug mediated toxicity.

(iii) Screening Against Intracellular Amastigotes:

The most widely used models for testing drugs against Leishmania species have involved either murine peritoneal macrophages (J-774) or human-monocyte tranformed macrophages (THP-1, U937, HL-60) as host cells. These models show species/strain variation in drug sensitivity (Escobar et al., 2002; Yardley and Croft, 2005). In these differentiated non-dividing macrophages, the rate of amastigote division in host cells and drug activity can be clearly assessed. The activity of test drug is measured by either microscopical counting of percentage of infected cells or number of amastigotes /macrophage or colorimetric or fluorometric methods (Neal and Croft, 1984). The slow rate of division of L. donovani and L. infantum amastigotes in this model is a limitation. Assays that use dividing host cells must ensure that the confounding effects of drug activity on both parasite and host cell number are considered (Croft et al., 2006, a). Many, if not all classical Screening methods are labour intensive and could not support automation.

a. Microscopical Method: In direct counting assays, drug activity are assessed towards intracellular amastigotes after Giemsa staining on chamber slide (Sereno et al, 2007). It is followed by evaluation of drug activity microscopically by determining the percentage of infected cells as well as the number of amastigotes per cell through examination of 100–300 macrophages. Counting cells is time consuming and may give inaccurate determination of IC_50, since determination of the parasite viability through a staining procedure is difficult.

b. Reporter Gene Assays: Over the past few years, many Leishmania parasites expressing reporter genes have been selected and used for testing drug activity. The main advantage of this technology is its rapidness and accuracy.

The term reporter gene is used to define a gene with a readily measurable phenotype that can be distinguished easily over a background of endogenous proteins. The use of such genes like the firefly luciferase (Ashutosh et al, 2005), β-galactosidase, chloramphenicol acetyltransferase (CAT), alkaline phosphatase or the green fluorescent protein (GFP) gene could considerably facilitate the screening of antimicrobial agents (Naylor, 1999). The reporter gene technology is generally more sensitive than the other previously mentioned classical methods. Moreover, reporter proteins bear or produce an easily detectable response that can be quantified even in intracellular conditions. In general, methods based on fluorescent proteins are less sensitive than methods using catalytic reporter genes like luciferase, β-galactosidase, and β-lactamase. A panel of recombinant parasites carrying a reporter gene either as an episomal copy or after its integration in a defined locus, generally the rDNA locus, is currently available.

Various strains of parasites expressing luciferase were recently developed and their susceptibility towards standard antileishmanial agents investigated (Roy et al., 2000, Sereno et al., 2001; Ashutosh et al, 2005). Drug discovery facilities at Central Drug Research Institute (CDRI), Lucknow have developed Leishmnia donovani cell lines expressing firefly luciferase reporter gene (luc.) as a part of episomal vector and established suitability of these cell lines for in vitro screening of antileishmanial agents (Ashutosh et al, 2005).This system has been adapted to evaluate compounds in a 96 well microplate format and is being employed (Sundru et al., 2006; Pandey et al., 2007;Gupta et al., 2007) for primary screening of novel synthetic compounds (Inhouse) and marine extracts (MoES project) and also for optimization of leads under DNDi supported consortium.

c. Limitations of Reporter Gene Assays: Reporter genes present several important limitations. Among them the antibiotic resistance allowing the selection of recombinant parasites could confer cross-resistance. Neomycin confers resistance towards paromomycin, a lead candidate drug supported by the Gates foundation. The development of method to create defined mutants lacking selectable markers could help to overcome this problem. The way by which the reporter gene is introduced could also have an impact on the screening. If, the reporters are part of plasmids, the relative output of reporter may depend on the copy number of the transfected plasmid (which vary from cell to cell) rather than on the activity of the drug. Secondly, transforming parasites could have biological consequences either by disrupting the genomic architecture or just by the presence of the foreign reporter protein. Thirdly, as previously mentioned for the β-galactosidase technology, the reporter could have some limitations (i.e. sensitivity, background activity from host macrophages) making it inaccurate for an in vitro determination of drug activity against intracellular amastigotes (Sereno et al., 2007).

d. Multiplexing: A versatile methodology that allows for multiple quantifications of drug toxicity against both the host cells and the intracellular amastigotes could represent a useful tool in the field of parasite pharmacology. To achieve this goal, reporters must use distinguishable signal from each other and compatible chemistries, like fluorophores emitting different wavelengths. Currently, there have been a growing number of examples using luminescence for multiplexing either in combination with: (i) other luminescent signals, (ii) fluorescence or (iii) β -galactose assay. Such methods could also help to directly compare experiments since the results are expressed as a ratio of the output signal emitted by the host cell on the one emitted by parasites (Grover et al., 2003; Young et al., 2004). The usefulness of these approaches for drug screening has to be evaluated on intracellular parasites like Leishmania.

6b. In vivo Assays

Animal models are expected to mimic the pathological features and immunological responses observed in humans when exposed to a variety of Leishmania spp. with different pathogenic characteristics. Many experimental models have been developed, each with specific features, but none accurately reproduces what happens in humans. For in vivo testing of new compounds several animal species have served as experimental host for VL. Important among them are BALB/c mice and Syrian golden hamster (primary tests), dogs (secondary tests) and monkeys viz., squirrel, vervet and Indian langur monkeys as tertiary screens. Animal models enable drug activity to be determined in relation to absorption (route of administration), distribution (different sites of infection), metabolism (pro-drugs, immunomodulators), and excretion and to give an early indication of the toxicity. A suitable laboratory host for the target parasite (L. donovani) is very important from the point of view of conducting research on various aspects including host-parasite interactions, pathogenesis, biochemical changes, prophylaxis, and maintenance of parasites and above all evaluation of antileishmanial action of newer compounds for development of new drugs.

Mouse Model

Mostly mice are being used as model for screening of new compounds, where a relatively low amount of compound is required, which are available as SPF and inbred strains enabling reproducible results with five animals per group. Mice are susceptible to most strains and species of Leishmania in both non-cure and self cure models. The aim of using the animal model is to find a drug that can be administered orally, be effective in a short course (< 10 days) and have no indication of toxicity at the highest doses tested (100 mg/kg). For visceral leishmaniasis inbred strains of mice are widely used with susceptible, resistant and intermediate strains. The BALB/c mouse is a commonly used strain, at 18- 20 g, with highly reproducible levels of infection when an amastigote inoculum is administered i.v. An assay in week two after infection examines the activity of the drug against the liver infection but not the spleen infection. The infection in each mouse strain needs to be characterized for each parasite strain used to ensure that drugs are tested appropriately. Athymic and scid mice provide a model for treatment of VL in immunosuppressed cases (Croft et al., 2006a). Hamster Model

Although many hamster species are susceptible to L. donovani infection (Smyly & Young, 1924) , the Syrian golden hamster (Mesocricetus auratus) establishes a good model for VL and provides a more synchronous infection in the liver and spleen that can develop into a chronic non-cure infection more similar to human VL (Farrell, 1976; Gifawesen & Farrell, 1989; Hommel et al., 1995). Gupta & Tiwari (2000) have reported the suitaibility and susceptibility of inbred hamsters in terms of parasite establishment and longer survival period as compaired to outbred hamsters.Very recently, Dea-Ayuela et al., (2007) have studied its suitability and established suitable immunobiological parameters for in vivo testing of new antileishmanial compounds in the golden hamster model of visceral leishmaniasis. The clinicopathological features of the hamster model of VL closely mimic active human disease. Systemic infection of the hamster with L. donovani results in a relentless increase in visceral parasite burden, progressive cachexia, hepatosplenomegaly, pancytopenia, hyper-gamma-globulinemia, and ultimately death (Gifawesen & Farrell, 1989). Biggest advantage is that biopsy is possible to monitor pre- & post treatment infection status and all antileishmanials are active against liver as well as spleen parasites.

A problem in all the models is the determination of drug activity upon necropsy or biopsy which has been dependent on microscopy to determine the level of infection. This is now being replaced by quantitative whole animal non-invasive imaging for parasites. Reporter genes have proved to be an excellent and promising tool for the detection of parasite stages in target tissues of animal hosts (Roy et al., 2000; Lang et al., 2005).

7. Conclusion

New treatments for visceral leishmaniasis have been introduced and others are undergoing clinical trials. The recent availability of oral miltefosine for VL has been the most significant development in the past few years. Care needs to be taken that resistance to these drugs does not develop and efficiency and safety of drug combinations in greater depth should be considered. Importantly, the cost of the treatment should be minimized to allow its dissemination and use mainly in poorer countries, where there is a high incidence of this disease. Efforts to find new leads and to select new targets will also contribute to the fight against leishmaniasis and the preparation of additional resources for the drug discovery pipeline.

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