Novel Agents against Miltefosine-Unresponsive Leishmania donovani Mousumi Das,a Gundappa Saha,a Anil K. Saikia,b Vikash Kumar Dubeya Department of Biosciences and Bioengineeringa and Department of Chemistry,b Indian Institute of Technology Guwahati, Assam, India
Visceral leishmaniasis is a deadly endemic disease. Unresponsiveness to the only available oral drug miltefosine poses a big challenge for the chemotherapy of the disease. We report a novel molecule, PS-203 {4-(4,4,8-trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2-yl)benzoic acid methyl ester}, as effective against a miltefosine-unresponsive strain of the parasite. Further, combinations of PS-203 with miltefosine were also evaluated and showed promising results against a miltefosine-unresponsive strain.
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vailable drugs against leishmaniasis exhibit high toxicity and do not prevent emergence of drug resistance. Miltefosine unresponsiveness is a major concern in India (1). Combinations of available drugs need to be considered apart from the discovery of novel drug candidates effective against prevalent drug-unresponsive strains (2). Combinations of imiquimod and meglumine antimonite for treatment of cutaneous leishmaniasis reduced the amount of meglumine antimonite needed, the time of treatment, and the occurrence of drug resistance due to antimony-based therapy (3). Further, a combination of miltefosine and paromomycin proved to be the most cost-effective treatment strategy in India in recent times (4). Several other combinations of the drugs were reported to be more efficient (5–10). However, not many attempts have been made to discover new drugs for killing miltefosine-unresponsive strains. The failure of miltefosine, the only available oral drug, is a big threat, especially in India (1). Miltefosine transporter protein LdMT, and, more specifically, its beta subunit LdRos3, is involved in the formation of miltefosine translocation machinery (7, 11). A single point mutation in the LdMT is reported to be responsible for miltefosine resistance in Leishmania (7, 11). However, there seem to be several other factors responsible for miltefosine unresponsiveness. We have earlier reported that miltefosine-unresponsive strains are better able to resist reactive oxygen species (ROS) (12). Thus, to tackle miltefosine-unresponsive strains, much better ROS-producing drug candidates are required. Our group previously reported the oxabicyclo derivative PS-203 {4-(4,4,8-trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2yl)-benzoic acid methyl ester} as a good antileishmanial agent with low toxicity in vitro and in vivo (13, 14). PS-203 disturbs the redox homeostasis of the parasite. In this report, we evaluated the efficacy of PS-203 against a miltefosine-unresponsive strain in vitro. Further, combinations of PS-203 with other antileishmanial compounds were tested against the miltefosine-unresponsive strain. Parasites, cell lines, and chemicals. The Leishmania donovani (MHOM/IN/10/BHU1081) and miltefosine-unresponsive Leishmania donovani (MHOM/IN/10/BHU1155) strains were obtained from Shyam Sundar (Banaras Hindu University, India) and were cultivated in an M199 liquid medium supplemented with 15% heat-inactivated fetal bovine serum (FBS), 100 U ml⫺1 penicillin, and 100 g ml⫺1 streptomycin. BHU-1155 is a miltefosine-unresponsive isolate that was obtained from the splenic biopsy specimen of a patient after a month of miltefosine treatment and was also used in our earlier studies (12). The human macrophage cell line U937, which was used in this study, was taken from the National Centre For Cell Science (NCCS), Pune, India, and was cul-
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tured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U ml⫺1 penicillin, and 100 g ml⫺1 streptomycin. All of the chemicals used in the experiments were procured from Sigma-Aldrich or Merck. PS-203 was synthesized in our laboratory (15). Antileishmanial activity assay on promastigote cells and viability assay on human macrophage cells. Antileishmanial effects on promastigote cells and the viability of human macrophage cells were investigated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and the 50% inhibitory concentration (IC50) was calculated as described earlier (14, 16). Antileishmanial activity assay on amastigote cells. The human macrophage cell line U937 was made adherent by using 100 ng/ml phorbol 12-myristate 13-acetate (PMA) treatment overnight (17). Macrophages were then infected with miltefosine-unresponsive promastigotes for 48 h. Different concentrations of PS-203 were added and further incubated for 24 h. After incubation, cells were fixed in methanol and were Giemsa stained. Antileishmanial effect of PS-203 toward the intracellular amastigotes was evaluated by microscopic counting of 100 infected macrophage cells and was compared with an untreated control. The parasite density in treated cells was expressed as a percentage of the control. Effect of PS-203 in combination with miltefosine was evaluated for the miltefosine-unresponsive promastigote strain. The fractional inhibitory concentration (FIC) index was calculated by the following formula: FIC index ⫽ [A]/IC50A ⫹ [B]/IC50B, where IC50A and IC50B are the IC50s of miltefosine and PS-203, respectively, when tested alone, and [A] and [B] are the IC50s of the miltefosine or PS-203 when treatment was carried out in combination. An FIC index of ⱕ0.5 indicates synergy while an index of ⬎4 indicates antagonism, and an index in a range between 0.5 and 4 indicates indifference (18, 19). The miltefosine-responsive and miltefosine-unresponsive strains were grown in our lab, and sensitivity toward miltefosine was reconfirmed by MTT assay. The data show a biphasic
Received 21 April 2015 Returned for modification 8 June 2015 Accepted 11 September 2015 Accepted manuscript posted online 21 September 2015 Citation Das M, Saha G, Saikia AK, Dubey VK. 2015. Novel agents against miltefosine-unresponsive Leishmania donovani. Antimicrob Agents Chemother 59:7826 –7829. doi:10.1128/AAC.00928-15. Address correspondence to Vikash Kumar Dubey, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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FIG 1 Effect of miltefosine or PS-203 on Leishmania promastigotes. (A) Miltefosine treatment for a miltefosine-unresponsive strain of L. donovani promastigote
Ld1155. The IC50 for the Ld1155 strain is 100 M. The data confirms that the Ld1155 strain is a miltefosine-unresponsive strain. (B) Effect of the PS-203 compound on Ld1081 and Ld1155 strains of L. donovani. The data indicate that the compound is equally effective on the two strains. The IC50 of PS-203 against the MHOM/IN/10/BHU1081 and MHOM/IN/10/BHU1155 strains is 4.0 ⫾ 0.5 M, which is marginally low compared to our earlier studies (14). The chemical structure of the PS-203 compound {4-(4,4,8-trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2-yl-benzoic acid methyl ester} is shown in the inset of panel B. Data represent the mean ⫾ standard deviation (SD) of the results of three independent experiments.
death pattern in the killing of miltefosine-unresponsive Leishmania donovani (BHU-1155) upon treatment with miltefosine, and the IC50 was found to be higher than that for the wild-type strain, which is 100 M (Fig. 1A). The data reconfirm that the strain is miltefosine unresponsive in laboratory conditions. The effect of PS-203 on miltefosine-unresponsive Leishmania donovani (BHU-1155) as well as miltefosine-responsive Leishmania
donovani (BHU-1081) was also evaluated in similar laboratory conditions for comparison (Fig. 1B). The data indicate that PS203 is equally effective against the two strains of miltefosine, with an IC50 of 4.0 ⫾ 0.5 M. The IC50 of PS-203 against the miltefosine-responsive strain observed was marginally lower than our previous report (20). We also explored a combination of PS-203 with miltefosine in
FIG 2 Effect of the miltefosine and PS-203 combination on the miltefosine-unresponsive strain of L. donovani promastigote (BHU 1155) as well as on the miltefosine-responsive strain of L. donovani promastigote (BHU 1081). (A) PS-203 shows significantly improved efficacy in the presence of miltefosine against the miltefosine-responsive strain. (B) The miltefosine-unresponsive strain treated with PS-203 alone and with PS-203 (2.5 M) and miltefosine (10 M, 50 M, or 100 M) in combination. The combination of the drugs has shown improved efficacy. Data represent the mean ⫾ SD of the results of three independent experiments.
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by the emergence of drug resistance, which is a matter of concern. Miltefosine (hexadecylphosphocholine) was a major breakthrough for the treatment of leishmaniasis. Miltefosine unresponsiveness is a greater challenge in the management of the disease. We have identified the PS-203 compound as very effective against a miltefosine-unresponsive strain. Further, our data suggest that a combination of PS-203 and miltefosine may enhance the efficacy of miltefosine against unresponsive strains as well as responsive strains of Leishmania donovani. ACKNOWLEDGMENTS Research fellowships awarded to M.D. and G.S. by IIT Guwahati are acknowledged. Financial support by the Department of Biotechnology, government of India, in the form of research grants (project no. BT/01/IYBA/ 2009 and BT/PR3409/MED/29/326/2011) to V.K.D. is also acknowledged.
REFERENCES FIG 3 Effect of PS-203 on amastigote cells of a miltefosine-unresponsive strain. (A) Percentage of amastigotes present in a human macrophage after different doses of PS-203 in infected macrophages. Data represent the mean ⫾ SD of the results of three independent experiments. The statistical significance of differences between the two groups was analyzed by unpaired Student’s t test using SigmaPlot software. Data having P values of ⬍0.05 (*) and ⬍0.001 (**) were considered to have statistically significant differences.
the miltefosine-responsive and miltefosine-unresponsive strains of Leishmania (Fig. 2). The data show that PS-203 shows improved efficacy in combination with miltefosine. The IC50 of PS-203 was ⬎1 M with 25 M miltefosine for the miltefosineresponsive stain (Fig. 2A). Interestingly, as shown in Fig. 2A, 25 M miltefosine without PS-203 (0 M on the x axis) can kill only 50% of Leishmania promastigotes, but in the presence of 1 M PS-203, its efficacy is significantly higher. Similarly, combination of PS-203 with miltefosine decreased the viability of miltefosineunresponsive promastigotes. The data shown in Fig. 2B were used for the calculation of the FIC index obtained for the miltefosine and PS-203 combination. The combination of PS-203 (1 M) and miltefosine (100 M) kills 50% of miltefosine-unresponsive promastigotes. The IC50 of PS-203 in the presence of 100 M miltefosine was 1 M. Likewise, the IC50 of miltefosine in the presence of 1 M PS-203 was found to be 100 M (Fig. 2B). The calculated FIC index for the miltefosine-unresponsive promastigotes was found to be 1.25, suggesting that the combination shows an additive response. Similarly, the FIC index for miltefosine-responsive promastigotes was calculated to be ⬃0.8, suggesting the combination was additive. The effects of PS-203 on the amastigote stage of the parasite inside human macrophages and on the antileishmanial activity were studied microscopically. A significant decrease was observed in parasite burden in infected macrophage cells treated with PS203. Nearly 50% of the amastigotes were inhibited at 5.7 g/ml of PS-203 treatment (Fig. 3). Infected macrophages without PS-203 treatment were used as a control. There is a decrease in the number of amastigotes with an increased dose of PS-203 treatment that suggests an antiproliferative effect on amastigotes. In our earlier studies, PS-203 was found to be safe in an in vivo model (13). Further, our current studies show no effect of PS-203 on human macrophage cells (cell line U937) even at higher concentrations up to 100 M. Control of the worldwide spread of leishmaniasis is challenged
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1. Croft SL, Sundar S, Fairlamb AH. 2006. Drug resistance in leishmaniasis. Clin Microbiol Rev 19:111–126. http://dx.doi.org/10.1128/CMR.19.1.111 -126.2006. 2. Maltezou HC. 2010. Drug resistance in visceral leishmaniasis. J Biomed Biotechnol 2010:617521. 3. Arevalo I, Ward B, Miller R, Meng TC, Najar E, Alvarez E, Matlashewski G, Llanos-Cuentas A. 2001. Successful treatment of drugresistant cutaneous leishmaniasis in humans by use of imiquimod, an immunomodulator. Clin Infect Dis 33:1847–1851. http://dx.doi.org/10 .1086/324161. 4. Meheus F, Balasegaram M, Olliaro P, Sundar S, Rijal S, Faiz MA, Boelaert M. 2010. Cost effectiveness analysis of combination therapies for visceral leishmaniasis in the Indian subcontinent. PLoS Negl Trop Dis 4(9):e818. http://dx.doi.org/10.1371/journal.pntd.0000818. 5. Bryceson A. 2001. A policy for leishmaniasis with respect to the prevention and control of drug resistance. Trop Med Int Health 6:928 –934. http: //dx.doi.org/10.1046/j.1365-3156.2001.00795.x. 6. Jha TK. 2006. Drug unresponsiveness and combination therapy for kalaazar. Indian J Med Res 123:389 –398. 7. Seifert K, Pérez-Victoria FJ, Stettler M, Sánchez-Cañete MP, Castanys S, Gamarro F, Croft SL. 2007. Inactivation of the miltefosine transporter, LdMT, causes miltefosine resistance that is conferred to the amastigote stage of Leishmania donovani and persists in vivo. Int J Antimicrob Agents 30:229 –235. http://dx.doi.org/10.1016/j.ijantimicag.2007.05.007. 8. van Griensven J, Balasegaram M, Meheus F, Alvar J, Lynen L, Boelaert M. 2010. Combination therapy for visceral leishmaniasis. Lancet Infect Dis 10:184 –194. http://dx.doi.org/10.1016/S1473-3099(10)70011-6. 9. Freitas-Junior LH, Chatelain E, Kim HA, Siqueira-Neto JL. 2012. Visceral leishmaniasis treatment: what do we have, what do we need and how to deliver it? Int J Parasitol: Drugs Drug Resist 2:11–19. http://dx.doi.org /10.1016/j.ijpddr.2012.01.003. 10. Purkait B, Kumar A, Nandi N, Sardar AH, Das S, Kumar S, Pandey K, Ravidas V, Kumar M, De T, Singh D, Das P. 2012. Mechanism of amphotericin B resistance in clinical isolates of Leishmania donovani. Antimicrob Agents Chemother 56:1031–1041. http://dx.doi.org/10.1128 /AAC.00030-11. 11. Seifert K, Matu S, Javier Pérez-Victoria F, Castanys S, Gamarro F, Croft SL. 2003. Characterisation of Leishmania donovani promastigotes resistant to hexadecylphosphocholine (miltefosine). Int J Antimicrob Agents 22:380 –387. http://dx.doi.org/10.1016/S0924-8579(03)00125-0. 12. Das M, Saudagar P, Sundar S, Dubey VK. 2013. Miltefosineunresponsive Leishmania donovani has a greater ability than miltefosineresponsive L. donovani to resist reactive oxygen species. FEBS J 280:4807– 4815. http://dx.doi.org/10.1111/febs.12449. 13. Saudagar P, Mudavath SL, Saha P, Saikia AK, Sundar S, Dubey VK. 2014. In vivo assessment of antileishmanial property of 4-(4,4,8Trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2-yl)-benzoic acid methyl ester, an oxabicyclo[3.3.1]nonanones. Lett Drug Des Discov 11:937–939. http://dx.doi.org/10.2174/1570180811666140423203826. 14. Saudagar P, Saha P, Saikia AK, Dubey VK. 2013. Molecular mechanism underlying antileishmanial effect of oxabicyclo[3.3.1]nonanones: inhibition of key redox enzymes of the pathogen. Eur J Pharm Biopharm 85: 569 –577. http://dx.doi.org/10.1016/j.ejpb.2013.08.014.
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15. Saha P, Reddy UC, Bondalapati S, Saikia AK. 2010. A novel synthesis of oxabicyclo[3.3.1]nonanone via (3,5)-oxonium-ene reaction. Org Lett 12: 1824 –1826. http://dx.doi.org/10.1021/ol1004092. 16. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55– 63. http://dx.doi.org/10.1016/0022-1759(83)90303-4. 17. Takashiba S, Van Dyke TE, Amar S, Murayama Y, Soskolne AW, Shapira L. 1999. Differentiation of monocytes to macrophages primes cells for lipopolysaccharide stimulation via accumulation of cytoplasmic nuclear factor kappaB. Infect Immun 67:5573–5578. 18. Pastor J, García M, Steinbauer S, Setzer WN, Scull R, Gille L, Monzote
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L. 2015. Combinations of ascaridole, carvacrol, and caryophyllene oxide against Leishmania. Acta Trop 145:31–38. http://dx.doi.org/10.1016/j .actatropica.2015.02.002. 19. de Morais-Teixeira E, Gallupo MK, Rodrigues LF, Romanha AJ, Rabello A. 2014. In vitro interaction between paromomycin sulphate and four drugs with leishmanicidal activity against three new world Leishmania species. J Antimicrob Chemother 69:150 –154. http://dx.doi.org /10.1093/jac/dkt318. 20. Saudagar P, Dubey VK. 2014. Carbon nanotube based betulin formulation shows better efficacy against Leishmania parasite. Parasitol Int 63: 772–776. http://dx.doi.org/10.1016/j.parint.2014.07.008.
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Visceral leishmaniasis is a deadly endemic disease. Unresponsiveness to the only available oral drug miltefosine poses a big challenge for the chemotherapy of the disease. We report a novel molecule, PS-203 {4-(4,4,8-trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2-yl)benzoic acid methyl ester}, as effective against a miltefosine-unresponsive strain of the parasite. Further, combinations of PS-203 with miltefosine were also evaluated and showed promising results against a miltefosine-unresponsive strain.
A
vailable drugs against leishmaniasis exhibit high toxicity and do not prevent emergence of drug resistance. Miltefosine unresponsiveness is a major concern in India (1). Combinations of available drugs need to be considered apart from the discovery of novel drug candidates effective against prevalent drug-unresponsive strains (2). Combinations of imiquimod and meglumine antimonite for treatment of cutaneous leishmaniasis reduced the amount of meglumine antimonite needed, the time of treatment, and the occurrence of drug resistance due to antimony-based therapy (3). Further, a combination of miltefosine and paromomycin proved to be the most cost-effective treatment strategy in India in recent times (4). Several other combinations of the drugs were reported to be more efficient (5–10). However, not many attempts have been made to discover new drugs for killing miltefosine-unresponsive strains. The failure of miltefosine, the only available oral drug, is a big threat, especially in India (1). Miltefosine transporter protein LdMT, and, more specifically, its beta subunit LdRos3, is involved in the formation of miltefosine translocation machinery (7, 11). A single point mutation in the LdMT is reported to be responsible for miltefosine resistance in Leishmania (7, 11). However, there seem to be several other factors responsible for miltefosine unresponsiveness. We have earlier reported that miltefosine-unresponsive strains are better able to resist reactive oxygen species (ROS) (12). Thus, to tackle miltefosine-unresponsive strains, much better ROS-producing drug candidates are required. Our group previously reported the oxabicyclo derivative PS-203 {4-(4,4,8-trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2yl)-benzoic acid methyl ester} as a good antileishmanial agent with low toxicity in vitro and in vivo (13, 14). PS-203 disturbs the redox homeostasis of the parasite. In this report, we evaluated the efficacy of PS-203 against a miltefosine-unresponsive strain in vitro. Further, combinations of PS-203 with other antileishmanial compounds were tested against the miltefosine-unresponsive strain. Parasites, cell lines, and chemicals. The Leishmania donovani (MHOM/IN/10/BHU1081) and miltefosine-unresponsive Leishmania donovani (MHOM/IN/10/BHU1155) strains were obtained from Shyam Sundar (Banaras Hindu University, India) and were cultivated in an M199 liquid medium supplemented with 15% heat-inactivated fetal bovine serum (FBS), 100 U ml⫺1 penicillin, and 100 g ml⫺1 streptomycin. BHU-1155 is a miltefosine-unresponsive isolate that was obtained from the splenic biopsy specimen of a patient after a month of miltefosine treatment and was also used in our earlier studies (12). The human macrophage cell line U937, which was used in this study, was taken from the National Centre For Cell Science (NCCS), Pune, India, and was cul-
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tured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U ml⫺1 penicillin, and 100 g ml⫺1 streptomycin. All of the chemicals used in the experiments were procured from Sigma-Aldrich or Merck. PS-203 was synthesized in our laboratory (15). Antileishmanial activity assay on promastigote cells and viability assay on human macrophage cells. Antileishmanial effects on promastigote cells and the viability of human macrophage cells were investigated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and the 50% inhibitory concentration (IC50) was calculated as described earlier (14, 16). Antileishmanial activity assay on amastigote cells. The human macrophage cell line U937 was made adherent by using 100 ng/ml phorbol 12-myristate 13-acetate (PMA) treatment overnight (17). Macrophages were then infected with miltefosine-unresponsive promastigotes for 48 h. Different concentrations of PS-203 were added and further incubated for 24 h. After incubation, cells were fixed in methanol and were Giemsa stained. Antileishmanial effect of PS-203 toward the intracellular amastigotes was evaluated by microscopic counting of 100 infected macrophage cells and was compared with an untreated control. The parasite density in treated cells was expressed as a percentage of the control. Effect of PS-203 in combination with miltefosine was evaluated for the miltefosine-unresponsive promastigote strain. The fractional inhibitory concentration (FIC) index was calculated by the following formula: FIC index ⫽ [A]/IC50A ⫹ [B]/IC50B, where IC50A and IC50B are the IC50s of miltefosine and PS-203, respectively, when tested alone, and [A] and [B] are the IC50s of the miltefosine or PS-203 when treatment was carried out in combination. An FIC index of ⱕ0.5 indicates synergy while an index of ⬎4 indicates antagonism, and an index in a range between 0.5 and 4 indicates indifference (18, 19). The miltefosine-responsive and miltefosine-unresponsive strains were grown in our lab, and sensitivity toward miltefosine was reconfirmed by MTT assay. The data show a biphasic
Received 21 April 2015 Returned for modification 8 June 2015 Accepted 11 September 2015 Accepted manuscript posted online 21 September 2015 Citation Das M, Saha G, Saikia AK, Dubey VK. 2015. Novel agents against miltefosine-unresponsive Leishmania donovani. Antimicrob Agents Chemother 59:7826 –7829. doi:10.1128/AAC.00928-15. Address correspondence to Vikash Kumar Dubey, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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FIG 1 Effect of miltefosine or PS-203 on Leishmania promastigotes. (A) Miltefosine treatment for a miltefosine-unresponsive strain of L. donovani promastigote
Ld1155. The IC50 for the Ld1155 strain is 100 M. The data confirms that the Ld1155 strain is a miltefosine-unresponsive strain. (B) Effect of the PS-203 compound on Ld1081 and Ld1155 strains of L. donovani. The data indicate that the compound is equally effective on the two strains. The IC50 of PS-203 against the MHOM/IN/10/BHU1081 and MHOM/IN/10/BHU1155 strains is 4.0 ⫾ 0.5 M, which is marginally low compared to our earlier studies (14). The chemical structure of the PS-203 compound {4-(4,4,8-trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2-yl-benzoic acid methyl ester} is shown in the inset of panel B. Data represent the mean ⫾ standard deviation (SD) of the results of three independent experiments.
death pattern in the killing of miltefosine-unresponsive Leishmania donovani (BHU-1155) upon treatment with miltefosine, and the IC50 was found to be higher than that for the wild-type strain, which is 100 M (Fig. 1A). The data reconfirm that the strain is miltefosine unresponsive in laboratory conditions. The effect of PS-203 on miltefosine-unresponsive Leishmania donovani (BHU-1155) as well as miltefosine-responsive Leishmania
donovani (BHU-1081) was also evaluated in similar laboratory conditions for comparison (Fig. 1B). The data indicate that PS203 is equally effective against the two strains of miltefosine, with an IC50 of 4.0 ⫾ 0.5 M. The IC50 of PS-203 against the miltefosine-responsive strain observed was marginally lower than our previous report (20). We also explored a combination of PS-203 with miltefosine in
FIG 2 Effect of the miltefosine and PS-203 combination on the miltefosine-unresponsive strain of L. donovani promastigote (BHU 1155) as well as on the miltefosine-responsive strain of L. donovani promastigote (BHU 1081). (A) PS-203 shows significantly improved efficacy in the presence of miltefosine against the miltefosine-responsive strain. (B) The miltefosine-unresponsive strain treated with PS-203 alone and with PS-203 (2.5 M) and miltefosine (10 M, 50 M, or 100 M) in combination. The combination of the drugs has shown improved efficacy. Data represent the mean ⫾ SD of the results of three independent experiments.
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by the emergence of drug resistance, which is a matter of concern. Miltefosine (hexadecylphosphocholine) was a major breakthrough for the treatment of leishmaniasis. Miltefosine unresponsiveness is a greater challenge in the management of the disease. We have identified the PS-203 compound as very effective against a miltefosine-unresponsive strain. Further, our data suggest that a combination of PS-203 and miltefosine may enhance the efficacy of miltefosine against unresponsive strains as well as responsive strains of Leishmania donovani. ACKNOWLEDGMENTS Research fellowships awarded to M.D. and G.S. by IIT Guwahati are acknowledged. Financial support by the Department of Biotechnology, government of India, in the form of research grants (project no. BT/01/IYBA/ 2009 and BT/PR3409/MED/29/326/2011) to V.K.D. is also acknowledged.
REFERENCES FIG 3 Effect of PS-203 on amastigote cells of a miltefosine-unresponsive strain. (A) Percentage of amastigotes present in a human macrophage after different doses of PS-203 in infected macrophages. Data represent the mean ⫾ SD of the results of three independent experiments. The statistical significance of differences between the two groups was analyzed by unpaired Student’s t test using SigmaPlot software. Data having P values of ⬍0.05 (*) and ⬍0.001 (**) were considered to have statistically significant differences.
the miltefosine-responsive and miltefosine-unresponsive strains of Leishmania (Fig. 2). The data show that PS-203 shows improved efficacy in combination with miltefosine. The IC50 of PS-203 was ⬎1 M with 25 M miltefosine for the miltefosineresponsive stain (Fig. 2A). Interestingly, as shown in Fig. 2A, 25 M miltefosine without PS-203 (0 M on the x axis) can kill only 50% of Leishmania promastigotes, but in the presence of 1 M PS-203, its efficacy is significantly higher. Similarly, combination of PS-203 with miltefosine decreased the viability of miltefosineunresponsive promastigotes. The data shown in Fig. 2B were used for the calculation of the FIC index obtained for the miltefosine and PS-203 combination. The combination of PS-203 (1 M) and miltefosine (100 M) kills 50% of miltefosine-unresponsive promastigotes. The IC50 of PS-203 in the presence of 100 M miltefosine was 1 M. Likewise, the IC50 of miltefosine in the presence of 1 M PS-203 was found to be 100 M (Fig. 2B). The calculated FIC index for the miltefosine-unresponsive promastigotes was found to be 1.25, suggesting that the combination shows an additive response. Similarly, the FIC index for miltefosine-responsive promastigotes was calculated to be ⬃0.8, suggesting the combination was additive. The effects of PS-203 on the amastigote stage of the parasite inside human macrophages and on the antileishmanial activity were studied microscopically. A significant decrease was observed in parasite burden in infected macrophage cells treated with PS203. Nearly 50% of the amastigotes were inhibited at 5.7 g/ml of PS-203 treatment (Fig. 3). Infected macrophages without PS-203 treatment were used as a control. There is a decrease in the number of amastigotes with an increased dose of PS-203 treatment that suggests an antiproliferative effect on amastigotes. In our earlier studies, PS-203 was found to be safe in an in vivo model (13). Further, our current studies show no effect of PS-203 on human macrophage cells (cell line U937) even at higher concentrations up to 100 M. Control of the worldwide spread of leishmaniasis is challenged
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