A RE-PURPOSED DRUG COMBINATION FOR ENHANCED TREATMENT OF VISCERAL LEISHMANIASIS AND PREVENTION OF PKDL

Abstract

This invention provides novel pharmaceutical compositions and therapeutic methods for treating visceral leishmaniasis (VL) and preventing post-kala-azar dermal leishmaniasis (PKDL) through the re-purposing of existing drugs. The compositions incorporate Buparvaquone, Chloroquine, Disulfiram, and Artemisinin, either individually or in combination with Miltefosine, the standard anti-leishmanial treatment. In vitro and in vivo studies demonstrate that these re-purposed drugs show improved efficacy, including reduced viability of Leishmania donovani parasites and lower inhibitory concentrations compared to Miltefosine alone. Synergistic effects are observed when combining Disulfiram with Miltefosine and other agents, as shown by fractional inhibitory concentration (FIC) indices under 0.5. This synergy enables lower dosage requirements and improved therapeutic outcomes, which are critical in treating drug-resistant VL strains and mitigating PKDL risk. The invention also provides a methodology for evaluating the efficacy of drug combinations through checkerboard assays, allowing for the identification of effective combinations with enhanced anti-leishmanial activity. This approach offers a promising solution for improving treatment strategies against leishmaniasis and reducing relapse rates.

Specification

Description:[0020].The following description provides specific details of certain aspects of the disclosure illustrated in the drawings to provide a thorough understanding of those aspects. It should be recognized, however, that the present disclosure can be reflected in additional aspects and the disclosure may be practiced without some of the details in the following description.
[0021].The various aspects including the example aspects are now described more fully with reference to the accompanying drawings, in which the various aspects of the disclosure are shown. The disclosure may, however, be embodied in different forms and should not be construed as limited to the aspects set forth herein. Rather, these aspects are provided so that this disclosure is thorough and complete, and fully conveys the scope of the disclosure to those skilled in the art. In the drawings, the sizes of components may be exaggerated for clarity.
[0022].It is understood that when an element or layer is referred to as being "on," "connected to," or "coupled to" another element or layer, it can be directly on, connected to, or coupled to the other element or layer or intervening elements or layers that may be present. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
[0023].The subject matter of example aspects, as disclosed herein, is described with specificity to meet statutory requirements. However, the description itself is not intended to limit the scope of this patent. Rather, the inventor/inventors have contemplated that the claimed subject matter might also be embodied in other ways, to include different features or combinations of features similar to the ones described in this document, in conjunction with other technologies.
[0024].Visceral leishmaniasis (VL), also known as kala-azar, is a severe and often fatal parasitic disease caused by Leishmania donovani. VL is a significant public health issue, particularly in regions such as India, where endemic cases pose ongoing challenges to disease management and control. Traditional treatments primarily rely on drugs like Miltefosine; however, increasing cases of drug resistance, along with the frequent relapse of patients developing post-kala-azar dermal leishmaniasis (PKDL), underscore the need for improved therapeutic strategies.
[0025].PKDL represents a challenging disease manifestation that occurs in patients post-VL treatment and acts as a reservoir for disease transmission. The emergence of PKDL not only threatens patients' health but also complicates eradication efforts for leishmaniasis. Existing treatment regimens are often ineffective at fully eradicating Leishmania parasites, especially in Miltefosine-resistant strains, thereby contributing to the persistence and spread of the disease.
[0026].Drug re-purposing has gained significant attention in recent years as a cost-effective, efficient method to combat diseases by finding new uses for existing drugs. This approach can expedite the development of improved therapies, leveraging established safety profiles and manufacturing processes of known drugs. Preliminary studies have shown promise in using re-purposed drugs such as Buparvaquone, Chloroquine, Disulfiram, and Artemisinin, either individually or in combination, to improve anti-leishmanial efficacy and lower the likelihood of PKDL onset.
[0027].The current invention focuses on the therapeutic potential of these re-purposed drugs in combination with Miltefosine. By targeting Leishmania with synergistic drug combinations, this approach aims to enhance treatment effectiveness, reduce required dosages, and minimize adverse effects.
[0028].This invention introduces novel therapeutic methods and pharmaceutical compositions utilizing re-purposed drugs to address significant challenges in the treatment of visceral leishmaniasis (VL) and prevention of post-kala-azar dermal leishmaniasis (PKDL). VL, caused by Leishmania donovani, is a severe parasitic disease that poses a substantial global health burden, particularly in regions where it is endemic, such as South Asia and East Africa. Left untreated, VL is fatal, while treatment with conventional drugs, especially Miltefosine, the standard oral therapy, has become less effective due to the emergence of drug-resistant Leishmania strains. Furthermore, a considerable percentage of treated VL patients experience relapse in the form of PKDL, a cutaneous manifestation that can serve as a long-term reservoir for transmission. This invention targets these issues by offering a new approach through re-purposing drugs with known efficacy against Leishmania, either alone or in combination with Miltefosine, to enhance therapeutic outcomes, address drug resistance, and reduce the relapse of PKDL.
[0029].The key drugs identified for re-purposing include Buparvaquone, Chloroquine, Disulfiram, and Artemisinin, which are well-documented for their primary applications against other diseases, yet demonstrate promising activity against Leishmania in preliminary studies. Each drug has unique pharmacological properties that contribute to anti-leishmanial efficacy. For example, Buparvaquone, an anti-parasitic drug typically used to treat protozoal infections in animals, has shown efficacy in reducing Leishmania parasite load. Chloroquine, widely used as an antimalarial, has demonstrated activity against various protozoan infections and offers potential synergistic effects when combined with other anti-leishmanial agents. Disulfiram, primarily used in treating alcohol dependence, possesses inhibitory effects on the parasite's metabolism and viability. Artemisinin, known for its use in malaria, has shown anti-leishmanial effects when used at specific dosages, offering an alternative mechanism for targeting the parasite.
[0030].This invention focuses on optimizing these drugs either as monotherapies or in combination therapies with Miltefosine. Combination therapies provide a multi-targeted approach, capitalizing on the distinct mechanisms of each drug to enhance overall efficacy and combat resistance more effectively than single-drug regimens. By using a checkerboard assay, the invention evaluates various drug combinations through the fractional inhibitory concentration (FIC) index, which measures the degree of interaction between two drugs, classifying the interactions as synergistic, additive, or antagonistic. Synergistic combinations are particularly valuable in this context, as they allow for lower doses of each drug to be used while achieving equal or greater efficacy, thereby minimizing the risk of side effects and improving patient tolerability. For instance, the combination of Disulfiram and Miltefosine demonstrated a significant reduction in IC50 values, suggesting enhanced potency in both drug-sensitive and drug-resistant Leishmania isolates. This synergy enables lower overall dosing, which can reduce adverse effects and potentially improve patient adherence to the treatment regimen.
[0031].The methodology further includes in vivo studies using murine and hamster models, providing an essential validation step by evaluating the therapeutic effects of these drug combinations in a living organism. These animal models closely mimic the human infection process, allowing for assessment of the drugs' efficacy in reducing parasite burden, decreasing the risk of PKDL, and evaluating any adverse effects. The in vivo testing includes both dose-response studies and time-course analyses, offering insights into optimal dosing regimens and potential adjustments for human application.
[0032].To ensure safe and effective translation to human therapy, the invention incorporates cytotoxicity testing using an MTT assay, which measures the viability of human macrophages exposed to varying concentrations of each drug. This is critical for identifying safe therapeutic windows, as it ensures that the drugs or drug combinations are non-toxic at the concentrations necessary for anti-leishmanial efficacy. Drugs that show significant cytotoxicity at therapeutic doses are excluded from consideration, while those demonstrating high anti-leishmanial activity with minimal impact on host cells are prioritized for further development.
[0033].A major advantage of the proposed re-purposed drug combinations is their ability to overcome limitations posed by resistance to Miltefosine. By combining drugs with complementary mechanisms, this invention provides alternative pathways to target Leishmania parasites. For example, Buparvaquone disrupts mitochondrial function, while Disulfiram inhibits critical metabolic enzymes, each attacking the parasite through unique mechanisms. In cases where Miltefosine resistance is present, these combinations enhance parasite inhibition and are effective even at lower concentrations than would be required for each drug individually. This multi-targeted strategy minimizes the likelihood of resistance development and provides a robust treatment option for VL patients, including those with resistant infections.
[0034].The potential impact of this invention extends beyond VL treatment alone. The prevention of PKDL through these optimized therapies could have significant public health implications, especially in endemic areas. PKDL often manifests after VL treatment, contributing to sustained Leishmania transmission cycles in regions where healthcare access and disease monitoring are limited. By reducing the likelihood of PKDL, this invention not only improves individual patient outcomes but also supports broader efforts to control and eliminate Leishmania transmission.
[0035].In addition, the economic and logistical advantages of re-purposing existing drugs make this approach particularly suitable for low-resource settings where VL is prevalent. Manufacturing processes and safety profiles of the drugs are already established, expediting regulatory approvals and reducing production costs. This is crucial in regions where healthcare infrastructure may not support the development and distribution of entirely new drugs. By providing a feasible, cost-effective treatment approach, this invention holds the potential to enhance access to effective therapy, especially in endemic regions that are most in need of improved leishmaniasis treatment options.
[0036].Overall, this invention represents a strategic and innovative approach to VL and PKDL management by utilizing synergistic drug combinations of re-purposed medications. The results of the checkerboard assay, in vivo model testing, and cytotoxicity assessments offer a comprehensive framework for the identification and validation of effective therapeutic combinations. This approach not only strengthens the efficacy of existing treatment regimens but also addresses critical gaps in managing drug-resistant and recurrent Leishmania infections, ultimately contributing to the global effort to control and eradicate leishmaniasis. , Claims:1.A method of treating visceral leishmaniasis and preventing post-kala-azar dermal leishmaniasis (PKDL), comprising:
a) Administering a pharmaceutical composition to a patient in need thereof, wherein the composition includes one or more re-purposed drugs selected from Buparvaquone, Chloroquine, Disulfiram, and Artemisinin;
b) Wherein the re-purposed drugs exhibit improved efficacy compared to Miltefosine in reducing the viability of Leishmania donovani parasites in vitro and in vivo.
2.The method as claimed in claim 1, wherein the pharmaceutical composition further comprises Miltefosine in combination with at least one of Buparvaquone, Chloroquine, Disulfiram, or Artemisinin, with the combination providing a synergistic therapeutic effect against Leishmania donovani, demonstrated by a fractional inhibitory concentration (FIC) index of less than or equal to 0.5.
3.The method as claimed in claim 2, wherein the combination includes Disulfiram and Miltefosine with FIC indices resulting in a synergistic interaction characterized by IC50 values of Disulfiram and Miltefosine in combination being significantly lower than the IC50 values of each drug alone.
4.A pharmaceutical composition for treating visceral leishmaniasis, comprising:
a) One or more drugs selected from Buparvaquone, Chloroquine, Disulfiram, and Artemisinin, either alone or in combination;
b) Wherein the composition exhibits an IC50 concentration effective against Leishmania donovani isolates that are resistant to Miltefosine.
5.The pharmaceutical composition as claimed in claim 4, wherein the composition is formulated for in vivo administration in a dosage effective to reduce parasitic infection in murine and hamster models, and is adaptable for treatment in human patients based on comparative cytotoxicity profiles for macrophage cells exposed to varying concentrations of the drug.
6.A method for evaluating the efficacy of drug combinations against Leishmania donovani comprising:
a) Conducting in vitro drug sensitivity tests using a checkerboard assay to determine the FIC index for combinations of Disulfiram, Miltefosine, Chloroquine, Buparvaquone, and/or Artemisinin;
b) Identifying combinations with FIC index values indicating synergistic or additive interactions that enhance anti-leishmanial activity.

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