Vol. 36, No. 2
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1992, p. 326-330
0066-4804/92/020326-05$02.00/0 Copyright © 1992, American Society for Microbiology
In Vitro and In Vivo Activities of the Hydroxynaphthoquinone 566C80 against the Cyst Form of Toxoplasma gondii FAUSTO G. ARAUJO,1 JAYNE
HUSKINSON-MARK,l WINSTON E. GUTTERIDGE,2 REMINGTON' 3*
AND JACK S.
Department of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto, California 94301'; The Wellcome Foundation Ltd., Beckenham, Kent BR3 3BS, England2; and Department of Medicine, Division of Infectious Diseases, Stanford University School of Medicine, Stanford, California 943053 Received 1 October 1991/Accepted 8 November 1991 The in vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against the cyst form of Toxoplasma gondii were evaluated. In vitro treatment (100 ,Ig of 566C80 per ml for 3 days) of cysts isolated from brains of mice infected for 1, 2, 3, 4, or 9 months resulted in loss of viability of the cysts and did not reveal any influence of the duration of in vivo infection on sensitivity to the drug. In vivo experiments to determine the effect of prolonged treatment with 200 mg of 566C80 per kg of body weight per day on cysts in brains of CBA/Ca mice infected with strain ME49 revealed a steady and significant decline in the numbers of cysts compared with the numbers in untreated controls. Histopathology of brains from control mice revealed inflammatory infiltrates around capillaries and in the parenchymas and meninges which were consistently less evident in the brains of treated mice. In addition, cysts were rarely observed in treated mice, whereas extensive inflammation and large numbers of cysts were found throughout the entire brain in control mice infected for the same period. The reduction in the numbers of cysts was evident as early as day 5 of treatment but was more marked at 8 weeks of treatment. The numbers of cysts in the brains of Swiss Webster mice infected for 3 or 6 months also significantly decreased following treatment for 15 or 30 days with the same dose of 566C80. Our results indicate that 566C80 has excellent activity against cysts of T. gondii both in vivo and in vitro and that sensitivity of the cysts to 566C80 is not affected by the duration of the infection in vivo.
Two forms of Toxoplasma gondii are found in infected humans, tachyzoites, which are present during active infection, and cysts, which are noted during chronic infection. Quiescent cysts in brains and other tissues of immunocompromised individuals, particularly in the brains of AIDS patients, have been implicated as an important source of parasites for reactivation of infection and development of toxoplasmic encephalitis (4, 13, 14). At present, the treatment of choice for toxoplasmosis, including toxoplasmic encephalitis, is the combination pyrimethamine-sulfonamide (3, 12, 13, 16). The general consensus, however, is that cysts of T. gondii are not affected by treatment with this drug combination (3, 13, 14). Thus, any drug or drug combination with activity against the cyst form of the parasite would be important for treatment and perhaps prevention of toxoplasmic encephalitis. When following up in-house data which showed that the hydroxynaphthoquinone 566C80 (Wellcome Foundation Limited, Beckenham, Kent, United Kingdom) had experimental activity against T. gondii (6), we demonstrated that this compound has remarkable protective activity against death due to acute toxoplasmosis in mice and that it appeared to reduce the number of cysts of T. gondii in brains of mice (2). In further work we found that 566C80 was one of the most active drugs against cysts of T. gondii in an in vitro assay (10). In the present studies we have examined the effect of prolonged treatment of chronically infected mice with 566C80 and have employed our in vitro assay (10) to examine whether duration of the infection in vivo affects susceptibility of T. gondii cysts to the drug.
MATERIALS AND METHODS Mice. Inbred female CBA/Ca mice weighing 18 to 20 g each were used in experiments to determine the effects of long-term treatment on the numbers of cysts and on the inflammatory response in the brain. Outbred Swiss Webster (SW) female mice weighing approximately 20 g each at the beginning of the experiment were used to determine the effects of treatment on reduction in the numbers of cysts in brains of mice infected 3 or 6 months earlier. T. gondii. Cysts were isolated (2, 10) from brains of mice
*
CONTIROL
|E31 TREATED| 1000 0
cn
0
m10
OE 5D IOD 15D 4W 6W 8W 1OW 12WK DAYS (D) OR WEEKS (W) AFrER INiTIATION OF TREATMENT
FIG. 1. Effect of 566C80 on T. gondii cysts in brains of chronically infected mice. Oral treatment with doses of 200 mg/kg/day was started 5 weeks after infection and lasted for 12 weeks. Controls *
were treated with the 566C80 diluent. Differences between control and treated groups were significant for each day or week (P < 0.01).
Corresponding author. 326
VOL. 36, 1992
566C80 IS ACTIVE AGAINST CYSTS OF T. GONDII
327
TABLE 1. In vitro activity of 566C80 against tissue cysts of T. gondii Duration of infection (mo) and group
Concn of 566C80
(Wg/ml)a
Incubation
AO-EB
(days)
stainingb
Days to deathc
Control A B C Control A B C
0 5 50 100
G G G/O
0 5 50 100
3 3 3 3 6 3 3 3
0 0
11/11 11/13 16/s s/s s/s s/s s/s s/s
Control A B C Control A B C
0 5 50 100 0 5 50 100
3 3 3 3 6 6 6 6
G G G/O 0 G G 0 0
13/13 11/11 s/s s/s 12/13 17/s s/s s/s
Control A B C Control A B C
0
3 3 3 3 6 6 6 6
G G 0 0 G G G 0
10/s 12/12 s/s s/s S/S S/S S/S S/S
3 3 3 3 6 6 6 6
G G G 0 G G G 0
3 3 3 3 7 7 7 7
G G G/O 0 G G 0 0
Cysts in
Serologic testse
braind
DT D
pf N/Ng P/P P/P N/N N/N
p N/N P/P P/P N/N N/N
p N/N P/P P/P N/N N/N
P/P N/N
P/P N/N
P/P N/N
p N/N N/N
p
N/N N/N
p N/N N/N
p
p
p
P/P N/N P/P N/N N/N
P/P N/N P/P P/P N/N N/N
P/P N/N P/P P/P N/N N/N
S/S 12/s s/s s/s s/s s/s N/P S/S
P/P p P/P N/N P/P P/P N/P N/N
P/P p P/P N/N P/P P/P N/P N/N
P/P p P/P N/N P/P P/P N/P N/N
9/9 9/10 13/14 s/s 13/s s/s s/s s/s
N/N
N/N
N/N
p P/P N/N N/N
p P/P N/N N/N
P/P N/N N/N
ELISA LS
1
0 G G/O
2
3
5 50 100 0 5 50 100
P/P
4
Control A B C Control A B C
0
5 50 100 0 5 50 100
9
Control A B C Control A B
C
0 5
50 100 0 S 50 100
p
a Amount of drug added to 1 ml of medium. Because of the relative insolubility of 566C80, the final concentrations may have been lower, as discussed in reference 10. b G, green; 0, orange; G/0, most cysts green but some orange; AO-EB, acridine orange-ethidium bromide. C There were two mice injected with treated or control cysts. Values indicate the day each mouse died; s, mouse survived. d Values before shills indicate result for first mouse; values after shills indicate result for second mouse. I ELISA, enzyme-linked immunosorbent assay; DT, Sabin-Feldman dye test. f P, cysts detected in brain and/or positive titer by the Sabin-Feldman dye test or by enzyme-linked immunosorbent assay. g N, cysts not detected in brain and/or negative titer by the Sabin-Feldman dye test or by enzyme-linked immunosorbent assay.
chronically infected with strain ME49 (1). CBA/Ca mice were infected intraperitoneally with 10 cysts each and SW mice were infected with 20 cysts each. CBA/Ca mice infected with this strain of T. gondii develop a progressive toxoplasmic encephalitis which results in death unless the animals are treated. Large numbers of T. gondii cysts are present in the brains of CBA/Ca mice after 3 to 4 weeks of
infection (2). SW mice also develop numerous cysts in their brains after 3 to 4 weeks of infection, but in contrast to the case with CBA/Ca mice, there is very little inflammatory response and SW mice rarely die as a result of the infection. In vitro experiments. To determine whether the in vitro sensitivities of cysts vary with their age in vivo, cysts were isolated from brains of mice infected 1, 2, 3, 4 or 9 months
328
ARAUJO ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 2. (A) Section of brain of infected mouse treated orally with 566C80 for 12 weeks. A significant reduction in the inflammatory response and an absence of T. gondii cysts were observed. (B) Section of brain of control mouse at 12 weeks. Widespread inflammatory exudate in the parenchyma and meninges and around blood vessels can be seen, and numerous cysts of T. gondii are present throughout the brain. Magnification, x 220. Hematoxylin-eosin staining was used.
earlier and treated with 566C80 as previously described (10). Briefly, isolated cysts were resuspended in RPMI culture medium containing 10% fetal bovine serum and either no 566C80 or a concentration of 5, 50, or 100 ,ug of the drug per ml. Because of the relative insolubility of 566C80, the final concentrations of the drug in the assay may have been lower, as previously discussed (10). For this reason, the concentrations are listed in Table 1 as A (5 ,ug/ml), B (50 ,ug/ml), and C (100 p,g/ml) as in our previous report (10). Thereafter, the cyst suspensions were placed in microcentrifuge tubes and incubated at 37°C in a 5% CO2 atmosphere for 3 or 6 days. Each experiment was conducted in triplicate. After incubation, cysts were examined after being stained with a solution of acridine orange and ethidium bromide as previously described (10). Viable cysts stain bright green. Cysts af-
fected by treatment present a variable degree of staining from yellowish green to orange-red and red (10). The remaining suspension of treated or control cysts was then inoculated intraperitoneally into two SW mice which had been previously treated with antisera to gamma interferon to enhance their susceptibility to T. gondii (19). The mice were observed for 30 days. The peritoneal fluids of those showing signs of acute toxoplasmosis or those dying during the observation period were examined for the presence of tachyzoites. Surviving animals were bled, their brains were examined for the presence of cysts, and their sera were examined for toxoplasma antibodies by using the SabinFeldman dye test (18) and an enzyme-linked immunosorbent assay performed with sonically disrupted cysts (Cyst ELISA) (10).
566C80 IS ACTIVE AGAINST CYSTS OF T. GONDII
VOL. 36, 1992
*
CONTROL 15 DAYS
1 TREATED 15 DAYS *
CONTROL 30 DAYS
El TREATED 30 DAYS 100
100
6
3
MONTHS AFTR
INFECTION
FIG. 3. Effect of 200 mg of 566C80 per kg per day administered orally for 15 or 30 days on T. gondii cysts in brains of mice infected for 3 or 6 months. Controls were treated with the 566C80 diluent. Differences between control and treated groups were significant for each day or week (P < 0.01).
In vivo experiments. To determine the effects of long-term treatment on the numbers of cysts and on the inflammatory response in the brain, CBA/Ca mice were infected as described above and treated with 200 mg of 566C80 per kg of
body weight per day, administered by gavage. The drug was dissolved in phosphate-buffered saline, pH 7.2, and treatment was initiated at 5 weeks of infection and continued for 12 weeks. Control mice, infected similarly, were treated with
phosphate-buffered saline only. At days 5 and 10 and at weeks 2, 4, 6, 8, 10, and 12 after initiation of treatment, three treated and three control mice were euthanized and their brains were processed individually for determination of numbers of cysts, as previously described (2). Briefly, brains were removed and cut in half longitudinally. One half was triturated individually in 1 ml of phosphate-buffered saline, and the numbers of cysts in five samples of 20 p1l each were determined under light microscopy with a 1Ox objective and ocular. The other half was immediately fixed in buffered formalin for processing for histopathology (11). The results are reported as the mean number of cysts in the brains of three mice ± the standard deviation at a determined period of time after initiation of treatment. To determine whether 566C80 is active in vivo against cysts in the brains of mice infected for prolonged periods of time, SW mice were infected intraperitoneally with 20 cysts each, as described above. Three and six months thereafter, groups of five mice each were treated with 566C80 as described above for either 15 or 30 days. All mice in each group were euthanized at the completion of the treatment, and their brains were processed for determination of the numbers of cysts as described above. The results are reported as the mean number of cysts in five brains ± the standard deviation.
RESULTS
Prolonged duration of infection did not alter the susceptibility of the cysts to in vitro treatment with 566C80 (Table 1). Cysts collected from mice infected for either 1 or 9 months were incubated with a concentration of 566C80 achieved by solubilizing 100 jig of the drug in 1 ml of medium. This treatment resulted in loss of viability of the cysts. This was evidenced by survival of the mice inoculated with treated
329
cysts and by the absence of T. gondii cysts in the brains of these mice and of antibodies to the parasite (Table 1). Lower concentrations of 566C80 were also active but were dependent on a longer incubation period in vitro. Prolonged incubation appeared to have a deleterious effect on the cysts. Thus, some loss of viability in cysts incubated for 6 days with medium only was demonstrated, as evidenced by the acridine orange-ethidium bromide staining and confirmed by mouse inoculation (Table 1). The in vivo experiments revealed a steady decline in the numbers of cysts in the brains of treated mice compared with the numbers in the brains of controls (Fig. 1). The reduction in the number of cysts was evident even 5 days after initiation of treatment, and by the eighth week of treatment the numbers of cysts had been reduced by almost 2 logs in the treated mice compared with the numbers in controls. Histopathology of the brains of mice treated for 8 weeks or longer revealed a complete clearing of perivascular inflammatory infiltrates and of infiltrates in the meninges and parenchyma (Fig. 2A). In addition, cysts were rarely seen in the histopathology sections of treated mice. In contrast, brains of control mice revealed an extensive inflammatory response throughout the entire brain, and cysts were easily seen in all the sections (Fig. 2B). A significant reduction in cyst numbers in brains of mice infected for 3 or 6 months and treated for either 15 or 30 days (Fig. 3) was also noted, suggesting that duration of the chronic infection (and age of the cysts) in vivo did not result in increased resistance to 566C80. DISCUSSION The above results confirm previous observations (10) on the excellent in vitro activity of the hydroxynaphthoquinone 566C80 against the cyst form of T. gondii and indicate that treatment in vivo is effective, significantly reducing the numbers of cysts as well as diminishing the inflammatory response in the brains of infected mice. The in vivo results are of particular interest since the antimicrobial drugs now in use for treatment of toxoplasmasmic encephalitis have not unequivocally been demonstrated to have activity in vivo against the cyst forms of T. gondii. A few reports have suggested that prolonged in vivo treatment with the combination pyrimethamine-sulfonamide may result in a reduction in the numbers of tissue cysts during latent infection (5, 20). Prolonged treatment is necessary for toxoplasmosis in AIDS patients and is frequently associated with untoward side effects when conventional pyrimethamine-sulfonamide therapy is used (3, 13, 16, 17). Although the effects of prolonged treatment with 566C80 are not yet known, it is of interest that human volunteers with Pneumocystis carinii pneumonia who were treated for 12 or 21 days with oral doses of 566C80 as high as 3,000 mg/day did not experience side effects that would force discontinuation of the treatment (9). 566C80 was originally developed as an antimalarial drug (7) and is currently in phase I/III clinical trials for this indication in Thailand and Zambia. Subsequently it was shown to have excellent activity against P. carinii (8), which, together with T. gondii, is a major cause of opportunistic infections in patients with AIDS (15). The results of the present study in conjunction with the facts that 566C80 is remarkably active against systemic murine toxoplasmosis (2) and that it has been used successfully in clinical trials for treatment of P. carinii infection in humans (9) suggested its use for treatment of human toxoplasmosis. Kovacs and his colleagues at the National Institutes of Health in Bethesda,
330
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Md., have studied 566C80 for treatment of toxoplasmic encephalitis in AIDS patients in a salvage protocol. The results are quite promising (13a). In addition to its promise for treatment of toxoplasmosis in patients with AIDS, 566C80 should also be evaluated for treatment of congenital and ocular toxoplasmosis. ACKNOWLEDGMENTS We thank Teri Lin and Gail Covaro for excellent technical assistance. This work was supported in part by grants A130230 and A104717 from the National Institutes of Health. REFERENCES 1. Araujo, F. G. 1991. Depletion of L3T4+ (CD4+) T lymphocytes prevents development of resistance to Toxoplasma gondii in mice. Infect. Immun. 59:1614-1619. 2. Araujo, F. G., J. Huskinson, and J. S. Remington. 1991. Remarkable in vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against tachyzoites and tissue cysts of Toxoplasma gondii. Antimicrob. Agents Chemother. 35:293-299. 3. Frenkel, J. K. 1971. Toxoplasmosis: mechanisms of infection, laboratory diagnosis and management. Curr. Top. Pathol. 54:
28-75. 4. Frenkel, J. K., and A. Escajadillo. 1987. Cyst rupture as a pathogenic mechanism of toxoplasmic encephalitis. Am. J. Trop. Med. Hyg. 36:517-522. 5. Gill, H. S., and 0. Prakash. 1972. Chemotherapy and chemoprophylaxis of experimental toxoplasmosis. Indian J. Med. Res. 60:1022-1027. 6. Hudson, A. T., M. Dickins, C. D. Ginger, W. E. Gutteridge, T. Holdich, D. B. A. Hutchinson, M. Pudney, A. W. Randall, and V. S. Latter. 566C80: a potent broad spectrum anti-infective agent with activity against malaria and opportunistic infections in AIDS patients. Drugs Exp. Clin. Res., in press. 7. Hudson, A. T., A. W. Randall, C. D. Ginger, B. Hill, V. S. Latter, N. McHardy, and R. B. Williams. 1985. Novel antimalarial hydroxynaphthoquinones with potent broad spectrum antiprotozoal activity. Parasitology 90:45-55. 8. Hughes, W. T., V. L. Gray, W. E. Gutteridge, V. S. Latter, and M. Pudney. 1990. Efficacy of a hydroxynaphthoquinone, 566C80, in experimental Pneumocystis carinii pneumonitis.
ANTIMICROB. AGENTS CHEMOTHER. Antimicrob. Agents Chemother. 34:225-228. 9. Hughes, W. T., W. Kennedy, J. Shenep, P. M. Flynn, S. V. Hetherington, G. Fullen, D. J. Lancaster, D. S. Stein, S. Palte, D. Rosenbaum, S. H. T. Liao, M. R. Blum, and M. D. Rogers. 1991. Safety and pharmacokinetics of 566C80, a hydroxynaphthoquinone with anti-Pneumocystis carinii activity: a phase I study in human immunodeficiency virus (HIV)-infected men. J. Infect. Dis. 163:843-848. 10. Huskinson-Mark, J., F. G. Araujo, and J. S. Remington. 1991. Evaluation of the effect of drugs on the cyst form of Toxoplasma gondii. J. Infect. Dis. 164:170-177. 11. Israelski, D. M., F. G. Araujo, F. K. Conley, Y. Suzuki, S. D. Sharma, and J. S. Remington. 1989. Treatment with anti-L3T4 (CD4) monoclonal antibody reduces the inflammatory response in toxoplasmic encephalitis. J. Immunol. 142:954-958. 12. Israelski, D. M., B. R. Dannemann, and J. S. Remington. 1990. Toxoplasmosis in patients with AIDS, p. 241-264. In M. A. Sande and P. A. Volberding (ed.), The medical management of AIDS, 2nd ed. The W. B. Saunders Co., Philadelphia. 13. Israelski, D. M., and J. S. Remington. 1988. Toxoplasmic encephalitis in patients with AIDS. Infect. Dis. Clin. N. Am. 2:429-445. 13a.Kovacs et al. Submitted for publication. 14. Luft, B. J., and J. S. Remington. 1988. AIDS commentary: toxoplasmic encephalitis. J. Infect. Dis. 157:1-6. 15. Mills, J. 1986. Pneumocystis carinii and Toxoplasma gondii infections in patients with AIDS. Rev. Infect. Dis. 8:1001-1011. 16. Remington, J. S., and G. Desmonts. 1990. Toxoplasmosis, p. 89-195. In J. S. Remington and J. 0. Klein (ed.), Infectious diseases of the fetus and newborn infant, 3rd ed. The W. B. Saunders Co., Philadelphia. 17. Ruskin, J., and J. S. Remington. 1976. Toxoplasmosis in the compromised host. Ann. Intern. Med. 84:193-199. 18. Sabin, A. B., and H. A. Feldman. 1948. Dyes as microchemical indicators of a new phenomenon affecting a protozoan parasite (Toxoplasma). Science 108:660-663. 19. Suzuki, Y., and J. S. Remington. 1989. A method for obtaining large numbers of trophozoites of avirulent strains of Toxoplasma gondii using an antibody to interferon-gamma. J. Parasitol. 75:174-176. 20. Werner, H., K. N. Masihi, I. Tisher, and E. Adusu. 1977. The effect of chemoimmunotherapy with SDDS, pyrimethamine, and anti-toxoplasma serum on Toxoplasma gondii cysts in latent infected NMRI mice. Tropenmed. Parasitol. 28:528-532.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1992, p. 326-330
0066-4804/92/020326-05$02.00/0 Copyright © 1992, American Society for Microbiology
In Vitro and In Vivo Activities of the Hydroxynaphthoquinone 566C80 against the Cyst Form of Toxoplasma gondii FAUSTO G. ARAUJO,1 JAYNE
HUSKINSON-MARK,l WINSTON E. GUTTERIDGE,2 REMINGTON' 3*
AND JACK S.
Department of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto, California 94301'; The Wellcome Foundation Ltd., Beckenham, Kent BR3 3BS, England2; and Department of Medicine, Division of Infectious Diseases, Stanford University School of Medicine, Stanford, California 943053 Received 1 October 1991/Accepted 8 November 1991 The in vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against the cyst form of Toxoplasma gondii were evaluated. In vitro treatment (100 ,Ig of 566C80 per ml for 3 days) of cysts isolated from brains of mice infected for 1, 2, 3, 4, or 9 months resulted in loss of viability of the cysts and did not reveal any influence of the duration of in vivo infection on sensitivity to the drug. In vivo experiments to determine the effect of prolonged treatment with 200 mg of 566C80 per kg of body weight per day on cysts in brains of CBA/Ca mice infected with strain ME49 revealed a steady and significant decline in the numbers of cysts compared with the numbers in untreated controls. Histopathology of brains from control mice revealed inflammatory infiltrates around capillaries and in the parenchymas and meninges which were consistently less evident in the brains of treated mice. In addition, cysts were rarely observed in treated mice, whereas extensive inflammation and large numbers of cysts were found throughout the entire brain in control mice infected for the same period. The reduction in the numbers of cysts was evident as early as day 5 of treatment but was more marked at 8 weeks of treatment. The numbers of cysts in the brains of Swiss Webster mice infected for 3 or 6 months also significantly decreased following treatment for 15 or 30 days with the same dose of 566C80. Our results indicate that 566C80 has excellent activity against cysts of T. gondii both in vivo and in vitro and that sensitivity of the cysts to 566C80 is not affected by the duration of the infection in vivo.
Two forms of Toxoplasma gondii are found in infected humans, tachyzoites, which are present during active infection, and cysts, which are noted during chronic infection. Quiescent cysts in brains and other tissues of immunocompromised individuals, particularly in the brains of AIDS patients, have been implicated as an important source of parasites for reactivation of infection and development of toxoplasmic encephalitis (4, 13, 14). At present, the treatment of choice for toxoplasmosis, including toxoplasmic encephalitis, is the combination pyrimethamine-sulfonamide (3, 12, 13, 16). The general consensus, however, is that cysts of T. gondii are not affected by treatment with this drug combination (3, 13, 14). Thus, any drug or drug combination with activity against the cyst form of the parasite would be important for treatment and perhaps prevention of toxoplasmic encephalitis. When following up in-house data which showed that the hydroxynaphthoquinone 566C80 (Wellcome Foundation Limited, Beckenham, Kent, United Kingdom) had experimental activity against T. gondii (6), we demonstrated that this compound has remarkable protective activity against death due to acute toxoplasmosis in mice and that it appeared to reduce the number of cysts of T. gondii in brains of mice (2). In further work we found that 566C80 was one of the most active drugs against cysts of T. gondii in an in vitro assay (10). In the present studies we have examined the effect of prolonged treatment of chronically infected mice with 566C80 and have employed our in vitro assay (10) to examine whether duration of the infection in vivo affects susceptibility of T. gondii cysts to the drug.
MATERIALS AND METHODS Mice. Inbred female CBA/Ca mice weighing 18 to 20 g each were used in experiments to determine the effects of long-term treatment on the numbers of cysts and on the inflammatory response in the brain. Outbred Swiss Webster (SW) female mice weighing approximately 20 g each at the beginning of the experiment were used to determine the effects of treatment on reduction in the numbers of cysts in brains of mice infected 3 or 6 months earlier. T. gondii. Cysts were isolated (2, 10) from brains of mice
*
CONTIROL
|E31 TREATED| 1000 0
cn
0
m10
OE 5D IOD 15D 4W 6W 8W 1OW 12WK DAYS (D) OR WEEKS (W) AFrER INiTIATION OF TREATMENT
FIG. 1. Effect of 566C80 on T. gondii cysts in brains of chronically infected mice. Oral treatment with doses of 200 mg/kg/day was started 5 weeks after infection and lasted for 12 weeks. Controls *
were treated with the 566C80 diluent. Differences between control and treated groups were significant for each day or week (P < 0.01).
Corresponding author. 326
VOL. 36, 1992
566C80 IS ACTIVE AGAINST CYSTS OF T. GONDII
327
TABLE 1. In vitro activity of 566C80 against tissue cysts of T. gondii Duration of infection (mo) and group
Concn of 566C80
(Wg/ml)a
Incubation
AO-EB
(days)
stainingb
Days to deathc
Control A B C Control A B C
0 5 50 100
G G G/O
0 5 50 100
3 3 3 3 6 3 3 3
0 0
11/11 11/13 16/s s/s s/s s/s s/s s/s
Control A B C Control A B C
0 5 50 100 0 5 50 100
3 3 3 3 6 6 6 6
G G G/O 0 G G 0 0
13/13 11/11 s/s s/s 12/13 17/s s/s s/s
Control A B C Control A B C
0
3 3 3 3 6 6 6 6
G G 0 0 G G G 0
10/s 12/12 s/s s/s S/S S/S S/S S/S
3 3 3 3 6 6 6 6
G G G 0 G G G 0
3 3 3 3 7 7 7 7
G G G/O 0 G G 0 0
Cysts in
Serologic testse
braind
DT D
pf N/Ng P/P P/P N/N N/N
p N/N P/P P/P N/N N/N
p N/N P/P P/P N/N N/N
P/P N/N
P/P N/N
P/P N/N
p N/N N/N
p
N/N N/N
p N/N N/N
p
p
p
P/P N/N P/P N/N N/N
P/P N/N P/P P/P N/N N/N
P/P N/N P/P P/P N/N N/N
S/S 12/s s/s s/s s/s s/s N/P S/S
P/P p P/P N/N P/P P/P N/P N/N
P/P p P/P N/N P/P P/P N/P N/N
P/P p P/P N/N P/P P/P N/P N/N
9/9 9/10 13/14 s/s 13/s s/s s/s s/s
N/N
N/N
N/N
p P/P N/N N/N
p P/P N/N N/N
P/P N/N N/N
ELISA LS
1
0 G G/O
2
3
5 50 100 0 5 50 100
P/P
4
Control A B C Control A B C
0
5 50 100 0 5 50 100
9
Control A B C Control A B
C
0 5
50 100 0 S 50 100
p
a Amount of drug added to 1 ml of medium. Because of the relative insolubility of 566C80, the final concentrations may have been lower, as discussed in reference 10. b G, green; 0, orange; G/0, most cysts green but some orange; AO-EB, acridine orange-ethidium bromide. C There were two mice injected with treated or control cysts. Values indicate the day each mouse died; s, mouse survived. d Values before shills indicate result for first mouse; values after shills indicate result for second mouse. I ELISA, enzyme-linked immunosorbent assay; DT, Sabin-Feldman dye test. f P, cysts detected in brain and/or positive titer by the Sabin-Feldman dye test or by enzyme-linked immunosorbent assay. g N, cysts not detected in brain and/or negative titer by the Sabin-Feldman dye test or by enzyme-linked immunosorbent assay.
chronically infected with strain ME49 (1). CBA/Ca mice were infected intraperitoneally with 10 cysts each and SW mice were infected with 20 cysts each. CBA/Ca mice infected with this strain of T. gondii develop a progressive toxoplasmic encephalitis which results in death unless the animals are treated. Large numbers of T. gondii cysts are present in the brains of CBA/Ca mice after 3 to 4 weeks of
infection (2). SW mice also develop numerous cysts in their brains after 3 to 4 weeks of infection, but in contrast to the case with CBA/Ca mice, there is very little inflammatory response and SW mice rarely die as a result of the infection. In vitro experiments. To determine whether the in vitro sensitivities of cysts vary with their age in vivo, cysts were isolated from brains of mice infected 1, 2, 3, 4 or 9 months
328
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FIG. 2. (A) Section of brain of infected mouse treated orally with 566C80 for 12 weeks. A significant reduction in the inflammatory response and an absence of T. gondii cysts were observed. (B) Section of brain of control mouse at 12 weeks. Widespread inflammatory exudate in the parenchyma and meninges and around blood vessels can be seen, and numerous cysts of T. gondii are present throughout the brain. Magnification, x 220. Hematoxylin-eosin staining was used.
earlier and treated with 566C80 as previously described (10). Briefly, isolated cysts were resuspended in RPMI culture medium containing 10% fetal bovine serum and either no 566C80 or a concentration of 5, 50, or 100 ,ug of the drug per ml. Because of the relative insolubility of 566C80, the final concentrations of the drug in the assay may have been lower, as previously discussed (10). For this reason, the concentrations are listed in Table 1 as A (5 ,ug/ml), B (50 ,ug/ml), and C (100 p,g/ml) as in our previous report (10). Thereafter, the cyst suspensions were placed in microcentrifuge tubes and incubated at 37°C in a 5% CO2 atmosphere for 3 or 6 days. Each experiment was conducted in triplicate. After incubation, cysts were examined after being stained with a solution of acridine orange and ethidium bromide as previously described (10). Viable cysts stain bright green. Cysts af-
fected by treatment present a variable degree of staining from yellowish green to orange-red and red (10). The remaining suspension of treated or control cysts was then inoculated intraperitoneally into two SW mice which had been previously treated with antisera to gamma interferon to enhance their susceptibility to T. gondii (19). The mice were observed for 30 days. The peritoneal fluids of those showing signs of acute toxoplasmosis or those dying during the observation period were examined for the presence of tachyzoites. Surviving animals were bled, their brains were examined for the presence of cysts, and their sera were examined for toxoplasma antibodies by using the SabinFeldman dye test (18) and an enzyme-linked immunosorbent assay performed with sonically disrupted cysts (Cyst ELISA) (10).
566C80 IS ACTIVE AGAINST CYSTS OF T. GONDII
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CONTROL 15 DAYS
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CONTROL 30 DAYS
El TREATED 30 DAYS 100
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INFECTION
FIG. 3. Effect of 200 mg of 566C80 per kg per day administered orally for 15 or 30 days on T. gondii cysts in brains of mice infected for 3 or 6 months. Controls were treated with the 566C80 diluent. Differences between control and treated groups were significant for each day or week (P < 0.01).
In vivo experiments. To determine the effects of long-term treatment on the numbers of cysts and on the inflammatory response in the brain, CBA/Ca mice were infected as described above and treated with 200 mg of 566C80 per kg of
body weight per day, administered by gavage. The drug was dissolved in phosphate-buffered saline, pH 7.2, and treatment was initiated at 5 weeks of infection and continued for 12 weeks. Control mice, infected similarly, were treated with
phosphate-buffered saline only. At days 5 and 10 and at weeks 2, 4, 6, 8, 10, and 12 after initiation of treatment, three treated and three control mice were euthanized and their brains were processed individually for determination of numbers of cysts, as previously described (2). Briefly, brains were removed and cut in half longitudinally. One half was triturated individually in 1 ml of phosphate-buffered saline, and the numbers of cysts in five samples of 20 p1l each were determined under light microscopy with a 1Ox objective and ocular. The other half was immediately fixed in buffered formalin for processing for histopathology (11). The results are reported as the mean number of cysts in the brains of three mice ± the standard deviation at a determined period of time after initiation of treatment. To determine whether 566C80 is active in vivo against cysts in the brains of mice infected for prolonged periods of time, SW mice were infected intraperitoneally with 20 cysts each, as described above. Three and six months thereafter, groups of five mice each were treated with 566C80 as described above for either 15 or 30 days. All mice in each group were euthanized at the completion of the treatment, and their brains were processed for determination of the numbers of cysts as described above. The results are reported as the mean number of cysts in five brains ± the standard deviation.
RESULTS
Prolonged duration of infection did not alter the susceptibility of the cysts to in vitro treatment with 566C80 (Table 1). Cysts collected from mice infected for either 1 or 9 months were incubated with a concentration of 566C80 achieved by solubilizing 100 jig of the drug in 1 ml of medium. This treatment resulted in loss of viability of the cysts. This was evidenced by survival of the mice inoculated with treated
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cysts and by the absence of T. gondii cysts in the brains of these mice and of antibodies to the parasite (Table 1). Lower concentrations of 566C80 were also active but were dependent on a longer incubation period in vitro. Prolonged incubation appeared to have a deleterious effect on the cysts. Thus, some loss of viability in cysts incubated for 6 days with medium only was demonstrated, as evidenced by the acridine orange-ethidium bromide staining and confirmed by mouse inoculation (Table 1). The in vivo experiments revealed a steady decline in the numbers of cysts in the brains of treated mice compared with the numbers in the brains of controls (Fig. 1). The reduction in the number of cysts was evident even 5 days after initiation of treatment, and by the eighth week of treatment the numbers of cysts had been reduced by almost 2 logs in the treated mice compared with the numbers in controls. Histopathology of the brains of mice treated for 8 weeks or longer revealed a complete clearing of perivascular inflammatory infiltrates and of infiltrates in the meninges and parenchyma (Fig. 2A). In addition, cysts were rarely seen in the histopathology sections of treated mice. In contrast, brains of control mice revealed an extensive inflammatory response throughout the entire brain, and cysts were easily seen in all the sections (Fig. 2B). A significant reduction in cyst numbers in brains of mice infected for 3 or 6 months and treated for either 15 or 30 days (Fig. 3) was also noted, suggesting that duration of the chronic infection (and age of the cysts) in vivo did not result in increased resistance to 566C80. DISCUSSION The above results confirm previous observations (10) on the excellent in vitro activity of the hydroxynaphthoquinone 566C80 against the cyst form of T. gondii and indicate that treatment in vivo is effective, significantly reducing the numbers of cysts as well as diminishing the inflammatory response in the brains of infected mice. The in vivo results are of particular interest since the antimicrobial drugs now in use for treatment of toxoplasmasmic encephalitis have not unequivocally been demonstrated to have activity in vivo against the cyst forms of T. gondii. A few reports have suggested that prolonged in vivo treatment with the combination pyrimethamine-sulfonamide may result in a reduction in the numbers of tissue cysts during latent infection (5, 20). Prolonged treatment is necessary for toxoplasmosis in AIDS patients and is frequently associated with untoward side effects when conventional pyrimethamine-sulfonamide therapy is used (3, 13, 16, 17). Although the effects of prolonged treatment with 566C80 are not yet known, it is of interest that human volunteers with Pneumocystis carinii pneumonia who were treated for 12 or 21 days with oral doses of 566C80 as high as 3,000 mg/day did not experience side effects that would force discontinuation of the treatment (9). 566C80 was originally developed as an antimalarial drug (7) and is currently in phase I/III clinical trials for this indication in Thailand and Zambia. Subsequently it was shown to have excellent activity against P. carinii (8), which, together with T. gondii, is a major cause of opportunistic infections in patients with AIDS (15). The results of the present study in conjunction with the facts that 566C80 is remarkably active against systemic murine toxoplasmosis (2) and that it has been used successfully in clinical trials for treatment of P. carinii infection in humans (9) suggested its use for treatment of human toxoplasmosis. Kovacs and his colleagues at the National Institutes of Health in Bethesda,
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Md., have studied 566C80 for treatment of toxoplasmic encephalitis in AIDS patients in a salvage protocol. The results are quite promising (13a). In addition to its promise for treatment of toxoplasmosis in patients with AIDS, 566C80 should also be evaluated for treatment of congenital and ocular toxoplasmosis. ACKNOWLEDGMENTS We thank Teri Lin and Gail Covaro for excellent technical assistance. This work was supported in part by grants A130230 and A104717 from the National Institutes of Health. REFERENCES 1. Araujo, F. G. 1991. Depletion of L3T4+ (CD4+) T lymphocytes prevents development of resistance to Toxoplasma gondii in mice. Infect. Immun. 59:1614-1619. 2. Araujo, F. G., J. Huskinson, and J. S. Remington. 1991. Remarkable in vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against tachyzoites and tissue cysts of Toxoplasma gondii. Antimicrob. Agents Chemother. 35:293-299. 3. Frenkel, J. K. 1971. Toxoplasmosis: mechanisms of infection, laboratory diagnosis and management. Curr. Top. Pathol. 54:
28-75. 4. Frenkel, J. K., and A. Escajadillo. 1987. Cyst rupture as a pathogenic mechanism of toxoplasmic encephalitis. Am. J. Trop. Med. Hyg. 36:517-522. 5. Gill, H. S., and 0. Prakash. 1972. Chemotherapy and chemoprophylaxis of experimental toxoplasmosis. Indian J. Med. Res. 60:1022-1027. 6. Hudson, A. T., M. Dickins, C. D. Ginger, W. E. Gutteridge, T. Holdich, D. B. A. Hutchinson, M. Pudney, A. W. Randall, and V. S. Latter. 566C80: a potent broad spectrum anti-infective agent with activity against malaria and opportunistic infections in AIDS patients. Drugs Exp. Clin. Res., in press. 7. Hudson, A. T., A. W. Randall, C. D. Ginger, B. Hill, V. S. Latter, N. McHardy, and R. B. Williams. 1985. Novel antimalarial hydroxynaphthoquinones with potent broad spectrum antiprotozoal activity. Parasitology 90:45-55. 8. Hughes, W. T., V. L. Gray, W. E. Gutteridge, V. S. Latter, and M. Pudney. 1990. Efficacy of a hydroxynaphthoquinone, 566C80, in experimental Pneumocystis carinii pneumonitis.
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