Jyoti Joshi and Sukhbir Kaur. / International Journal of Biopharmaceutics. 2015; 6(1): International Journal of Biopharmaceutics

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1 13 e- ISSN Print ISSN International Journal of Biopharmaceutics Journal homepage: IJB TO EVALUATE THE CURATIVE POTENTIAL OF SINGLE DOSE OF CISPLATIN IN COMBINATION WITH IMMUNOTHERAPY IN INBRED BALB/C MICE AGAINST VISCERAL LEISHMANIASIS Jyoti Joshi and Sukhbir Kaur* Department of Zoology, Panjab University, Chandigarh , India. ABSTRACT Visceral leishmaniasis (VL) is a severe chronic systemic disease caused by the protozoan parasites of the genus Leishmania. The conventional drugs for treatment of VL have limitations including unresponsiveness, relapse, specific toxicities and parenteral administration lasting for long durations. In this context we evaluated the short course (single dose) therapy using cisplatin and Killed Leishmania donovani (KLD) antigen/78kda antigen along with MPL-A for its protective efficacy and immunomodulatory activity. Animals were infected with 10 7 promastigotes of L. donovani and a month after infection, these animals were given a single dose of either cisplatin or cisplatin+kld+mpl-a or cisplatin+78kda+mpl-a. To check their curative potential, the treated BALB/c mice were then sacrificed on 15 and 30 d.p.t. respectively. It was found that animals treated with cisplatin along with immunotherapy not only resulted in parasite elimination but the immune profile was greatly enhanced (IFN- and IL-2 ) to protective Th1 type and the profile of IL-10 and IL-4 was downregulated. This emphasised the use of single dose of cisplatin along with immunotherapy which can be an alternative for treatment of visceral leishmaniasis. Key words: Visceral leishmaniasis, cisplatin, Leishmania donovani. INTRODUCTION Visceral leishmaniasis caused by Leishmania donovani represents a major public health problem in tropical and subtropical regions of the world. With an estimated 300,000 cases per year, India carries the largest VL burden (Sundar and Chakravarty, 2013). In active VL, macrophages host the replicating amastigotes in phagolysosomal compartments leading to splenomegaly, hepatomegaly, hyperglobulinemia, anemia, weight-loss, incessant fever and ultimately death if not treated (Banjara, 2013). No effective vaccine is yet available against this parasite and its control relies primarily on chemotherapy. Corresponding Author Sukhbir Kaur puzoology@yahoo.com Current challenges in anti-leishmanial chemotherapy include the increasing resistance of parasites to the frontline drugs, high costs, toxicity, and a limited repertoire of new available drugs (Mukhopadhyay et al., 2012). Since the discovery of the pentavalent antimonials, until today, the search for newer drugs with antileishmanial activity, without toxicity, and the drugs able to overcome the emergence of drug resistant strains, still remains the current goal. So far no antileishmanial therapy is available which does not have any side effects. It is well known that VL causes immune supression and the effective treatment is dependent on the generation of protective Th2 type of immune response. Because of this synergy between chemotherapy and protective immunity, it was thought that immunochemotherapy can be promising in the treatment

2 14 of visceral leishmaniasis. A recent study with cisplatin, and KLD/78kDa antigen along with MPL-A (double dose), reported successful in BALB/c mice with established L. donovani infection (Joshi and Kaur, 2014, Joshi et al., 2014). Here we have evaluated lower doses of cisplatin immunochemotherapy (single dose) for their efficacy in curing murine VL. MATERIALS AND METHODS Animals and parasites BALB/c mice (4-6 weeks old) used in the experiments were obtained from Institute of Microbial Technology, Chandigarh, India and then maintained in the Central Animal House, Panjab University, Chandigarh. This study was carried out according to the guidelines of the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA, Registration No. 45/1999/ CPCSEA). L. donovani strain Dd8 (MHOM/IN/80/Dd8), originally obtained from London School of Hygiene and Tropical Medicine, London, was maintained in vitro at 22 o C + 1 o C in modified NNN medium by serial culturing after every h (Rao et al., 1984). Experimental Infection In vivo infection and treatment Twelve mice from each group were infected intracardially with 10 7 promastigotes/0.1ml (Kaur et al., 2010). The infected animals after 30 days post infection were divided into four different groups. Group 1 served as infected animals. Group 2 animals received intraperitoneal injection of cisplatin at a dose of 0.5 mg/kg for five days. Group 3 and 4 animals were given a combination of drug (cisplatin) and a subcutaneous injection of vaccine (KLD+MPL-A/78kDa+MPL-A) for Figure 1. Hepatic parasite load in terms of Leishman Donovan Units (LDU) in different groups of animals one day only. The protocol for preparing antigens and drugs were followed as per the method of Joshi et al., Further, the animals were then sacrificed on 15 and 30 days post treatment. Parameters studied Assessment of Infection Livers of all the animals were aseptically removed and their impression smears 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 cell nuclei X weight of organ in milligrams (Bradley and Kirkley 1977). Cytokine responses The lymphocytes from spleens of infected and drug treated mice was cultured in 24 well plates in 1 ml of RPMI-1640 containing 20 mm NaHCO3, 10 mm HEPES, 10 U/ml of penicillin, 100µg/ml streptomycin and 2mM L-glutamine and 10% FCS. Cells were stimulated with 50µg/ml of the parasite antigen and then cells were incubated at 37ºC for 72h and supernatants were collected and stored at -20ºC. This was then assayed for IL-2, IL-10,IL-4 and IFN-γ by using ELISA kits (BenderMed Systems, Diaclone, France) (Kaur et al., 2008). Statistical Analysis The statistical significance of the difference between various groups was determined by PostHoc test and ANOVA. Differences were considered statistically significant for p<0.05. Figure 2a. IFN- levels in different groups of animals. * p value Infected vs Infected+cisplatin/ Infected * p value Infected vs Infected+cisplatin/ Infected

3 15 Figure 2b. IL-2 levels in different groups of animals. Figure 3a. IL-4 levels in different groups of animals. * p value Infected vs Infected+cisplatin/ Infected * p value Infected vs Infected+cisplatin/ Infected Figure 3b. IL-10 levels in different groups of animals. * p value Infected vs Infected+cisplatin/ Infected RESULTS Parasite Load Treatment of animals with immuno chemotherapy significantly (p<0.001) reduced the parasite load as compared to the infected controls. Maximum reduction in parasite load (p<0.001) was observed in the animals treated with cisplatin+78kda +MPL-A, and the parasite load lessened by 89.76% (437.32±48.23) respectively on 30 d.p.t (Fig 1). Cytokine Responses IFN- and IL-2 The protective TH1 cytokines (IFN- and IL-2) was found to be high in the treated animals as compared to the infected controls (p<0.001). Maximum levels of these cytokines were observed in animals treated with cisplatin+78kda+mpl-a followed by animals treated with cisplatin+kld+mpl-a on 15 d.p.t. respectively (Fig 2a,b) IL-4 And IL-10 The levels of Th2 regulated cytokines IL-4 and IL-10 were significantly lesser in treated animals as compared to the infected controls. Maximum levels of this cytokine were observed in the infected controls. Its levels were found to be significantly reduced (p<0.001) in immunochemotherapy treated animals as compared to the infected controls. Minimum levels of this cytokine were observed in animals treated with cisplatin+78kda+mpl-a (Fig 3a,b). DISCUSSION AND CONCLUSION Although for the control of leishmaniasis, a large number of new and improved drugs are becoming available, but none of the drugs so far meets the criteria of an efficient antileishmanial with little or no toxicity. Moreover, complete elimination of the parasite from current treatments is never achieved. Herein we investigated the curative efficacy and immunomodulatory potential of cisplatin along with 78 kda and KLD antigen formulated with an adjuvant (MPL-A). Our results show that maximum protection was observed in animals treated with immunochemotherapy (cisplatin+78kda+mpl-a). The results are in consistence with our earlier studies where imparting two doses of immunochemotherapy led

4 16 to a significant decline in parasite load (Joshi and Kaur, 2014). Approximately 100% protection against canine VL was achieved in dogs vaccinated with LiESAp vaccine (a 54 kda excreted protein of L.infantum) combined with muramyl dipeptide (Lemesre et al., 2007). However while crude parasite extracts together with Glucantime were only partially effective, purified antigen LiF2 (L.infantum-derived fraction 2) combined with Glucantime cured all dogs within 6 months (Santos et al., 2007). Moreover, longer progression-free survival with no treatment related deaths was observed in phase I/II clinical trials of advanced gastric cancer patients when treated with S-1 plus cisplatin (Koizumi et al., 2008). The fatal visceral disease in humans is the result of intramacrophage infection and the outcome of infection is largely dependent on the ability of the host to mount a Th1 or Th2 type of immune response (Kaur et al., 2011). It has been observed that the effector memory (CD45RA - CCR7 - ) CD4+ T cells are the main population of cells producing IFN-gamma in cured CL and ML individuals who responded in vitro to SLA (soluble L. braziliensis antigen) (Carvalho et al., 2013). Treatment of animals with different therapies induced preferential production of IFN- and a massive increase was seen in the splenocytes of immunochemotherapy treated animals. When cisplatin and UFT, which is a prodrug of 5-FU, were administered with an immunomodulator polysaccharide K (PSK) to ten patients with colorectal cancer, an increased concentration of IFN- was observed with reduced production of IL-10 after 2 months of treatment demonstrating the immunomodulatory potential of this combination (Shibata et al., 2002). Cisplatin is known to boost the cytotoxic T-lymphocyte mediated antitumor immunity which plays a key role in protection against Leishmania species. Therefore, cisplatin may enhance the CD8+ T cell mediated killing of the parasite. Substantial levels of IL-2, a principal T cell growth factor for Th1 type of immune response, was also produced by all the treated animals. A preliminary study suggested that low-dose CDDP and IL-2 in association with the pineal hormone MLT, given as a second line therapy, is an effective and well-tolerated treatment for patients with metastatic melanoma, with a clinical efficacy at least comparable to that obtained with a first-line therapy of dacarbazine plus interferon-alpha (Lissoni et al., 2002). Similarly, a study carried out by Toledo et al. (2001) where the continuous administration of Leishmania antigen in immunochemotherapy (Glucantime plus Leishvacin) treated ACL patients may have contributed to a greater decrease in IL-10 production which lead to a faster elimination of the parasites by IFN- activated macrophages. Hence, our results show that the immune modulation by immunochemotherapy may be responsible for the disease resolution in inbred BALB/c mice. In conclusion, we can say that treatment of L. donovani infected mice with single dose of immunochemotherapy resulted in hepatic parasite reduction without inflicting any liver and kidney toxicity (data not shown). This decline in parasite number was supported by a curative immune response with a significant fall in disease promoting cytokines (IL-10 and IL-4) and subsequent increase in protective cytokines (IL-2 and IFNγ). In the light of these observations, our study highlighted that single dose of immunochemotherapy can be a better option for treatment over other therapies in the control of visceral leishmaniasis. REFERENCES Banjara MR. Combination therapy for visceral leishmaniasis. Int J Infect Microbiol. 2013; 2: Bradley DJ and Kirkley J. 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