Journal of Antimicrobial Chemotherapy (1992) 30, 827-832
The activity of hydroxynaphthoquinones against Leishmania donovani S. L. Croft*, J. Hogg, W. E. Gutteridge, A. T. Hudson and A. W. RaodaU
Wellcome Research Laboratories, Beckenham, Kent, BR3 3BS, UK
Introduction
Leishmania donovani, a haemoflagcllatc protozoan parasite, is the causative agent of potentially-fatal visceral leishmaniasis (kala-azar). The parasite survives and multiplies in the mammalian host as an intracellular amastigote form in the macrophages of the liver, spleen and bone marrow. At present, clinical treatment for visceral leishmaniasis is dependent primarily upon pentavalent antimonials, which require long courses of treatment at high doses and parenteral administration. They also have toxic side-effects and variable efficacy (Bryceson, 1987). As part of a programme to find new drugs for the treatment of leishmaniasis, a series of hydroxynaphthoquinones were tested against L. donovani in vitro and in vivo. The anti-protozoal activity of hydroxynaphthoquinones has long been established (see Hudson, 1984), but attention has been re-focused on the activity of this group of compounds by the synthesis of a novel series of hydroxynaphthoquinones with remarkable activity against a range of sporozoan parasites, including Plasmodium spp., Theileria, Toxoplasma, Eimeria spp. (Hudson et al., 1985, 1986; Araujo, Huskinson & Remington, 1991) and Pneumocystis carinii (Hughes et al., 1990). Two of the series, parvaquone and buparvaquone, have been developed for the treatment of East Coast Fever in cattle (McHardy et al., 1983, 1985), whilst atovaquone (566C80) is being evaluated clinically for the treatment of malaria, P. carinii pneumonitis and toxoplasmosis (Hudson et al., 1991). Materials and methods Parasites L. donovani (MHOM/ET/67/L82; LV9) was maintained routinely in male golden hamsters (Wright's strain). Amastigotes were isolated six to eight weeks after infection from the spleen of infected hamsters. •Present address: London School of Hygiene of Tropical Medicine, Keppel Street, London, WC1E 7HT, UK. 827 0305-7453/92/120827+06 $08.00/0
© 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Otago on January 6, 2015
The hydroxynaphthoquinones, buparvaquone, 250C80 and 56W82, showed high activity in vitro against Leishmania donovani amastigotcs in mouse peritonea] macrophages, with EDy, values of O05, 2-95 and 13-82 (itt, respectively. Fourteen other hydroxynaphthoquinones were tested, of which only S66C80 and 608C86 showed significant activity against amastigotes. Buparvaquone, 250C80 and 56W82 were also highly-active against cultured promastigotes. In a BALB/c mouse model, treatment with 100 mg/kg/day for five days with buparvaquone, 250C80 and 566C80 (atovaquone) reduced liver amastigote numbers by 60%, 22% and 30-5%, respectively.
828
S. L. Croft et oL
Amastigote test
Promastigote test Promastigotes were cultivated at 26°C in Schneider's medium (Gibco), plus 20% heat-inactivated fetal calf serum, and adjusted to a concentration of 2 x 10* promastigotes/mL. Compounds were added to the same medium to give concentrations in a dilution series from 50 //M. The presence and motility of promastigotes was monitored microscopically for 48 h. The MIC was defined as the concentration at which these forms were no longer present. Jn-vivo test Male BALB/c mice were infected iv with 5 x 106 L. donovani amastigotes. One week after infection, mice were divided randomly into groups of five and treated with the various drug vehicles described in Results. All mice were killed after treatment for five days. Drug activity was calculated from the number of amastigotes/500 liver cells x liver weight (mg) in treated and untreated groups. Compounds Sodium stibogluconate (Pentostam; Wellcome, Dartford, UK) was used as the standard positive control in all experiments. Naphthoquinones were synthesized as described previously (Hudson & Randall, 1984; Hudson et al., 1986). Except for 568C80 (a 1:1 cis: trans mixture), all compounds capable of forming cis and trans isomers were obtained and tested as the trans form.
Results
Seventeen hydroxynaphthoquinones were tested against L. donovani amastigotes in the in-vitro macrophage model. Five of these compounds (Figure) showed significant antileishmanial activity (Table I). Buparvaquone was evaluated in a series of tests which gave EDj,, values ranging between 0-12 and 0-005 /JM. Only two other compounds, 250C80 and 56W82, cleared or nearly cleared all amastigotes from macrophages at the highest concentration tested (50 /XM). The antiprotozoal atovaquone, and its close analogue 608C86, showed low-grade activity, while other related compounds (Figure)
Downloaded from http://jac.oxfordjournals.org/ at University of Otago on January 6, 2015
The procedure followed that described in detail by Neal & Croft (1984). Briefly, mouse peritoneal macrophages were isolated from the peritoneal activity of outbred CD1 mice (Charles Rivers Ltd, Margate, UK). Macrophages were cultured in Labtek eight-well tissue chamber slides in RPMI 1640 medium, plus 10% heat-inactivated fetal calf serum (Gibco, Paisley, UK) at 37°C in a 5% CO2-air mixture. Macrophages infected with freshly-isolated amastigotes were incubated in the presence of drug-containing medium for seven days, with medium being replaced on days one, three and five after infection. Compounds were tested in a five-fold dilution series from 50 nu, with four replicates at each concentration. The proportion of infected macrophages in Giemsa-stained preparations was determined after a seven-day exposure to the drugs. EDy, values were calculated by sigmoidal analysis.
Anil lilnliiiiinlil hydroxym|iliUiuqiiliiooet
829
0 Activt Compound* R
No. 720C80 (buparvoquont)
-CH.-Q-CtCH.J,
250C86
-CH.{3^QC.
566C8O (atovaquonc) 608C86 Inactive Compounds
826C76
-«O
963C76
-CH.O
1X74 (menoctont)
-(CH.)4
The activity of hydroxynaphthoquinones against Leishmania donovani S. L. Croft*, J. Hogg, W. E. Gutteridge, A. T. Hudson and A. W. RaodaU
Wellcome Research Laboratories, Beckenham, Kent, BR3 3BS, UK
Introduction
Leishmania donovani, a haemoflagcllatc protozoan parasite, is the causative agent of potentially-fatal visceral leishmaniasis (kala-azar). The parasite survives and multiplies in the mammalian host as an intracellular amastigote form in the macrophages of the liver, spleen and bone marrow. At present, clinical treatment for visceral leishmaniasis is dependent primarily upon pentavalent antimonials, which require long courses of treatment at high doses and parenteral administration. They also have toxic side-effects and variable efficacy (Bryceson, 1987). As part of a programme to find new drugs for the treatment of leishmaniasis, a series of hydroxynaphthoquinones were tested against L. donovani in vitro and in vivo. The anti-protozoal activity of hydroxynaphthoquinones has long been established (see Hudson, 1984), but attention has been re-focused on the activity of this group of compounds by the synthesis of a novel series of hydroxynaphthoquinones with remarkable activity against a range of sporozoan parasites, including Plasmodium spp., Theileria, Toxoplasma, Eimeria spp. (Hudson et al., 1985, 1986; Araujo, Huskinson & Remington, 1991) and Pneumocystis carinii (Hughes et al., 1990). Two of the series, parvaquone and buparvaquone, have been developed for the treatment of East Coast Fever in cattle (McHardy et al., 1983, 1985), whilst atovaquone (566C80) is being evaluated clinically for the treatment of malaria, P. carinii pneumonitis and toxoplasmosis (Hudson et al., 1991). Materials and methods Parasites L. donovani (MHOM/ET/67/L82; LV9) was maintained routinely in male golden hamsters (Wright's strain). Amastigotes were isolated six to eight weeks after infection from the spleen of infected hamsters. •Present address: London School of Hygiene of Tropical Medicine, Keppel Street, London, WC1E 7HT, UK. 827 0305-7453/92/120827+06 $08.00/0
© 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Otago on January 6, 2015
The hydroxynaphthoquinones, buparvaquone, 250C80 and 56W82, showed high activity in vitro against Leishmania donovani amastigotcs in mouse peritonea] macrophages, with EDy, values of O05, 2-95 and 13-82 (itt, respectively. Fourteen other hydroxynaphthoquinones were tested, of which only S66C80 and 608C86 showed significant activity against amastigotes. Buparvaquone, 250C80 and 56W82 were also highly-active against cultured promastigotes. In a BALB/c mouse model, treatment with 100 mg/kg/day for five days with buparvaquone, 250C80 and 566C80 (atovaquone) reduced liver amastigote numbers by 60%, 22% and 30-5%, respectively.
828
S. L. Croft et oL
Amastigote test
Promastigote test Promastigotes were cultivated at 26°C in Schneider's medium (Gibco), plus 20% heat-inactivated fetal calf serum, and adjusted to a concentration of 2 x 10* promastigotes/mL. Compounds were added to the same medium to give concentrations in a dilution series from 50 //M. The presence and motility of promastigotes was monitored microscopically for 48 h. The MIC was defined as the concentration at which these forms were no longer present. Jn-vivo test Male BALB/c mice were infected iv with 5 x 106 L. donovani amastigotes. One week after infection, mice were divided randomly into groups of five and treated with the various drug vehicles described in Results. All mice were killed after treatment for five days. Drug activity was calculated from the number of amastigotes/500 liver cells x liver weight (mg) in treated and untreated groups. Compounds Sodium stibogluconate (Pentostam; Wellcome, Dartford, UK) was used as the standard positive control in all experiments. Naphthoquinones were synthesized as described previously (Hudson & Randall, 1984; Hudson et al., 1986). Except for 568C80 (a 1:1 cis: trans mixture), all compounds capable of forming cis and trans isomers were obtained and tested as the trans form.
Results
Seventeen hydroxynaphthoquinones were tested against L. donovani amastigotes in the in-vitro macrophage model. Five of these compounds (Figure) showed significant antileishmanial activity (Table I). Buparvaquone was evaluated in a series of tests which gave EDj,, values ranging between 0-12 and 0-005 /JM. Only two other compounds, 250C80 and 56W82, cleared or nearly cleared all amastigotes from macrophages at the highest concentration tested (50 /XM). The antiprotozoal atovaquone, and its close analogue 608C86, showed low-grade activity, while other related compounds (Figure)
Downloaded from http://jac.oxfordjournals.org/ at University of Otago on January 6, 2015
The procedure followed that described in detail by Neal & Croft (1984). Briefly, mouse peritoneal macrophages were isolated from the peritoneal activity of outbred CD1 mice (Charles Rivers Ltd, Margate, UK). Macrophages were cultured in Labtek eight-well tissue chamber slides in RPMI 1640 medium, plus 10% heat-inactivated fetal calf serum (Gibco, Paisley, UK) at 37°C in a 5% CO2-air mixture. Macrophages infected with freshly-isolated amastigotes were incubated in the presence of drug-containing medium for seven days, with medium being replaced on days one, three and five after infection. Compounds were tested in a five-fold dilution series from 50 nu, with four replicates at each concentration. The proportion of infected macrophages in Giemsa-stained preparations was determined after a seven-day exposure to the drugs. EDy, values were calculated by sigmoidal analysis.
Anil lilnliiiiinlil hydroxym|iliUiuqiiliiooet
829
0 Activt Compound* R
No. 720C80 (buparvoquont)
-CH.-Q-CtCH.J,
250C86
-CH.{3^QC.
566C8O (atovaquonc) 608C86 Inactive Compounds
826C76
-«O
963C76
-CH.O
1X74 (menoctont)
-(CH.)4
