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Antimicrobial Agents and Chemotherapy, November 2001, p. 3234-3237, Vol. 45, No. 11
Research Service, Veterans Affairs Medical
Center, Cincinnati, Ohio 45220,1 and
Division of Infectious Diseases, University of Cincinnati
College of Medicine, Cincinnati, Ohio 452672
Received 9 February 2001/Returned for modification 21 July
2001/Accepted 10 August 2001
Quinupristin-dalfopristin (Q-D), which is active against bacteria
and Toxoplasma gondii, was examined for its activity
against Pneumocystis carinii. After 72 h of
incubation with rat P. carinii in an ATP cytotoxicity
assay, the 50% inhibitory concentration of Q-D was 10.6 µg/ml, a
level that can be achieved in serum with high-dose administration. Q-D
administered intraperitoneally at doses of 50 to 200 mg per kg of body
weight per day in the treatment and 100 mg/kg/day three times per week
in the prophylaxis of pneumocystosis in immunosuppressed mice reduced
the organism burden up to 15- and 302-fold, respectively. We conclude
that Q-D has activity against P. carinii in vitro and in vivo.
Pneumocystis carinii is a
leading cause of pneumonia in patients infected with human
immunodeficiency virus and in other immunocompromised hosts.
Anti-P. carinii drugs in clinical use (e.g.,
trimethoprim-sulfamethoxazole [TMP-SXT]), which have been available
for many years, were originally developed to treat infections other
than pneumocystosis (16, 27). Their development for use
against P. carinii came mainly from rat and mouse models of
pneumocystosis, which are reliable predictors of activity against the
disease in humans (1, 9, 10, 22, 23, 28, 29, 30, 31).
Recently, progress has also been made with in vitro techniques to
screen drugs using rat P. carinii as the test organism
(12, 13, 17, 32). Problems associated with anti-P.
carinii agents include limited efficacy, increased toxicity, and
developing resistance (16, 18, 26, 27). Given the lack of
interest among pharmaceutical companies in developing new compounds,
another strategy has been to investigate existing drugs used for other
purposes for activity against the organism.
The present study is an example of the latter approach and involved
quinupristin-dalfopristin (Q-D), a fixed combination of semisynthetic
streptogramins which has been marketed primarily for the treatment of
serious resistant enterococcal infections. Q-D has a spectrum of
activity similar to that of azalides, ketolides, and macrolides
(2, 5, 15, 21). A recent study has shown that similar to
these compounds, Q-D is active against Toxoplasma gondii
(19). Since drugs active against T. gondii are
also active against P. carinii, we examined the effects of
Q-D on P. carinii by in vitro and in vivo techniques.
P. carinii organisms used in vitro studies were purified
from infected, immunosuppressed rats as described previously (8, 12, 13, 32). As determined by contour-clamped homogeneous electric field analysis, the organism preparations were predominantly P. carinii f. sp. carinii form 1;
microscopically, about 95% of the developmental stages were trophs,
and 5% were cysts. The organisms were cryopreserved, stored in liquid
nitrogen, and cultured for contaminants before use. Q-D in powdered
form for vitro testing was kindly provided by the manufacturer (Rhone
Poulenc Rorer, Collegeville, Pa.). Candidate drugs were prepared in
RPMI culture medium with dimethyl sulfoxide (final concentration,
<0.2% [vol/vol]). They were then evaluated for their effects on
P. carinii by the previously described ATP cytotoxicity
assay, a bioluminescence reaction that measures the viability of the
organism preparation maintained ex vivo for short periods of time.
Organisms (108/ml, measured as total nuclei) were
added with the drug or as controls (medium alone) in triplicate, and
the ATP content was sampled after 24, 48, and 72 h of incubation
at 35°C in 5% CO2. The ATP content was
measured by the luciferin-luciferase assay with an AutoLumat LB 953 luminometer (Wallac, Inc., Gaithersburg, Md.) and expressed as relative
light units. Activity of the drugs tested against P. carinii
was expressed as the concentration of the agent needed to lower the ATP
level by 50% compared to the level of the control
(IC50). Pentamidine (2 µg/ml) and ampicillin (10 µg/ml) were routinely included as positive and negative control drugs, respectively, in the assay. The drugs were also analyzed for
their potential human toxicity by studying their effects on A549 cells,
a human lung carcinoma cell line. The IC50 of the drug for the A549 cells was compared with the
IC50 for P. carinii. Based on their
IC50s, drugs were classified using an activity scale ranging from very marked (<0.1 µg/ml) to inactive ( Adult C3H/HeN mice (Charles River, Hollister, Calif.) were housed with
infected mice under barrier conditions and administered corticosteroids
to induce pneumocystosis as described (31, 32). When the
infection reached moderate intensity (after 6 to 7 weeks), the mice
were randomly divided into treatment and control groups. Q-D was
administered intraperitoneally (i.p.) once daily and compared with
TMP-SXT, the standard drug, which was given by oral gavage. The drugs
were continued for 3 weeks, during which time the mice remained on the
immunosuppressive regimen. Control animals receiving steroids (C-S)
received no treatment or a placebo. Prophylaxis studies were similar to
the treatment studies except that the drugs were administered
throughout immunosuppression.
Drug effectiveness was based on lowering organism burden rather than
survival, because some mice died from causes (e.g., drug toxicity)
other than pneumocystosis (28, 29, 31, 32). The animals
had to receive treatment for at least 7 days to be included in the data
analysis, because it usually takes this long to observe an effect. The
right lung was homogenized and stained with cresyl echt violet, which
selectively stains P. carinii cysts, and the organisms were
quantitated on a blinded basis. The lower limit of detection is
2.23 × 104 (log10
4.35) cysts/lung. The organism counts in the treatment and
prophylaxis groups were compared with those in the C-S group. Statistical analysis for normally distributed data consisted of analysis of variance followed by Student's Neuman-Keuls test for multiple comparisons using GraphPad software. Nonparametric
statistics were performed using the Kruskal-Wallis test followed by
Dunn's multiple comparison test. The Q-D reduced the ATP pools in P. carinii in a dose- and
time-dependent manner (Fig. 1A). Maximal
effect was seen at 72 h with an IC50 of 10.6 µg/ml, which amounts to slight to moderate activity on our scoring
scale. Toxicity evaluation revealed that the IC50 of Q-D for A549 cells was 122.0 µg/ml, compared with an
IC50 of 10.6 µg/ml for P. carinii at
72 h, a >10-fold difference (Fig. 1B).
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.11.3234-3237.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
In Vitro and In Vivo Effects of
Quinupristin-Dalfopristin against Pneumocystis
carinii
ABSTRACT
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50
µg/ml) (12, 32). Descriptive statistical analysis and
the histograms were developed using GraphPad (San Francisco, Calif.)
Software for Science.
value was set at 0.05. Drug
activity was also classified using a scale ranging from 1,000-fold
reduction (very marked) to <5-fold reduction (inactive) (28, 29,
32).
View larger version (54K):
[in a new window]
FIG. 1.
(A) Effects of different concentrations of Q-D on ATP
levels in P. carinii at different time points as
calculated by linear regression. The mean values of three separate
experiments are presented. The mean IC50s were 116.6 µg/ml at 24 h, 22.1 µg/ml at 48 h, and 10.6 µg/ml at
72 h. Pentamidine, the positive control drug, reduced the ATP
content by 90% at 24 h, 90% at 48 h, and 98% at 72 h
in the three experiments. Ampicillin, the negative control drug,
lowered the ATP content by 0% at 24 h, 2% at 48 h, and 2%
at 72 h. (B) Effects of Q-D on ATP levels in P.
carinii and the human lung carcinoma cell line A549 after
72 h of exposure. The IC50 of the drug for P.
carinii was 10.6 µg/ml, compared with an IC50 of
122.0 µg/ml for A549 cells.
Q-D doses of 50 and 100 mg per kg of body weight per day in the
treatment of pneumocystosis lowered the mean organism cyst count four-
to fivefold, from log10 7.49 ± 0.57/lung in
the C-S group to log10 6.82 ± 0.63 and
log10 6.87 ± 0.37/lung, respectively (P < 0.01) (Fig. 2A).
Q-D at 200 mg/kg/day reduced the organism count 15-fold (moderate
activity), to log10 6.32 ± 0.46/lung
(P < 0.001). This was greater than the reduction with
the 50- and 100-mg/kg/day doses (P < 0.05). TMP-SXT at
50/250 mg/kg/day lowered the organism count 851-fold (marked activity),
to log10 4.56 ± 0.38/lung
(P < 0.001). This was greater than that achieved with the 200-mg/kg/day dose of Q-D (P < 0.001).
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Nonparametric statistics were used to analyze the data for the
prophylaxis experiment. Q-D administered i.p. at 100 mg/kg/day three
times per week decreased the median P. carinii cyst count 302-fold (marked activity), from log10 8.01 cysts/lung in the C-S group to 5.53 organisms/lung
(P < 0.01). For comparison purposes, TMP-SXT at 50/250
mg/kg/day as treatment during the last 3 weeks of the study
lowered the median organism cyst count 4,570-fold (very marked
activity) to undetectable levels (
log10 4.35 cysts/lung) (P < 0.001). The differences between the results
obtained with Q-D and TMP-SXT were not statistically significant.
Q-D caused transient, dose-related lethargy in the mice for the first few days but was otherwise well tolerated in both experiments. Q-D at 500 mg/kg/day i.p. resulted in the death of the mice within minutes after administration.
The in vitro data in the present study suggest that Q-D is less active against P. carinii than against T. gondii in tissue culture (19) and is also less active than standard anti-P. carinii drugs against the organism in the ATP assay (12, 13). However, the activity of Q-D is similar to that of some inhibitors of sterol biosynthesis, which are potential P. carinii drug targets (10, 11, 17, 25). Q-D administered to humans at higher-than-clinically-used doses (12.6 to 29.4 mg/kg) achieves levels in serum (10.7 to 24.2 µg/ml) that exceed the IC50 of 10.6 µg/ml (3).
The slight to moderate activity of Q-D in the treatment of established pneumocystosis in mice correlated well with the in vitro data. Q-D was much more effective in P. carinii prophylaxis, a finding that is consistent with our previous experience of studying drugs in this manner (29). Our results with Q-D were obtained in a model of chronic infection, whereas most studies of the drug have been performed in models of acute infection (6, 7). However, since Q-D given subcutaneously at 120 mg/kg results in a level in serum of 13.2 µg/ml (4), it seems likely that we achieved levels in serum that exceed the IC50 for P. carinii with our i.p. doses of 100 to 200 mg/kg.
Judging from its IC50 and need for parenteral administration, it appears unlikely that Q-D will be developed for its anti-P. carinii properties in humans. The present report is important because it extends studies of Q-D to a new organism and new type of animal model. More potent anti-P. carinii agents may be discovered among other streptogramins and, with the aid of the P. carinii genome project (24), may bring new insights into the mechanism of drug action. There may also be increased understanding of other interesting properties of Q-D, such as its short half-life, long postantibiotic effect, concentration in macrophages, and suppression of cytokine responses (2, 3, 4, 5, 6, 7, 14, 20, 21).
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ACKNOWLEDGMENTS |
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This study was supported by the Medical Research Service, Department of Veterans Affairs, and by Public Service contract AI 75319 and grant RO1 HL64570 from the National Institutes of Health.
We thank Randy Thomas and Diane Gillotte for excellent assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Research Service (151), VA Medical Center, 3200 Vine St., Cincinnati, OH 45220. Phone: (513) 475-6328. Fax: (513) 475-6415. E-mail: peter.walzer{at}med.va.gov
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Antimicrobial Agents and Chemotherapy, November 2001, p. 3234-3237, Vol. 45, No. 11
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.11.3234-3237.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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