LEISHMANIA

The present application claims Priority benefit of Provisional Application No. 4962/CHE/2015 filed 17/09/2015, the content of which is incorporated herein by reference in its_entirety.

Field of the invention:

The present invention relates to an antileishmanial and immunomodulatory activity of Withaferin-A.

More particularly, it relates to a synergistic effect of Withaferin-A and Miltefosine in the treatment of Visceral Leishmaniasis (VL)

Background and Prior Art of the present invention:

Withaferin-A is potent anti cancer compound from Withania somnifera plant. The compound has been tested against various cancer types in-vitro (Yco et al., 2014; Li et al., 2015) and in-vivo (Lee et al., 2015; Yang et al., 2012). The antileishmanial potential of this compound in mice models has not been proved till date. Only one report has shown that Withaferin-A was able to inhibit protein kinase of the L donovani parasite in-vitro (Sen et al., 2007). First time, the Applicant have identified target protein in Leishmania donovani and used enzyme inhibition assay to prove that Withaferin-A is binding to the Pteridine Reductase-1 enzyme and inhibit its activity, which is essential for the growth of the parasites. Reference may be made from the article of Parasite Immunology, 2014, 36, 253-265 titled "Efficacy of Withania somnifera chemotypes NMITLI - 101 R, 1 18R and Withaferin A against experimental visceral leishmaniasis" by C. D. P. TRIPATHI, R. GUPTA, P. K. KUSHAWAHA, C. MANDAL, S. MISRA BHATTACHARYA & A. DUBE, which disclose The immunoprophylactic and therapeutic potentials of root extracts of Withania somnifera chemotypes (NMITLI-1 18, NMITLI-101 ) and pure withanolide-withaferin-A was investigated against Leishmania donovani infection in hamsters. The naive animals, fed orally with immunostimulatory doses of chemotypes 101 R, 1 18R (10 and 3 mg/kg) and withaferin-A (9 and 3 mg/kg) for five consecutive days and challenged with Leishmania parasites on day 6, were euthanized on days 30 and 45 p.c. for the assessment of parasite clearance, realtime analysis of mRNAs of Th1/Th2 cytokines (IFN-c, IL-12, TNF-a, iNOS/IL-4, IL-10 and TGF-b), NO production, reactive oxygen species (ROS) generation, lymphocyte transformation test and antibody responses. By day 45 p.c, there was a significant increase in the mRNA expression of iNOS, IFN-c, IL-12 and TNF-a but decrease in IL-4, IL-10 and TGF-b, an enhanced Leishmania-specific LTT response as well as ROS, NO and antileishmanial lgG2 levels in 101 R-treated hamsters followed by 1 18R- and withaferin A-treated ones, respectively.

Another reference may be made from the article of Grover et al. BMC Genomics 2012, 13(Suppl 7):S20 titled "Blocking Protein kinase C signaling pathway: mechanistic insights into the anti-leishmanial activity of prospective herbal drugs from Withania somnifera", which discloses the inhibitory property of withaferin-A as well as that of withanone at the molecular level. Another reference may be made from the US Patent No. 8,501 ,186, which relates to a plant fraction obtained from the roots of plant Wilhania somnifera. It also envisages a vaccine comprising novel adjuvant capable of invoking T cell dependent immune responses to T cell independent antigens such as polysaccharide. On the other hand, the Applicant has shown the utility of Withaferin-A as both antileishmanial and immunomodulatory compound against experimental visceral leishmaniasis in susceptible BALB/c mice model. Results demonstrate that Withaferin-A at a concentration of 2mg/kg/body weight was significantly decreased the parasite burden in spleen (**P<0.01)) and in liver (***P<0.001) respectively without causing any side effect to the mice. In addition to this, it was able to downregulate the expression of IL-10 cytokine and increasing the IFN- y/IL-10 expression ratio compared to untreated mice which is another important response require to clear the parasite from the host cells. This clearly shows the effectiveness of Withaferin-A against Visceral Leishmaniasis (VL). Withaferin-A has been known for its anti cancer properties and this compound has been shown to possess antileishmanial activity recently. But the parasite specific target for Withaferin-A has not been addressed till now. Few reports have shown the Leishmanial protein kinase C as one of the targets of Withaferin-A but they failed to report its efficacy in mouse models. This is the first time Applicant's addressing the target protein Pteridine Reductase-1 enzyme from L. donovani parasites, as the target for the Withaferin-A and this enzyme are exclusively present in parasites and not in humans. This gives an advantage of specificity for this compound against the parasites. Objects of the present invention:

It is an objective of the invention to provide antileishmanial activity of Withaferin-A through the inhibition of the Pteridine Reductase-1 enzyme of L donovani parasite, which is conserved only in parasites and not in humans. It is another objective of the invention to provide immunomodulatory role of Withaferin-A in mouse model of chronic visceral leishmaniasis.

It is yet another object of the invention to provide a Withaferin-A for the reduction of intracellular parasite load in dose dependent manner ex-vivo.

Further, it is yet another object of the invention to provide a Withaferin-A for the reduction of the parasite burden in spleen and liver of mice in chronic experimental visceral leishmaniasis.

Further, it is yet another object of the invention to provide a Withaferin-A for increase the lgG2a (Th1 ) antibody titers in serum of chronic experimental visceral leishmaniasis.

Still, it is another object of the invention to provide synergistic composition of Withaferin-A and Miltefosine for the treatment of visceral leishmaniasis

Summary of the invention:

The present invention discloses the potent antileishmanial activity of Withaferin-A alone and / or synergistic effects together with Miltefosine treatment.

Brief description of drawings:

The invention may be best understood by reference to the following description, taken in conjunction with the accompanying figures. These figures and the associated description are provided to illustrate some embodiments of the invention, and not to limit the scope of the invention. In the following the invention will be described in greater detail with reference to exemplary embodiments in accordance with the accompanying drawings, in which:

Figure 1 illustrates the antileishmanial effect of Withaferin-A on intracellular parasites were determined by microscopic count after 72hrs of treatment. The graph depicts the number of amastigotes/100 macrophage in the dose dependent concentrations of Withaferin-A.

Figure 2 illustrates the antileishmanial effect of Withaferin-A in BALB/C model of visceral leishmaniasis a) LDU in spleen b) LDU in liver of control (infected) and Withaferin-A treated mice groups after 45 days post infection/post treatment. The experiment was done with five mice per group and significance between control (infected) and treated groups were calculated using one-way ANOVA with dunnett's posttest. (Leishman donovan units= Number of amastigotes per 1000 nuclei x weight of the tissue in mg).

Figure 3 illustrates immunomodulatory effects of Withaferin-A in BALB/C mice spleen tissue using RT-qPCR analysis. mRNA expression analysis of Th1 , Th2 cytokines and iNOS gene expression was estimated in Withaferin-A treated mice. The experiment was done with five mice per group and significance between control (infected) and treated groups was calculated using one-way ANOVA with dunnett's post test.

Figure 4 illustrates effect of Withaferin-A on histological changes in liver. The picture depicts Hematoxylin and eosin stained liver sections of control and Withaferin-A treated mice. The arrows show the granuloma structures in liver sections.

Figure 5 illustrates the enzyme kinetics of LdPTRI . a) The recombinant Z.dPTR1 enzyme assay using Michealis-Menten was performed using 20mM sodium acetate buffer (pH 4.8) for the different concentrations of substrate biopterin. b) The enzyme inhibition studies were performed for Methotrexate and Withaferin-A using Lineweaver Burk plot. The Ki values for each compound were calculated from the graph.

Figure 6 illustrates the microscopic study which revealed the significant reduction of parasite burden in the spleen of mice suggesting that Withaferin-A have a potent antileishmanial activity alone or has synergistic effects together with Miltefosine treatment.

Figure 6 (a) illustrates the microscopic study of different drug treated groups of spleen cells; A- Infected control (Vehicle control) B- Intraperitoneal (IP) injection, C - Withferin-A (2mg/kg b.wt.,) Intraperitoneal (IP) injection, D- Withaferin -A (2mg/kg b.wt .) + Miltefosine (2.5mg/kg/b.wt.) both given orally, E- Withaferin-A (2 mg/kg b.wt.) IP injection and Miltefosine (2,5mg/kg/b.wt.,) orally, F- Miltefosine (5mg/kg/b.wt.) orally considered as Positive control.

Figure 6 (b) illustrates Numbers of LDUs/100 nucleated spleen cells.

Figure 7 illustrates the assessment of parasite burden in spleen by limiting dilution method wherein the live parasite in the spleen of Visceral Leishmaniasis mice treated with Withaferin-A alone and / or in combination with miltefosine were counted.

Figure 8 illustrates the enhanced lgG2a/lgG1 antibodies ratio in serum of different treated groups suggesting the synergistic effects of Withaferin-A upon Miltefosine treatment in BALBc mice Figure 9 illustrates the effect of Withaferin-A along with Miltefosine treatment which show that there is significantly decrease in the granuloma formation in the Liver of Visceral Leishmanial mice

Figure 9 (a) illustrates the liver section of different drug treated groups 10x & 40x images A- Control, B- Infected control, C-Withaferin-A, D-WA-Miltefosine (Both oral), E- WA (IP) & Miltefosine (Oral) and F- Miltefosine alone.

Figure 9 (b) illustrates the numbers of granuloma in different drug treated groups. Detailed description of the invention:

The following detailed description is merely exemplary in nature and is to enable any person skilled in the art to make and use the invention. The examples shown in description are not intended to limit the application and uses of the various embodiments. Various modifications to the disclosed invention will be readily apparent to those skilled in the art, and the methodology defined herein may be applied to other embodiments and applications without departing from the spirit and the scope of the present disclosure.

The foregoing objects of the present disclosure are accomplished and the problems and shortcomings associated with the prior art, and approaches are overcome by the present disclosure, as described below in the preferred embodiments.

According to the WHO statistics, 1 .5-2 million new leishmaniasis cases are being reported annually which affects 12 million people living in 88 countries. This disease is more prevalent in low-income groups; those are residing in the endemic region. Out of which 50% of the Visceral Leishmaniasis (VL) cases are from the Indian subcontinent. Current chemotherapy for leishmaniasis includes Pentavalent antimonials, Amphotericin-B and more recently introduced first oral drug Miltefosine. But the factors like the emergence of resistance, severe side effects, high cost and low efficacy have been hindering the usage of these drugs. The failure of these drugs the effective chemotherapy necessitates for the development of alternative drugs. In Applicant's previous study, Applicant showed the effective antileishmanial activity of fractions F5 and F6 through apoptotic like death mechanism in the parasite (Chandrasekaran et al. 2012). Withaferin-A is an abundant compound in the withanolides extracted from W. somnifera leaves extract fractions (F5 and F6). Therefore, to understanding of the novel biochemical interaction of Withaferin-A in the parasite clearly pave a way for developing selective antileishmanial drugs. Hence, Applicant explore the Pteridine Reductase-1 (PTR-1 ) as the target enzyme, which is exclusively present in Leishmania parasites and is essential for the growth of the parasites.

Exploiting the difference from host to host, Applicant have used L. donovani infected susceptible BALB/c mice model to mimic the visceral leishmaniasis. The mice were treated with Withaferin-A at a concentration of 2mg/kg/body weight was able to reduce the parasite burden in the spleen and liver as compared to untreated mice. It was also observed the immunomodulatory role of Withaferin-A in host cells gene expression and found that there was suppression of Th2 (IL-10, IL-4, TGF-β) cytokines and upregulation of Th1 (IL-12) cytokine.

Withaferin-A can suppress the expression of IL-10 (Th2) cytokine, which is essential for parasite propagation in host macrophages. There was no significant difference between the IFN-γ expression in the treated and untreated spleen, but the ratio of IFN-v/IL-10 is higher in treated as compared to untreated spleen. Therefore, the clearance of parasites in the spleen of VL mice was through antileishmanial and immunomodulatory properties of Withaferin-A. Accordingly, the present invention provides a novel synergistic composition for the treatment of visceral leishmaniasis which comprises an effective amount of Withaferin-A and Miltefosine.

In an embodiment of the present invention, the said composition comprises about 2mg/kg b.wt Withaferin-A and about 2.5mg/kg b.wt Miltefosine.

The present invention also provides the use of the composition for the preparation of medicines for the treatment of visceral leishmaniasis.

The present invention also provides the use of the Withaferin-A is for the treatment of visceral leishmaniasis.

The present invention also provides the method for treating a Visceral leishmaniasis, comprising administering a therapeutically effective amount of the composition of to a patient suffering from visceral leishmaniasis,

In an embodiment of the present invention, the dosage of the composition are used about Withaferin-A 2 mg and Miltefosine about 2.5mg/kg b.wt /day together) or in a single dose The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present invention.

Example - 1

Estimating the parasite number after the treatment with dose dependent increase in Withaferin-A concentration.

Ex-vivo assessment of antileishmanial activity was done with mouse peritoneal macrophages. PMM was harvested from BALB/C mice with injecting 4% thioglycollate broth in intraperitoneal cavity. After 48hrs the cells were recovered by flushing 4 ml of cold RPMI1640 medium into the peritoneal cavity. The cells were spun at 500g for 15 mins at 4°C and 2x10 ⁵ cells were plated on coverslips. After 24hrs of incubation, un adhered cells were washed and parasites were added at 1 :10 ratio. Infection was done for 16hrs and uningested parasites were washed three times with serum-free RPMI1640 medium. F5 (Ι δμς/ιτιΙ), F6 (1 (^g/ml) and Withaferin-A was added at different concentrations (0.5, 1 .0 and 1 .5μΜ) and incubated for additional 72 hrs. The coverslips were removed, fixed with methanol and Giemsa staining was done after 72 hrs of incubation. The amastigotes were counted per 100 macrophages and compared with control macrophages.

Example - 2

Effect of Withaferin A on chronic experimental visceral leishmaniasis mice model.

Female BALB/c mice (20-30 g) were injected via the tail vein with 1 x10 ⁷ L donovani promastigotes. Withaferin-A (2mg/kg/day for 14 days) was administered intraperitoneal^ starting from the 30th day of post-infection. Mice were euthanized and sacrifice after 14 days of post treatments and liver and spleen were assessed for visceral infection.

Example - 3

Estimating the parasite burden in spleen and liver of mice after the treatment. After 4 weeks of infection and 14 days of post treatment, mice were euthanized and liver and spleen touch biopsies were microscopically examined after fixing and staining the slides with Giemsa. In order to quantitate levels of infection, Leishman Donovan units (LDU) were calculated as: (number of amastigotes/number of organ nuclei) X weight of organ in grams

Example - 4

Expression analysis of Th1 and Th2 cytokines in mice spleen.

Quantitative real-time PCR (qRT-PCR) was performed to assess the expression of mRNAs for various cytokines and iNOS in splenic cells. Total RNA from splenic tissues was isolated using Nucleospin RNA kit (Machery-nagel) and cDNA was synthesized using a first-strand cDNA synthesis kit (Takara). The real time PCR analysis was carried for IFN-γ, IL-10, TGF-β and IL-4 genes and results were analysed using by the A2-ddCt method. Thus, all the values for experimental samples were expressed as fold differences between the sample mRNA and the calibrator (GAPDH) mRNA. The data are presented as the means ± SD of data from three independent experiments that yielded similar results.

Example - 5

PTR1 enzyme activity and in-vitro inhibition assay of recombinant enzyme (Lc/PTFM)

Reductase activity (/.dPTRI ) was assayed in 1 ml quartz cuvettes and the volume of each reaction was 500μΙ. The assay was initiated by the addition of substrate after incubation at 30°C for 45mins at t=0, and the rate were measured for 1 min intervals for 3 min. NADPH oxidation was monitored at 340 nm. The inhibition studies for /.dPTRI were performed at 30°C in the presence of NADPH (100μΜ) and several fixed concentrations (0 to 120μΜ) of substrate biopterin at pH 4.8 in 20 mM sodium acetate buffer (Sigma). Since pteridines exhibit absorbance changes when reduced, the extinction coefficient 7,230 M-1 cm-1 was used for the coupled oxidation/reduction of NADPH/biopterin. The kinetic parameters Km and Vmax for the Pteridine substrates were evaluated by fitting the Michaelis-Menten equation by nonlinear regression. For inhibition studies, recombinant LdPTRI was incubated with Withaferin-A and NADPH and the reaction initiated with the substrate biopterin. Lineweaver-Burk plots were used to determine the mode of inhibition by inhibitor, examining ligand competition under conditions where [inhibitor] « [enzyme] and [biopterin]. ln-vitro and ln-vivo studies reveal that Withaferin-A has antilieshmanial and immunomodulatory properties against L donovani parasite. In-silicon analysis of leishmania specific gene Pteridine Reductase-1 has strong binding energy with Withaferin-A, which inhibit the Folate biosynthesis in parasites. This enzyme is important for parasite survival in-vivo. Exploiting this, it could be help to better management of the disease with enhanced efficacy of treatment to control Visceral Leishmaniasis in endemic areas.

To our knowledge, this is first report showing the efficacy of Withaferin-A as antileishmanial and immunomodulatory compound in chronic visceral leishmaniasis mice model.

Withaferin-A has still to be evaluated at a higher concentration for more than 95% parasite reduction in spleen and liver. Additionally the safety index of this compound has to be estimated in the mice model.

Withaferin-A can be used alone or in combination with existing antileishmanial drugs like Miltefosine or Sodium Antimony Gluconate (SAG) or Amphotericin B to enhance the efficacy of these drugs in treating Visceral Leishmaniasis (VL).

Example 6

Antileishmanial activity of Withaferin-A (WA) alone and synergistic effect with Miltefosine drug treatment in VL mice Inbred BALB/c mice were infected with 1 x10 ⁷ stationary phase promastigotes of L donovani through the tail vein injection. After 30 days of post infection, mice were treated with Withaferin-A+Miltefosine for 14 days together with their respective drug control and route. Mice were Infected but not given any treated consider as infected control groups. Each group has 5 mice. The mice groups are taken as below;

1 . Infected control (Vehicle control) Intraperitoneal (IP) injection (300μΙ/ά3ν),

• 2. Withferin-A (2mg/kg b.wt./day) Intraperitoneal (IP) injection,

• 3. Withaferin -A (2mg/kg b.wt/day) + Miltefosine (2.5mg/kg/b.wt.) both given orally.

• 4. Withaferin-A (2 mg/kg b.wt./day) IP injection and Miltefosine (2,5mg/kg/b.wt.,) orally.

• 5. Miltefosine (5mg/kg/b.wt/day) orally considered as Positive control.

The microscopic study revealed the significant reduction of parasite burden in the spleen of mice suggesting that Withaferin-A has potent antileishmanial activity alone or has shown synergistic effects together with Miltefosine treatment.

Example 7

Assessment of parasite burden in spleen by limiting dilution method

Parasite burden in visceral organs was determined by limiting dilution assay (Day et al., 2013). Briefly, the spleen was homogenised in complete Medium-199 (cM199) by using 70μηι cell strainers (BD Biosciences). Total homogenates were spun down at 400xg for 5 min. The red blood cells (RBCs) of the spleen were lysed by incubating with RBC lysis buffer for 10 min at RT and followed by a wash with fresh medium at 400xg for 5 min. Finally, splenocytes were resuspended in 2ml of fresh cM199 media. For limiting dilution assay, 50 μΙ of each cell suspension was loaded separately into 1 ^st well of 96-well plates, which were preloaded with 200μΙ of fresh medium in all the wells. Next, the serial dilution was performed by mixing and transferring 50μΙ from 1 ^st well to 2 ^nd well and so on till 12 ^th well in a row. After 10- days of incubation at 25°C the plates were read under the light microscope for detection of the motile parasite in the positive well of highest dilution. It was observed that the treatments with Withaferin-A ( ^* p=0.01 ) alone or together with Miltefosine ( ^** p=0.006) significantly reduced the parasite burden in the spleen of each treated groups compared to infected control mice. Hence, the results clearly indicate that the Withaferin-A shown a potent antileishmanial activity alone or have synergistic effects together with Miltefosine treatment in VL mice.

Example 8

Quantitation of the serum lgG1 and lgG2a

Serum IgGs were quantified by standard indirect ELISA method. Briefly, 96-well ELISA plate were coated with of soluble Leishmania antigen (SLA) 0^g/10(^l/well and incubated for overnight at 4°C. Next day, the solution was discarded and washed for 3-times with 1 x phosphate buffer saline-tween20 (PBS-T) followed by blocking for 2h at RT with 150μΙ of 1 % bovine serum albumin (BSA) prepared in PBS. Plates were then washed and incubated with serum (1 :100 in dilution buffer) for 1 h at 37°C, washed again and incubated with anti-mouse lgG1 and lgG2a antibodies (Abeam ^® ) in 1 :10000 dilutions for 1 h at 37°C. Finally, the plate was washed for 3-times with 5 min interval and then incubated with 100μΙ of 1x TMB/H ₂ 0 ₂ substrate reagent for 30 min in the dark at RT. The reaction was terminated by adding 50μΙ of stop solution 1 N H ₂ S0 ₄ ) and the colour intensity was measured at A ₄₅ o _nm using microplate reader (TECAN).

Withanolides induces lgG2a response

The immune activation during the leishmaniasis determines the outcome of the disease and the humoral response in the form of antibody production which plays an important role to cure any infectious diseases. Hence, we estimated the amounts of lgG1 and lgG2a immunoglobulin's present in the mice sera from all groups. We found a significant increase of lgG2a in all treated groups (P<0.01 ) compared to infected control, which is a key marker of Th1 polarization and suppress the parasite burden in all the treated groups. Although the decrease of lgG1 in the treated group were insignificant as compared to the infected group. The ratio of lgG2a/lgG1 were significantly high in all treated groups compared to infected control. The results indicate that Withaferin-A induces the lgG2a antibodies mediated Th1 type of immune response in VL mice.

Enhanced lgG2a/lgG1 antibodies ratio in sera of different treated groups suggesting the synergistic effects of Withaferin-A upon Miltefosine treatment in BALBc mice.

Example 9

Decreased the granuloma formation in the Liver

Hepatic tissue from control and treated mice were isolated and fixed in 10% Formalin in PBS. Wax blocks were made and sections were stained with haematoxylin/eosin stain for histological studies. The number of granulomas per fifty fields was counted using a light microscope (Leica). It was observed that L. donovani infected mice showed infiltration of tissue macrophage & T lymphocytes in liver and develop into granulomatous structures harboring the amastigotes parasites; thereby the infection is cleared in the liver with time.

Withaferin-A in combination with Miltefosine treatment, mice exhibited the formation of very loose and scattered granuloma compared to the infected controls. The numbers of granuloma formation were significantly decreased in mice where combination (Withaferin-A+Miltefosine) treatment were given as compared to their alone treatment and infected control. The result indicates that Withaferin-A elicit the active granulomas formation in liver to clear the intracellular parasites.

Withaferin-A significantly decreased the granuloma formation in the Liver of VL mice along with Miltefosine treatment.