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Antimicrobial Agents and Chemotherapy, September 1998, p. 2284-2289, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Two 2-Hydroxy-3-Alkyl-1,4-Naphthoquinones with In
Vitro and In Vivo Activities against Toxoplasma
gondii
Anis A.
Khan,1,2
Mohamed
Nasr,3 and
Fausto G.
Araujo1,*
Department of Immunology and Infectious
Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto,
California 943011;
Division of AIDS,
National Institute of Allergy and Infectious Diseases, Bethesda,
Maryland 208923; and
Division of
Infectious Disease and Geographic Medicine, Department of Medicine,
Stanford University School of Medicine, Palo Alto, California
943052
Received 26 September 1997/Returned for modification 25 November
1997/Accepted 29 June 1998
 |
ABSTRACT |
Two 3-alkyl-substituted 2-hydroxy-1,4-naphthoquinones, NSC 113452 (NSC52) and NSC 113455 (NSC55), were evaluated for activity against
Toxoplasma gondii in vitro and in murine models of acute toxoplasmosis. In vitro, both NSC52 and NSC55 significantly inhibited intracellular replication of T. gondii. In vivo, each
compound was examined alone and in combination with other drugs
currently used for treatment of human toxoplasmosis. Although
none of the compounds protected mice against death when administered
orally, both were significantly protective when administered
intraperitoneally. In addition, a significant increase in survival was
observed when suboptimal doses of each compound were used in
combination with suboptimal doses of pyrimethamine or
sulfadiazine. These results indicate that combinations of NSC52 or
NSC55 with pyrimethamine or sulfadiazine have promising activity
against T. gondii.
| |
INTRODUCTION |
The therapy of choice for
toxoplasmosis is the synergistic combination of pyrimethamine plus a
sulfonamide or pyrimethamine plus clindamycin (14). This
combination is effective for treatment of immunocompetent patients
(7). However, it frequently fails in immunodeficient
individuals who develop side effects that are of sufficient severity to
require discontinuation of one or both of the drugs (13,
14). For this reason, a continued search for new therapies and/or
new therapeutic approaches for treatment of human toxoplasmosis,
particularly toxoplasmic encephalitis, has been a high priority in our
laboratory (1-6, 11). We have previously demonstrated that
the hydroxynaphthoquinone atovaquone is remarkably active against
Toxoplasma gondii, both in vitro and in vivo (1,
2). It also demonstrates enhanced activity when combined with
pyrimethamine or sulfadiazine (4).
Hydroxynaphthoquinones containing a cyclohexyl moiety are metabolized
via hydroxylation at the 4 position of the cyclohexyl ring. The
secondary alcohols resulting from this process are less active against
Plasmodium species. In contrast, a variety of lipophilic 4 substituents that avoided rapid metabolism demonstrated potent activity
against Plasmodium falciparum, Eimeria tenella,
and Theileria parva (9). Thus, we considered it
of interest to examine a number of related 3-alkyl-substituted
2-hydroxy-1,4-naphthoquinones for in vitro activity against T. gondii. Two of these compounds, a
3-(4-cycloheptylphenyl)propyl-2-hydroxy-1,4-naphthoquinone termed NSC
113452 (NSC52) and a
3-(4-cyclohexylphenyl)propyl-2-hydroxy-1,4-naphthoquinone termed NSC 113455 (NSC55) (Fig. 1),
significantly inhibited replication of intracellular T. gondii tachyzoites and were therefore chosen for further
evaluation by using murine models of acute toxoplasmosis. Because it is
unlikely that a single drug will be effective in all forms of
toxoplasmosis (10) and atovaquone previously demonstrated enhanced activity in combination with pyrimethamine or
sulfadiazine (4), we also examined combinations of the two
new hydroxynaphthoquinones with these drugs.
| |
MATERIALS AND METHODS |
T. gondii.
Tachyzoites of the RH strain were obtained
from the peritoneal cavities of mice infected for 2 days with the
parasite and were used for in vitro and in vivo evaluations as
previously described (1). Tissue cysts of the C56 strain
were obtained from the brains of chronically infected mice and also
used to evaluate the compounds following oral infection of mice as
previously described (6).
Cells.
Human foreskin fibroblasts (HFF; ATCC CRL1635) were
grown in Dulbecco's modified Eagle medium (DMEM; Gibco Bethesda
Research Laboratories, Grand Island, N.Y.) containing 100 U of
penicillin per ml; 1 µg of streptomycin per ml, and 10%
heat-inactivated fetal bovine serum (HyClone, Logan, Utah).
Mice.
Outbred female Swiss Webster mice (Simonsen
Laboratories, Gilroy, Calif.) weighing approximately 20 g at the
beginning of each experiment were used. Food and water were available
to the mice at all times.
Drugs.
NSC52 and NSC55 were provided by the Drug Synthesis
and Chemistry Branch, National Cancer Institute, Bethesda, Md.
Compounds were selected from a large pool of naphthoquinones by
utilizing computerized substructure searching of the National Cancer
Institute database files. Atovaquone and pyrimethamine were
obtained from Burroughs Wellcome Co. (Research Triangle Park, N.C.),
and sulfadiazine was obtained from City Chemical Co. (New York, N.Y.).
Each drug was dissolved in a small volume of dimethyl sulfoxide (DMSO)
and brought to the required volume with DMEM to prepare a 0.1 M stock solution for the in vitro assays. All dilutions were made with complete
DMEM to the desired concentration. The final DMSO concentration in
solution was less than 1%. For oral or intraperitoneal (i.p.) administration, both NSC52 and NSC55 were dissolved in sterile phosphate-buffered saline. Atovaquone was suspended in 0.25%
carboxymethyl cellulose and sonicated for oral administration.
Pyrimethamine was dissolved in 0.25% carboxymethyl cellulose for oral
administration. Sulfadiazine was dissolved in double-distilled water
and administered to mice in drinking water.
In vitro studies.
NSC52 and NSC55 were examined at
concentrations of 0.01 to 10 µM for both activity against T. gondii and toxicity to HFF cells. Atovaquone was used at the same
concentrations for comparison. In vitro activity was defined as the
capacity of the drug to inhibit intracellular replication of T. gondii and was determined by using a modification of the method
reported earlier (3, 11). Briefly, HFF cells were plated at
104/well in 96-well flat-bottom tissue culture microtiter
plates (Costar Corp., Cambridge, Mass.) and incubated at 37°C in a
5% CO2 incubator. After reaching confluence, monolayers
were infected with 4 × 104 tachyzoites/well. Four
hours following infection, monolayers were washed to remove
extracellular parasites and various concentrations of the test drugs
were added. Triplicate wells were used for each drug concentration.
Addition of the drugs to the wells marked the starting time point.
[3H]uracil (1 µCi/well) was added to each well 24 h prior to harvesting of the cells at 24, 48, and 72 h following
addition of the test drugs, and the plates were incubated at 37°C
until the endpoint was reached. At the time of harvest, medium was
removed from the wells, and 1% sodium dodecyl sulfate (Sigma Chemical
Co., St. Louis, Mo.) and 100-µg/ml unlabeled uracil (Sigma) were
added to each well and incubated for 30 min at room temperature. Cells were harvested following addition of 100 µl of a 10% trichloroacetic acid solution (kept at 4°C) to each well. Each well was washed with
an additional 100 µl of 10% trichloroacetic acid solution three
separate times and finally washed with 95% ethanol. The filters were
air dried, and radioactivity was counted in a scintillation counter
after addition of 5 ml of liquid scintillation cocktail (Ready Safe;
Beckman Instruments, Inc., Fullerton, Calif.). Infected monolayers
treated with medium that contained the respective drug diluent alone
served as a negative control, and atovaquone prepared as were NSC52 and
NSC55 was used as a positive control.
Drug toxicity to HFF cells was determined by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide cell
proliferation
assay by using a Cell Titer 96 Kit (Promega Corp.,
Madison, Wis.).
Briefly, cells were plated in triplicate wells at
10
3/well. Following a 4-h incubation at 37°C in a 5%
CO
2 incubator,
various dilutions of the test drugs were
added. Four hours before
each time point at 24, 48, and 72 h, 14 µl of the dye indicator
solution was added. Following an additional
4 h of incubation,
100 µl of the solubilization-stop solution
was added to each well.
Plates were kept overnight in a sealed
container with a humidified
atmosphere, and the
A570 read was in an automatic enzyme-linked
immunosorbent assay plate reader.
In vivo studies.
The activity of NSC52 and NSC55 alone or in
combination with sulfadiazine or pyrimethamine was investigated
by using two murine models of acute toxoplasmosis. In one model, mice
were infected i.p. with 2.5 × 103 tachyzoites of the
RH strain, and in the other, mice were infected orally with 10 cysts of
T. gondii C56 (3). Treatment of mice infected
i.p. was initiated 24 h after infection. In mice infected orally,
treatment was initiated 3 days after infection. In both groups,
treatment lasted for 10 days. NSC52 or NSC55 was administered i.p. or
orally; atovaquone and pyrimethamine were administered orally,
and sulfadiazine was administered in drinking water. Mice were observed
for 30 days from the day of infection for death and time to death.
Uninfected mice were treated with NSC52 and NSC55 in parallel for the
same duration as infected mice and observed for signs of drug toxicity
such as piloerection, lethargy, loss of weight, or death.
Statistical analysis was performed by using survival tools for StatView
version 4.02 (Abacus Concepts, Berkeley, Calif.).
P values
were obtained by the log-rank test of the Kaplan-Meier
product limited
survival analysis, and a value of
0.05 was considered
to be
statistically significant.
| |
RESULTS |
In vitro results.
In two separate experiments, NSC52 and NSC55
demonstrated significant inhibition of intracellular growth of T. gondii at concentrations of 0.01 to 10 µM following 24, 48, or
72 h of exposure. The 50% inhibitory concentrations of atovaquone
(as a control), NSC52, and NSC55 following 48 h of exposure were
0.13, 0.11, and 0.10 µM, respectively (Fig. 2A, C, and
E). Untreated parasites in the control
monolayers grew normally, as indicated by increased uptake of
[3H]uracil with time. Neither NSC52 nor NSC55 showed
significant toxicity to the host cells, except at the highest
concentration of 10 µM (Fig. 2B, D, and F). DMSO alone at
concentrations of up to 10 µM did not have any effect on
intracellular toxoplasmas.
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FIG. 2.
Inhibition of replication of tachyzoites of the T. gondii RH within HFF cells by exposure to atovaquone (A), NSC52
(C), or NSC55 (E). Effect of exposure to atovaquone (B), NSC52 (D), or
NSC55 (F) on the growth of HFF cells. Each point represents the mean
plus the standard deviation of measurements of three wells.
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|
In vivo results obtained with drugs administered alone.
Oral
treatment of mice infected with RH tachyzoites with either NSC52 at 10, 50, or 100 mg/kg/day or NSC55 at 25 or 50 mg/kg/day did not result in
significant protection against death (data not shown). In contrast,
when NSC52 and NSC55 were administered i.p., the results revealed
significant protection against death (Fig. 3A and
B). Treatment with a 50-mg/kg/day or
NSC52 resulted in 30% survival (P < 0.0001) (Fig.
3A). All control mice died by day 7 of infection. A 100-mg/kg/day dose
of NSC52 administered i.p. appeared to be toxic, since 50% of the
treated mice died at the same time as the untreated controls and the
remaining mice died a few days later. Treatment of RH-infected mice
with a 25- or 50-mg/kg/day dose of NSC55 administered i.p. resulted in
20% (P < 0.01) or 10% (P = 0.4)
survival, respectively (Fig. 3B). All control mice died by day 8 of
infection. A dose of 100 mg/kg/day caused earlier deaths of mice, as
was noted with NSC52.
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FIG. 3.
Effect of treatment with NSC52 (A) or NSC55 (B)
administered i.p. on survival of mice infected i.p. with tachyzoites of
T. gondii RH. Rx, drug; d, day.
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|
Treatment of mice infected orally with cysts of the C56 strain with
NSC52 at 25, 50, or 100 mg/kg/day administered i.p. resulted
in no
effect, a 4-day prolongation of the time to death (
P = 0.003),
or 50% survival (
P < 0.001), respectively
(Fig.
4A). Treatment
of similarly
infected mice with NSC55 at 25 or 50 mg/kg/day also
administered i.p.
resulted in no effect or a 6-day prolongation
of the time to death
(
P = 0.001), respectively (Fig.
4B). An NSC55
dose of
100 mg/kg/day produced only a 2-day prolongation of the
time to death
(
P = 0.86). Similar results were observed in two
separate experiments. A parallel experiment to determine the effect
of
i.p. treatment of normal mice with NSC52 revealed that an NSC52
dose of
100 mg/kg/day was slightly toxic (one mouse died at day
15 of
treatment). The same dose of NSC55 was toxic and caused
100% mortality
(data not shown).
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FIG. 4.
Effect of treatment with NSC52 (A) or NSC55 (B)
administered i.p. on survival of mice infected orally with cysts of
T. gondii C56. Rx, drug; d, day.
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|
Results of combination therapy.
Because both NSC52 and NSC55
affected the survival of mice infected either i.p. with tachyzoites or
orally with cysts similarly, we chose to evaluate drug combinations
only in mice infected orally with cysts. In these experiments, a
dose-response study revealed that an NSC52 dose of 5, 10, or 25 mg/kg/day caused a slight prolongation of the time to the death of
mice. In contrast, at least 60% of the mice survived when any of these
doses was used in combination with pyrimethamine at 10 mg/kg/day (Fig. 5A, B, and C). This dose of pyrimethamine administered alone protected only 20% of the mice. Treatment of mice with these same doses of NSC52 in combination with a sulfadiazine dose of 20 mg/liter resulted in 90, 30, and 40%
survival, respectively (Fig. 5D, E, and F). Treatment with this dose of
sulfadiazine administered alone resulted in a prolongation of the time
to death that was not significant. In a separate experiment, uninfected
mice were treated i.p. with NSC52 at 5 mg/kg/day plus sulfadiazine at
20 mg/liter in drinking water for 10 days to determine the toxicity of
the combination. No signs of toxicity were noted in these mice during
treatment and up to 20 days following its conclusion (data not shown).
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FIG. 5.
Effect of treatment with NSC52 administered i.p. in
combination with pyrimethamine (Pyr) administered orally (A, B,
and C) or in combination with sulfadiazine (Sulfa) administered in
drinking water (D, E, and F) on the survival of mice infected orally
with cysts of T. gondii C56. P values for the
combinations were 0.007, 0.0005, and 0.056, respectively, compared with
respective NSC52 doses and 0.246, 0.056, and 0.167, respectively,
compared with respective pyrimethamine doses (A, B, and C).
P values for the combinations were 0.0001, 0.025, and 0.003, respectively, compared with respective NSC52 doses and 0.0002, 0.378, and 0.002, respectively, compared with respective sulfadiazine doses
(D, E, and F). Rx, drug; d, day; L, liter.
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|
A dose-response experiment with NSC55 also revealed that doses of 5, 10, and 25 mg/kg/day caused only a slight prolongation
of the time to
the death of mice. In contrast, treatment with
these same doses in
combination with a pyrimethamine dose of 10
mg/kg/day resulted
in 70, 60, and 60% survival, respectively (Fig.
6A, B, and
C). This dose of pyrimethamine
administered alone protected
only 20% of the mice against death.
Treatment with these same
doses of NSC55 in combination with a
20-mg/liter dose of sulfadiazine
resulted in 50, 30, and 60% survival,
respectively (Fig.
6D, E,
and F). Sulfadiazine alone did not have a
protective effect.
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FIG. 6.
Effect of treatment with NSC55 administered i.p. in
combination with pyrimethamine (Pyr) administered orally (A, B,
and C) or in combination with sulfadiazine (Sulfa) administered in
drinking water (D, E, and F) on the survival of mice infected orally
with cysts of T. gondii C56. P values for the
combinations were 0.001, 0.007, and 0.0003, respectively, compared with
respective NSC55 doses and were not significant compared with
respective pyrimethamine doses (A, B, and C). P
values for the combinations were 0.0001, 0.078, and 0.0001, respectively, compared with respective NSC55 doses and 0.166, 0.762, and 0.0006, respectively, compared with respective sulfadiazine doses
(D, E, and F). Rx, drug; d, day; L, liter.
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|
| |
DISCUSSION |
Our results show that NSC52 and NSC55 had potent in vitro activity
against T. gondii without a significant cytotoxic effect on
host cells at concentrations that significantly inhibited intracellular multiplication of the parasite. Both compounds also demonstrated significant activities in protecting mice against death due to i.p. or
oral infection with T. gondii. However, protection was noted
only when the compounds were administered i.p.; oral administration was
not effective. The reasons for this observation are not clear. Both
compounds displayed toxicity to mice; an NSC52 dose of 100 mg/kg/day
administered i.p. to mice infected i.p. with tachyzoites caused earlier
deaths. Interestingly, this was not observed when the mice were
infected orally with cysts. In contrast, an NSC55 dose of 100 mg/kg/day caused earlier deaths in mice infected i.p. with tachyzoites
or orally with cysts.
Combination of doses of NSC52, NSC55, sulfadiazine, or
pyrimethamine that were not protective or produced only
marginal effects when administered alone were used to investigate
synergistic activity. Thus, doses of NSC52 and NSC55 that were not
effective and far smaller than the dose toxic for mice induced
significant protection when combined with ineffective doses of
pyrimethamine or sulfadiazine. Of interest was the finding
that the dose of sulfadiazine needed to protect mice against death due
to acute toxoplasmosis when used in combination with atovaquone was
significantly lower than that which had been reported (4).
Reduction of the dose of sulfadiazine may be important to eliminate the
side effects that this drug causes in immunocompromised patients
undergoing therapy for toxoplasmosis (14).
Drug combinations were investigated because it is highly unlikely that
a single drug will be efficacious against all forms of toxoplasmosis,
particularly in immunocompromised individuals. The best therapy against
toxoplasmosis in these individuals is still the combination of
pyrimethamine plus a sulfonamide (14). In addition,
drug combinations may allow the use of lower doses with comparable or
even better therapeutic results.
Drugs related to the 1,4-naphthoquinones NSC52 and NSC55,
including atovaquone, have been shown to have potent activity
against Theileria, Eimeria, and
Plasmodium species (8). Atovaquone is remarkably
active in murine toxoplasmosis and has been used to treat AIDS patients
with toxoplasmic encephalitis (12). However, its relatively
poor bioavailability causes variable levels in serum with consequent
variability in efficacy. Thus, new formulations of atovaquone or
related compounds with similar activity against T. gondii should be evaluated to find more efficient drugs for treatment of toxoplasmosis. Our results indicate that both NSC52 and
NSC55 are active against T. gondii, particularly when
combined with pyrimethamine or sulfadiazine. Further research
with these compounds may disclose their usefulness for treatment of
toxoplasmosis in humans.
| |
ACKNOWLEDGMENTS |
This work was supported by the Division of AIDS, National
Institute of Allergy and Infectious Diseases, under contract
N01-AI-35174.
We thank Teri Slifer, Limei Yang, Eddie Wehri, and Eric Ho for
excellent technical help.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Immunology and Infectious Diseases, Research Institute, Palo Alto
Medical Foundation, 860 Bryant St., Palo Alto, CA 94301. Phone: (650) 326-8120. Fax: (650) 329-9853. E-mail: faraujo{at}PAMFRI.ORG.
| |
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Antimicrobial Agents and Chemotherapy, September 1998, p. 2284-2289, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
