• View in gallery

    Isobolograms describing the interaction between artemisinin with indinavir (A) or nelfinavir (B) against P. falciparum isolate 3D7 and RKL 303. Each combination was done in triplicate and data point shows mean FIC of each drug in six different combination ratios with standard error bars. The line of additivity joins both axes at mean FIC50 values of combination 1 and 6.

  • 1.

    WHO, 1998. The Use of Artemisinin and its Derivatives as Antimalarial Drugs. (Report of a joint CTD/DMP/TDR informal consultation.) WHO/MAL/98.1086.

    • Search Google Scholar
    • Export Citation
  • 2.

    White NJ, 2008. Qinghaosu (artemisinin): the price of success. Science 320: 330334.

  • 3.

    WHO, 2007. Resistance to artemisinin derivatives along the Thai-Cambodian border. Wkly Epidemiol Rec 82: 360.

  • 4.

    French N, Nakiyingi J, Lugada E, Watera C, Whitworth JA, Gilks CF, 2001. Increasing rates of malarial fever with deteriorating immune status in HIV-1-infected Ugandan adults. AIDS 15: 899906.

    • Search Google Scholar
    • Export Citation
  • 5.

    Skinner-Adams TS, Andrews KT, Melville L, McCarthy J, Gardiner DL, 2007. Synergistic interactions of the antiretroviral protease inhibitors saquinavir and ritonavir with chloroquine and mefloquine against Plasmodium falciparum in vitro. Antimicrob Agents Chemother 51: 759762.

    • Search Google Scholar
    • Export Citation
  • 6.

    Joint United Nations Programme on HIV/AIDS (UNAIDS)/World Health Organization, 2005. AIDS Epidemic Update 2005. Available at: www.unaids.org/epi/2005/doc/report_pdf.asp.

    • Search Google Scholar
    • Export Citation
  • 7.

    Whitworth J, Morgan D, Quigley M, Smith A, Mayanja B, Eotu H, Omoding N, Okongo M, Malamba S, Ojwiya A, 2004. Effect of HIV-1 and increasing immunosuppression on malaria parasitaemia and clinical episodes in adults in rural Uganda: a cohort study. Lancet 356: 10511056.

    • Search Google Scholar
    • Export Citation
  • 8.

    Abu-Raddad LJ, Patnaik P, Kublin JG, 2006. Dual infection with HIV and malaria fuels the spread of both diseases in Sub-Saharan Africa. Science 314: 16031606.

    • Search Google Scholar
    • Export Citation
  • 9.

    Kim HH, Daar ES, 2009. Newer antiretroviral agents and how to use them. Curr HIV/AIDS Rep 6: 5562.

  • 10.

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193: 673675.

  • 11.

    Nguyen-Dinh P, Payne D, 1980. Pyrimethamine sensitivity in Plasmodium falciparum: determination in vitro by a modified 48-hour test. Bull World Health Organ 58: 909912.

    • Search Google Scholar
    • Export Citation
  • 12.

    Lambros C, Vanderberg JP, 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65: 418420.

  • 13.

    Parikh S, Gut J, Istvan E, Goldberg DE, Havlir DV, Rosenthal PJ, 2005. Antimalarial activity of human immunodeficiency virus type 1 protease inhibitors. Antimicrob Agents Chemother 49: 29832985.

    • Search Google Scholar
    • Export Citation
  • 14.

    Fivelman QL, Adagu IS, Warhurt DC, 2004. Modified fixed ratio isobologram method for studying in vitro interactions between atovaquone and proguanil or dihydroartemisinin against drug resistant strains of Plasmodium falciparum. Antimicrob Agents Chemother 48: 40974102.

    • Search Google Scholar
    • Export Citation
  • 15.

    Bhattacharya A, Mishra LC, Bhasin VK, 2008. In vitro activity of artemisinin in combination with clotrimazole or heat-treated amphotericin B against Plasmodium falciparum. Am J Trop Med Hyg 78: 721728.

    • Search Google Scholar
    • Export Citation
  • 16.

    Gupta S, Thapar MM, Wernsdorfer WH, Bjorkman A, 2002. In vitro interactions of artemisinin with atovaquone, quinine, and mefloquine against Plasmodium falciparum. Antimicrob Agents Chemother 46: 15101515.

    • Search Google Scholar
    • Export Citation
  • 17.

    Odds FC, 2003. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 52: 1.

  • 18.

    Chawira AN, Warhurst DC, 1987. The effect of artemisinin combined with standard antimalarials against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum in vitro. J Trop Med Hyg 90: 18.

    • Search Google Scholar
    • Export Citation
  • 19.

    Department of Health and Human Services and the Henry J. Kaiser Family Foundation, 1998. Guidelines for the use of antiretroviral agents in HIV-infected adults and adolescents. Ann Intern Med 128: 10791100.

    • Search Google Scholar
    • Export Citation
  • 20.

    Bryson YJ, Mirochnick M, Stek A, Mofenson LM, Connor J, Capparelli E, Watts DH, Huang S, Hughes MD, Kaiser K, Purdue L, Asfaw Y, Keller M, Smith E, The PACTG 353 Team, 2008. Pharmacokinetics and safety of nelfinavir when used in combination with zidovudine and lamivudine in HIV-infected pregnant women: Pediatric AIDS Clinical Trials Group (PACTG) Protocol 353. HIV Clin Trials 9: 115125.

    • Search Google Scholar
    • Export Citation
  • 21.

    Andrews KT, Fairlie DP, Madala PK, Ray J, Wyatt DM, Hilton PM, Melville LA, Beattie L, Gardiner DL, Reid RC, Stoermer MJ, Skinner-Adams T, Berry C, McCarthy JS, 2006. Potencies of human immunodeficiency virus protease inhibitors in vitro against Plasmodium falciparum and in vivo against murine malaria. Antimicrob Agents Chemother 50: 639648.

    • Search Google Scholar
    • Export Citation
  • 22.

    Meshnick SR, 1994. The mode of action of antimalarial endoperoxides. Trans R Soc Trop Med Hyg 88: 3132.

  • 23.

    Ludwig UE, Webb RJ, van Goethem IDA, East JM, Lee AG, Kimura M, O'Neill PM, Bray PG, Ward SA, Krishna S, 2003. Artemisinins target the SERCA of Plasmodium falciparum. Nature 424: 957961.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

HIV Protease Inhibitors, Indinavir or Nelfinavir, Augment Antimalarial Action of Artemisinin in vitro

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  • Department of Zoology, University of Delhi, Delhi, India

Most malaria endemic regions are co-infested with HIV infection. Treatment of one may affect outcome of the other in co-infected individuals. HIV protease inhibitors, indinavir or nelfinavir, are important antiretroviral drugs and artemisinin is central to malaria treatment. We show these protease inhibitors augment the antimalarial activity of artemisinin against P. falciparum in vitro.

Menace of malaria looms over one-fifth of the world population in more than 80 malaria endemic countries. Over one million people die annually due to Plasmodium falciparum malaria.1 Most of these deaths occur in resource poor settings. The increased morbidity and mortality has been mainly due to widespread emergence of drug resistant P. falciparum strains. After the emergence of chloroquine resistance in P. falciparum, the only sure cure of malaria rests largely on artemisinin based combination therapies.1 Artemisinin is now the only vital drug for effective treatment of malaria. Any resistance to it could be a fatal setback to malaria control programs. Recent appearance of artemisinin tolerant parasites at the Thai-Cambodia border is a cause of concern.2,3 Most of the malaria-endemic countries are also burdened and co-infested by pandemic human immunodeficiency virus.4,5 An estimated 40 million HIV-infected cases exist in Africa alone with an annual mortality of over 3 million people.6 Malaria is more prevalent, severe, and recurrent in HIV infected people than those without the virus.4,7 This co-infection would also augment the spread of geographic boundaries of malaria infection in HIV prevalent areas.8 HIV infection is long lasting, requiring almost life long antiretroviral treatment, whereas malaria is self limiting. Treatment of one infection may affect outcome of the other in co-infected individuals. Novel antiretroviral treatments are being steadily introduced.9 The effect of HIV protease inhibitors on antimalarial potency of artemisinin has not been reported. This prompted us to evaluate the interaction of HIV protease inhibitors, indinavir or nelfinavir, on the antimalarial efficacy of artemisinin on chloroquine sensitive (3D7) and chloroquine resistant (RKL 303) strains of P. falciparum in vitro.

Erythrocytic stages of chloroquine-sensitive P. falciparum, strain 3D7, and chloroquine-resistant RKL 303 strain were cultured and maintained as stocks on human B+ erythrocytes by candle-jar method10 at 37°C. Antimalarial potential of eight antiretroviral drugs (Matrix Laboratories Ltd., Secunderabad, India) and artemisinin (Sigma-Aldrich, St. Louis, MO) (Table 1) was evaluated by exposing parasites to graded concentration of drugs by the modified 48 hour test11 with the aid of candle jar method.10 Synchronized ring stage parasites12 were used in all the experiments. Percentage parasitemia and inhibition of parasite multiplication rate in relation to control was determined at the end of experiment by examining Giemsa-stained blood films. Counting of parasites was done in random adjacent microscopic fields, equivalent to about 3,000 erythrocytes at 1,000× magnification. The complete culture medium was prepared by adding sterile 5% sodium bicarbonate to Roswell Park Memorial Institute-1640 (Sigma-Aldrich) and supplemented with 10% B+ serum. Drug dilutions were prepared in antibiotic-free complete culture medium from the drug stock solution of 1 mg/mL in dimethyl sulfoxide (DMSO) 50% inhibitory concentration (IC50) values were computed from log dose-response curve (Microsoft Excel, Microsoft Corp., Redmond, WA) and presented in Table 1. HIV protease inhibitor drugs, indinavir and nelfinavir, show promising antimalarial efficacy at adequate (clinically relevant) concentrations. These in vitro results on inhibition of parasite multiplication rate by antiretroviral drugs reported here are comparable with that of other.13 Each Shouldn't there be more than one reference cited? “These in vitro results on inhibition of parasite multiplication rate by antiretroviral drugs reported here are comparable with those of others.13”of these drugs was further investigated for its antimalarial interaction in combination with artemisinin in vitro using fixed ratio method. For combination assay, artemisinin was combined with either of the drugs in six fixed-ratios of 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5.14,15 The first and the last ratio of these six preparations had the artemisinin and antiretroviral drug alone at a concentration approximately 8× their respective IC50 value against a particular parasite strain. Further dilutions of these combinations have been described in plate preparation elsewhere.14,15 After 48 hours, thin blood film slides from each triplicate experimental well and from control were prepared separately for counting the parasitemia. The fractional inhibitory concentration (FIC) for each drug in triplicate for six fixed-dose ratios was calculated by the following formula:
DE1

The sum FICs (Σ FIC) value for drugs of the six fixed-dose preparations were determined to classify the drug-drug interaction and isobologram construction. The Σ FIC < 1 represents synergism, Σ FIC ≥ 1 and < 2 represents additive interaction, Σ FIC ≥ 2 and < 4 represents slight antagonism, whereas Σ FIC ≥ 4 represents marked antagonism.1517 The isobolograms obtained from fixed ratio combinations against two strains of P. falciparum are presented in Figure 1. The points appearing below the additive line of concentration 1 and 6 represent synergism (Σ FIC < 1). The drug-drug interaction studies have previously shown antagonism between known antimalarial drugs, artemisinin and pyrimethamine;18 using this method we have also found synergism between pyrimethamine and sulfadoxine (data not presented). Indinavir synergizes the antimalarial action of artemisinin irrespective of the parasite strains (Figure 1A, Table 2), in most combination ratios. Similarly artemisinin and nelfinavir interaction shows synergism in nearly all combinations against both chloroquine resistant and chloroquine sensitive P. falciparum parasites (Figure 1B, Table 2), except at combination 4 (i.e., 2:3 drug ratios) where the interaction was additive.

Figure 1.
Figure 1.

Isobolograms describing the interaction between artemisinin with indinavir (A) or nelfinavir (B) against P. falciparum isolate 3D7 and RKL 303. Each combination was done in triplicate and data point shows mean FIC of each drug in six different combination ratios with standard error bars. The line of additivity joins both axes at mean FIC50 values of combination 1 and 6.

Citation: The American Society of Tropical Medicine and Hygiene 82, 1; 10.4269/ajtmh.2010.09-0427

Malaria and HIV infection are among the most important infectious diseases of man. HIV protease inhibitors reduce viral load in patients. Guidelines for the optimal use of antiretroviral therapy include the use of protease inhibitors, indinavir and nelfinavir, as antiretroviral agents for treatment of advanced HIV infection on the basis of their strong clinical benefit and/or restrained sustainability of plasma viral load.19 Combination antiretroviral regimens containing nelfinavir are commonly recommended to HIV-infected pregnant women.20 Risk of antimalarial treatment failure persists in immune suppressed patients. Effect of these HIV inhibitors on the antima'larial efficacy of drugs is best studied using in vitro P. falciparum culture system, devoid of immune interference. Synergistic interactions of other HIV protease inhibitors in combination with chloroquine or mefloquine have been reported.5 Intraerythrocytic parasite digests hemoglobin and converts the toxic heme generated into non-toxic crystalline hemozoin. The P. falciparum genome sequence has revealed the existence of 10 aspartic proteases (plasmepsins), some of which are involved in hemoglobin digestion.5,13 Plasmepsins inhibition by HIV protease inhibitors has already been suggested.13 However, which specific enzymes are inhibited remains to be determined. In silico docking experiments with some HIV protease inhibitors have been shown to fit in the active site of P. falciparum aspartyl proteases.21 An endoperoxide bridge in artemisinin structure interacts with iron in the infected erythrocyte to form a free toxic radical that destroys proteins in nanodomain neighborhood, that are crucial to the parasite's survival, leading to demise of the parasite.22 Artemisinin inhibits an essential calcium adenosine triphosphatase enzyme on endoplasmic reticulum of falciparum, disrupting calcium homeostasis.23 This multi-targeted attack on the parasite by artemisinin may even be responsible for inhibiting proteolytic activity of some of the hemoglobin digesting enzymes. The exact mode of action of artemisinin on parasites remains elusive as does that of indinavir or nelfinavir. Synergistic interaction observed between these two different groups of drugs may be due to protease inhibition of different antimalarial metabolic steps in hemoglobin digestion. In conclusion, indinavir or nelfinavir aid artemisinin in antimalarial activity in vitro.

  • 1.

    WHO, 1998. The Use of Artemisinin and its Derivatives as Antimalarial Drugs. (Report of a joint CTD/DMP/TDR informal consultation.) WHO/MAL/98.1086.

    • Search Google Scholar
    • Export Citation
  • 2.

    White NJ, 2008. Qinghaosu (artemisinin): the price of success. Science 320: 330334.

  • 3.

    WHO, 2007. Resistance to artemisinin derivatives along the Thai-Cambodian border. Wkly Epidemiol Rec 82: 360.

  • 4.

    French N, Nakiyingi J, Lugada E, Watera C, Whitworth JA, Gilks CF, 2001. Increasing rates of malarial fever with deteriorating immune status in HIV-1-infected Ugandan adults. AIDS 15: 899906.

    • Search Google Scholar
    • Export Citation
  • 5.

    Skinner-Adams TS, Andrews KT, Melville L, McCarthy J, Gardiner DL, 2007. Synergistic interactions of the antiretroviral protease inhibitors saquinavir and ritonavir with chloroquine and mefloquine against Plasmodium falciparum in vitro. Antimicrob Agents Chemother 51: 759762.

    • Search Google Scholar
    • Export Citation
  • 6.

    Joint United Nations Programme on HIV/AIDS (UNAIDS)/World Health Organization, 2005. AIDS Epidemic Update 2005. Available at: www.unaids.org/epi/2005/doc/report_pdf.asp.

    • Search Google Scholar
    • Export Citation
  • 7.

    Whitworth J, Morgan D, Quigley M, Smith A, Mayanja B, Eotu H, Omoding N, Okongo M, Malamba S, Ojwiya A, 2004. Effect of HIV-1 and increasing immunosuppression on malaria parasitaemia and clinical episodes in adults in rural Uganda: a cohort study. Lancet 356: 10511056.

    • Search Google Scholar
    • Export Citation
  • 8.

    Abu-Raddad LJ, Patnaik P, Kublin JG, 2006. Dual infection with HIV and malaria fuels the spread of both diseases in Sub-Saharan Africa. Science 314: 16031606.

    • Search Google Scholar
    • Export Citation
  • 9.

    Kim HH, Daar ES, 2009. Newer antiretroviral agents and how to use them. Curr HIV/AIDS Rep 6: 5562.

  • 10.

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193: 673675.

  • 11.

    Nguyen-Dinh P, Payne D, 1980. Pyrimethamine sensitivity in Plasmodium falciparum: determination in vitro by a modified 48-hour test. Bull World Health Organ 58: 909912.

    • Search Google Scholar
    • Export Citation
  • 12.

    Lambros C, Vanderberg JP, 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65: 418420.

  • 13.

    Parikh S, Gut J, Istvan E, Goldberg DE, Havlir DV, Rosenthal PJ, 2005. Antimalarial activity of human immunodeficiency virus type 1 protease inhibitors. Antimicrob Agents Chemother 49: 29832985.

    • Search Google Scholar
    • Export Citation
  • 14.

    Fivelman QL, Adagu IS, Warhurt DC, 2004. Modified fixed ratio isobologram method for studying in vitro interactions between atovaquone and proguanil or dihydroartemisinin against drug resistant strains of Plasmodium falciparum. Antimicrob Agents Chemother 48: 40974102.

    • Search Google Scholar
    • Export Citation
  • 15.

    Bhattacharya A, Mishra LC, Bhasin VK, 2008. In vitro activity of artemisinin in combination with clotrimazole or heat-treated amphotericin B against Plasmodium falciparum. Am J Trop Med Hyg 78: 721728.

    • Search Google Scholar
    • Export Citation
  • 16.

    Gupta S, Thapar MM, Wernsdorfer WH, Bjorkman A, 2002. In vitro interactions of artemisinin with atovaquone, quinine, and mefloquine against Plasmodium falciparum. Antimicrob Agents Chemother 46: 15101515.

    • Search Google Scholar
    • Export Citation
  • 17.

    Odds FC, 2003. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 52: 1.

  • 18.

    Chawira AN, Warhurst DC, 1987. The effect of artemisinin combined with standard antimalarials against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum in vitro. J Trop Med Hyg 90: 18.

    • Search Google Scholar
    • Export Citation
  • 19.

    Department of Health and Human Services and the Henry J. Kaiser Family Foundation, 1998. Guidelines for the use of antiretroviral agents in HIV-infected adults and adolescents. Ann Intern Med 128: 10791100.

    • Search Google Scholar
    • Export Citation
  • 20.

    Bryson YJ, Mirochnick M, Stek A, Mofenson LM, Connor J, Capparelli E, Watts DH, Huang S, Hughes MD, Kaiser K, Purdue L, Asfaw Y, Keller M, Smith E, The PACTG 353 Team, 2008. Pharmacokinetics and safety of nelfinavir when used in combination with zidovudine and lamivudine in HIV-infected pregnant women: Pediatric AIDS Clinical Trials Group (PACTG) Protocol 353. HIV Clin Trials 9: 115125.

    • Search Google Scholar
    • Export Citation
  • 21.

    Andrews KT, Fairlie DP, Madala PK, Ray J, Wyatt DM, Hilton PM, Melville LA, Beattie L, Gardiner DL, Reid RC, Stoermer MJ, Skinner-Adams T, Berry C, McCarthy JS, 2006. Potencies of human immunodeficiency virus protease inhibitors in vitro against Plasmodium falciparum and in vivo against murine malaria. Antimicrob Agents Chemother 50: 639648.

    • Search Google Scholar
    • Export Citation
  • 22.

    Meshnick SR, 1994. The mode of action of antimalarial endoperoxides. Trans R Soc Trop Med Hyg 88: 3132.

  • 23.

    Ludwig UE, Webb RJ, van Goethem IDA, East JM, Lee AG, Kimura M, O'Neill PM, Bray PG, Ward SA, Krishna S, 2003. Artemisinins target the SERCA of Plasmodium falciparum. Nature 424: 957961.

    • Search Google Scholar
    • Export Citation

Author Notes

*Address correspondence to Virendra K. Bhasin, Department of Zoology, University of Delhi, North Campus, Delhi-110007, India. E-mail: virendrabhasin@hotmail.com† These authors contributed equally to this work.

Financial support: This work was supported in part by the Department of Biotechnology, Ministry of Science and Technology, Government of India and University of Delhi, Delhi. LCM and MS received fellowship from the CSIR and UGC, New Delhi, respectively. AB received support from Ramjas College, North Campus, and University of Delhi.

Authors' addresses: Lokesh C. Mishra, Amit Bhattacharya, Manish Sharma, and Virendra K. Bhasin, Department of Zoology, University of Delhi, Delhi, India, E-mails: lokesh20sept@yahoo.co.in, amit4dec@yahoo.com, manish24jan@yahoo.co.in, and virendrabhasin@hotmail.com.

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