Trimethoprim-sulfamethoxazole (TMP-SMX) is the first-line drug for both curative and preventive treatment of Pneumocystis carinii pneumonia (PCP) (1), and recent clinical observations have shown a preventive efficacy against opportunistic toxoplasmosis in HIV-infected patients (2). This drug consists of five parts of sulfamethoxazole (SMX) to one part of trimethoprim (TMP) and was formulated in this way to produce an in vivo ratio of 20:1, resulting in synergistic activity against gram-positive and gram-negative bacteria (3,4). The use of the drug with this ratio in the setting of PCP and toxoplasmosis was empirical, because it was the only commercialized formulation.
The high incidence of adverse effects in HIV-infected patients receiving TMP-SMX (5-8) warrants the evaluation of other strategies in the use of this drug. Although the mechanism of intolerance to TMP-SMX is poorly known (9), some evidences have demonstrated dose-related intolerance. The respective roles of SMX and TMP are unclear even though that of SMX appears predominant. Therefore, it is important to assess the efficacy of reduced doses of both components and/or to modify their ratio. The administration of lower doses of TMP-SMX appeared to be better tolerated, without loss of prophylactic efficacy against PCP (10-12). Although experimental studies have demonstrated the efficacy of low-dose TMP-SMX against PCP (13-18), data are lacking for toxoplasmosis. Another approach is to reexamine the activity of TMP and SMX alone and/or combined at different ratios, in an attempt to identify the lower dose of each compound yielding optimal efficacy for treatment and prophylaxis of both PCP and toxoplasmosis.
For PCP, in vitro studies have been performed with TMP-SMX (19). In vivo, previous studies suggest the interest in altering the TMP-SMX ratio. Walzer et al. have shown that the results obtained with TMP (100 mg/kg/day) and SMX (3 mg/kg/day) were comparable to those achieved with the standard regimen of TMP at 50 mg/kg/day and SMX at 250 mg/kg/day after 10 days of treatment of experimental murine PCP (17).
For toxoplasmosis, TMP-SMX ratios ranging from 1:140 to 2:1 were found to be synergistic in experiments in vitro (20,21), and acutely infected mice were fully protected with TMP combined with SMX at ratios ranging between 1:8 and 8:1 (22).
This study evaluated different doses and ratios of TMP-SMX in a newly developed rat model of concurrent P. carinii and Toxoplasma gondii infections (23) that better mimics the clinical situation observed in HIV-infected patients.
MATERIALS AND METHODS
Induction of Pneumocystis carinii and Toxoplasma gondii Infections
The animal protocol that we used has been previously described in detail (23). In brief, P. carinii pneumonia was induced in male Wistar rats (Janvier Breeding Laboratories, Le Genest St Isle, France), weighing ≈200 g, by immunosuppression (IS): 25 mg of cortisone acetate (Hydrocortisone; Hoechst-Roussel, Paris, France) injected subcutaneously twice weekly and a low-protein (8%) diet (Usine Alimentation Rationelle, Villemoisson, France). After 5 weeks of IS, rats were intraperitoneally inoculated with 107 tachyzoites of the virulent RH strain of T. gondii.
At the end of the 7-week study period, and 24 h after the last drug administration, rats were anesthetized with pentobarbital (Penthotal; Abbott, Orsay, France) and exsanguinated via the abdominal aorta by using a Vacutainer (Becton Dickinson, Meylan, France). The lungs, brain, spleen, and liver were removed aseptically and weighed. Bacterial infections in lungs were determined by smears on plates agar.
Assessment of Pneumocystis carinii and Toxoplasma gondii Infections
Pneumocystis carinii cysts were counted in lung tissue after enzymatic digestion and O toluidine blue staining, as previously described (23). The number of cysts per gram of lung was expressed as a mean log value ± 1 SD.
Toxoplasma gondii infection was assessed by determination of parasitic burdens in brain, liver, lungs, spleen, and pleural fluid, by using a tissue culture method and an indirect immunofluorescence assay, as previously described (24). For each organ, the result of parasitic burdens was expressed as a mean log value ± 1 SD.
TMP-SMX reference combination (Roche, Neuilly-sur-Seine, France) was the pediatric solution form; TMP and SMX (Sigma, Paris, France) (powder form) were prepared in 1% carboxyme-thylcellulose in sterile distilled water, sonicated, and administered by gavage.
Drugs were administered from the beginning of the corticosteroid administration, 5 days a week for 5 weeks, and then every day after T. gondii inoculation until the rats died or were killed. The doses of each combination that had shown efficacy in PCP prophylaxis were selected: reference combination 20 mg/kg TMP plus 100 mg/kg SMX (13,21,22,25). In the first experiment, TMP (20 mg/kg) and SMX (100 mg/kg) were studied alone. In the second experiment, the effects of different doses of TMP (1, 5, and 20 mg/kg) were studied in combination with those of a low dose of SMX (5 mg/kg), and the effects of a high dose of TMP (100 mg/kg) were studied in combination with those of low doses of SMX (5 and 20 mg/kg) (17) (unpublished data).
Drugs were administered per os, every day, 48 h after T. gondii inoculation until the rats died or were killed. The reference combination of TMP (20 mg/kg) plus SMX (100 mg/kg) and the reversed combination of TMP (100 mg/kg) plus SMX (20 mg/kg) were selected.
TMP, SMX, and N4-acetylsulfamethoxazole (N-SMX) were determined in serum and organ tissues by reversed-phase high-performance liquid chromatography (26). Determinations were made 24 h after the last drug administration and when the rats were killed.
Pneumocystis carinii cysts and T. gondii parasitic burdens were processed by one-way analysis of variance, and each interesting pair of groups was compared using the Bonferroni adjusted t test. Differences in values were considered significant if p was ≤ 0.05 or 0.05 divided by the number of tests. Data were expressed as mean ± 1 SD. Correlation and regression coefficients were calculated using the least-squares method.
Prophylactic Activity of TMP, SMX, and TMP-SMX Reference Combinations (Table 1)
Pneumocystis carinii development was monitored in 15 rats: the level of the latent infection (log 3.7 ± 0.6 cysts per gram of lung tissue) increased to log 4.9 ± 0.3 cysts and to log 6.7 ± 0.5 cysts/g lung tissue after 5 and 7 weeks of IS, respectively.
All control coinfected rats (Table 1) died of pleural effusion 5.7 ± 3.1 days after T. gondii inoculation. Active P. carinii and T. gondii infections were found.
The same results were obtained in the group of rats treated with TMP alone at a dose of 20 mg/kg.
All rats treated with SMX (100 mg/kg) were alive at the end of the study; no pleural fluid was found. At autopsy, a low T. gondii burden was found in all organs except the liver. In lungs, the number of P. carinii cysts was significantly lower than that of untreated control rats (p < 0.001), demonstrating efficacy.
All rats treated with the reference regimen TMP (20 mg/kg) plus SMX (100 mg/kg) were alive at the end of the study; no pleural fluid was found. When the rats were killed, a low parasitic burden was detected in only one. PCP was prevented in all rats.
Prophylactic Activity of Various Ratios of TMP and SMX (Table 2)
Pneumocystis carinii development was monitored in 30 rats: the level of the latent infection (log 3.6 ± 1.0 cysts/g lung tissue) increased to log 6.7 ± 0.5 cysts and to log 7.1 ± 0.3 cysts/g lung tissue after 5 and 7 weeks of IS, respectively.
All control coinfected rats (Table 2) died of pleural effusion 4.4 ± 1.4 days after T. gondii inoculation. Active P. carinii and T. gondii infections were found.
TMP plus SMX at 1 + 5-mg/kg and 5 + 5-mg/kg doses were not effective in preventing toxoplasmosis (data not shown). The efficacy improved when TMP doses increased to 20 and 100 mg/kg, with a fixed dose of 5 mg/kg SMX (Table 2). Toxoplasma gondii burdens were log 5.1 and 4.5, respectively, in the brains of two rats treated with TMP-SMX (100 and 5 mg/kg) that died on days 11 and 13 after T. gondii inoculation; the third rat died on day 14 after bacterial infection (Staphylococcus aureus). Toxoplasmic prevention was also achieved with the combination of 100 mg/kg TMP and 20 mg/kg SMX: only one rat had T. gondii in the brain (log 3.3) and in the liver (log 3.75), and no bacterial infection was found. In all groups, P. carinii infection was prevented.
Two of 10 rats treated with the reference regimen TMP (20 mg/kg) plus SMX (100 mg/kg) died of bacterial infection (Staphylococcus aureus) on day 6 after T. gondii inoculation. The other rats were alive at the end of the study; no pleural fluid was found. When the rats were killed, a low parasitic burden was detected in only one rat. PCP was prevented in all rats.
Therapeutic Treatment (Table 3)
Pneumocystis carinii development was monitored in 15 rats: the level of the latent infection (log 3.2 ± 0.3 cysts/g lung tissue) increased to log 6.8 ± 0.4 cysts and to log 7.1 ± 0.3 cysts/g lung tissue after 5 and 7 weeks of IS, respectively.
All untreated coinfected control rats exhibited active dual infection.
TMP plus SMX at 20 + 100-mg/kg doses was fully effective against both toxoplasmosis and PCP. In contrast, the reversed TMP-SMX ratio was poorly effective: two of five rats died of disseminated toxoplasmosis, and the reduction of P. carinii was only partial.
Trimethoprim and Sulfamethoxazole Dosages (Table 4)
After 7 weeks of prophylactic treatment and 24 h after the last drug administration, TMP was not detected in serum of IS-surviving rats, and low levels were obtained in organs with each ratio of TMP-SMX. A correlation was found between the doses of SMX administered and the concentration of this compound in serum (r = 0.80, p < 0.001), lung (r = 0.79, p < 0.001), and brain (r = 0.81, p < 0.001). Globally, the serum concentration was highly related to the lung concentration (r = 0.89, p < 0.001) and to the brain concentration (r = 0.99, p < 0.001).
N4-Acetylsulfamethoxazole was mainly detected in serum and in lung tissue with the highest dose of TMP-SMX (20 + 100 mg/kg).
TMP-SMX is a combination of a dihydrofolate inhibitor (TMP) thought to potentiate the activity of sulfonamide (SMX) by inhibition of folic-acid synthesis. Several investigators have already examined the efficacy of different combinations of the two compounds against T. gondii and P. carinii, but no definitive conclusion could be reached on the optimum dose that should be selected for treatment or prevention of both infections. The study by Remington (27) on experimental T. gondii infection in mice concluded that the combination of TMP and SMX had no greater activity than did SMX. In other studies, combinations of TMP and SMX at various ratios were examined for their efficacy against toxoplasmosis (21); however, because of the very short half-life of TMP in mice, the authors considered the mouse model inadequate for predicting the efficacy of the combination of TMP and SMX in humans. More relevant results were obtained in a rat model of PCP in which the drug synergy between TMP and SMX could be assessed for prophylaxis and treatment of PCP (13,17). In our laboratory, this model was extended in order to realize a concurrent P. carinii and T. gondii infection (23), and it provided a unique opportunity to assess for the efficacy of TMP and SMX at various doses, alone or in combination, against both pneumocystosis and toxoplasmosis.
Administration of TMP at 20 mg/kg prevented neither toxoplasmosis nor PCP. These results are consistent with the fact that TMP has almost no activity on dihydrofolate reductase of P. carinii(28) and confirm previous observations in the rat model of PCP (29). The inefficacy of TMP on toxoplasmosis might be related to the low dose that was administered and correlates with the absence of detectable TMP in serum and low levels in tissue. Although higher doses of TMP alone were not examined in our study, it failed to provide any protection in other murine models of acute toxoplasmosis (21,22,27). This lack of efficacy against T. gondii in vivo is surprising because TMP inhibits T. gondii in vitro with a parasiticidal activity (20); however, such an effect is observed only for concentrations ≥2 mg/L, which may not be reached in infected tissue.
By contrast, the administration of SMX alone (100 mg/kg) provided significant protection against PCP and toxoplasmosis, a result that is consistent with the inhibitory effect of this drug against T. gondii and P. carinii in vitro (20,30) and its partial efficacy in separate murine models of toxoplasmosis or PCP (17,21,22,27). These results obtained with various combinations of TMP and SMX assessed the synergy of the two compounds in the prophylaxis and treatment of both PCP and toxoplasmosis, but marked differences were observed, depending on the doses and ratios of each compound.
Against T. gondii, the drug synergism developed in a dose-dependent manner: with the combination of 1 mg/kg TMP plus 5 mg/kg SMX (ratio, 1:5), 94% of rats died of toxoplasmosis; the number of surviving rats and the delay of death increased proportionally to TMP doses from 1 to 100 mg/kg and with low doses of SMX (5 and 20 mg/kg). The reference regimen consisting of 20 mg/kg TMP plus 100 mg/kg SMX (rati, 1:5) as well as the reversed ratio of 100 mg/kg TMP plus 20 mg/kg SMX prevented toxoplasmosis.
For prophylaxis of PCP, all combinations tested were found to be effective, even when low dosages of SMX and TMP were combined. In our study, control groups treated with SMX (1 or 5 mg/kg) or TMP (1 or 5 mg/kg) were not examined, and therefore we could not assess the synergistic role of a low dosage of TMP. However, most of the effect could be attributed to SMX, since TMP alone at 20 mg/kg was found to be ineffective and SMX at 3 mg/kg was previously found to have a mild activity for treatment of PCP (17).
When TMP and SMX were administered in combination for treatment of PCP and toxoplasmosis, the reference regimen consisting of 20 mg/kg TMP plus 100 mg/kg SMX was remarkably effective against both pathogens, as already noted in previous experiments (23). By contrast, the reversed ratio was poorly effective against toxoplasmosis. For PCP, we observed only a significant reduction of parasite presence when compared with controls, but cyst numbers remained at a higher level than that of rats treated with the reference regimen.
Taken together, these results indicate that if SMX accounts for most of the activity shown by the combination against P. carinii and T. gondii in both prophylactic and therapeutic situations, an increase in the dose of TMP may allow a reduction in the dose of SMX without significant loss of prophylactic activity. This finding may be clinically relevant in case of intolerance to SMX. However, administration of a lower dose of SMX, even when combined with high doses of TMP, does not represent a valuable alternative for curative treatment, because a high dose of SMX is required for efficacy against toxoplasmosis. Therefore, clinical evaluation of altered TMP-SMX ratios in the setting of prophylaxis of both PCP and toxoplasmosis is warranted. Furthermore, SMX alone could be sufficient for prevention of only PCP.
Acknowledgment: This study was supported in part by a grant from the Agence Nationale pour la Recherche sur le SIDA. We thank Jean-Jacques Pocidalo for helpful discussions at the outset of this work, and Premavathy Rajagopalan-Levasseur for help in preparing the manuscript.
1. Masur H. Prevention and treatment of Pneumocystis
pneumonia. N Engl J Med
2. Carr A, Tindall B, Brew BJ, Marriott DJ, et al. Low-dose trimethoprim
-sulfamethoxazole prophylaxis for toxoplasmosis encephalitis in patients with AIDS. Ann Intern Med
3. Brumfitt W, Hamilton-Miller JTM. Reassessment of the rationale for the combinations of sulfonamides with diaminopyrimidines. J Chemother
4. Zinner SH, Mayer H. Sulfonamides and trimethoprim
. In: Mandel GL, Douglas RG, Bennett JE, eds. Principles and practice of infectious diseases
. New York: John Wiley and Sons, 1994:354-64.
5. Gordin FM, Simon GL, Wofsy CB, Mills J. Adverse reactions to trimethoprim
-sulfamethoxazole in patients with the acquired immunodeficiency syndrome. Ann Intern Med
6. Jaffe HS, Abrams DI, Amman AJ, Lewis BJ, Golden JA. Complications of co-trimoxazole in treatment of AIDS-associated Pneumocystis carinii
pneumonia in homosexual men. Lancet
7. Kovacs JA, Hiemenz JW, Macher AM, et al. Pneumocystis carinii
pneumonia: a comparison between patients with the acquired immunodeficiency syndrome and patients with other immunodeficiencies. Ann Intern Med
8. Mitsuyasu R, Groopman J, Volberding P. Cutaneous reaction to trimethoprim
-sulfamethoxazole in patients with AIDS and Kaposi's sarcoma. N Engl J Med
9. Koopmans PP, Van der Ven AJ, Vree TB, Van der Meer JWM. Pathogenesis of hypersensitivity reactions to drugs in patients with HIV infection: allergic or toxic? AIDS
10. May T, Beuscart C, Reynes J, et al. Trimethoprim
-sulfamethoxazole versus aerosolized pentamidine for primary prophylaxis of Pneumocystis carinii
pneumonia: a prospective, randomized, controlled clinical trial. J Acquir Immune Defic Syndr
11. Ruskin J, Lariviere M. Low-dose co-trimoxazole for prevention of Pneumocystis carinii
pneumonia in human immunodeficiency virus disease. Lancet
12. Wormser GP, Horowitzh HW, Ducanson FP, et al. Lowdose intermittent trimethoprim
-sulfamethoxazole for prevention of Pneumocystis carinii
pneumonia in patients with human immunodeficiency virus infection. Arch Intern Med
13. Brun-Pascaud M, Girard PM, Pocidalo JJ. Low-dose trimethoprim
-sulfamethoxazole alone and in association with zidovudine for prevention and treatment of murine Pneumocystis carinii
pneumonia. Antimicrob Agents Chemother
14. Hughes WT, Killmar JT, Oz HS. Relative potency of 10 drugs with anti-Pneumocystis carinii
activity in animal model. J Infect Dis
15. Hughes WT, Smith BL. Intermittent chemoprophylaxis for Pneumocystis carinii
pneumonia. Antimicrob Agents Chemother
16. Shear HL, Valladares G, Narachi MA. Enhanced treatment of Pneumocystis carinii
pneumonia in rats with interferon-γ and reduced doses of trimethoprim
/sulfamethoxazole. J Acquir Immune Defic Syndr
17. Walzer PD, Foy J, Steele P, White M. Synergic combinations of RO 1-8958 and other dihydrofolate reductase inhibitors with sulfamethoxazole and dapsone for therapy of experimental pneumocystosis. Antimicrob Agents Chemother
18. Walzer PD, Kim CK, Foy JM, Zhang J. Furazolidone and nitrofurantion in the treatment of experimental Pneumocystis carinii
pneumonia. Antimicrob Agents Chemother
19. Cushion MT, Stanforth D, Linke MJ, Walzer PD. Method of testing the susceptibility of Pneumocystis carinii
to antimicrobial agents in vivo. Antimicrob Agents Chemother
20. Derouin F, Chastang C. In vitro effect of folate inhibitors on Toxoplasma gondii. Antimicrob Agents Chemother
21. Grossman PL, Remington JS. The effect of trimethoprim
and sulfamethoxazole on Toxoplasma gondii
in vitro and in vivo. Am J Trop Med Hyg
22. Vischer WA. Comparative study of the efficacy of some sulphanilamide derivatives in clinical use against acute toxoplasmosis in the mouse. Zentralblatt Bakteriol Hyg
23. Brun-Pascaud M, Chau F, Simonpoli AM, Girard PM, Derouin F, Pocidalo JJ. Experimental evaluation of combined prophylaxis against murine pneumocystosis and toxoplasmosis. J Infect Dis
24. Piketty C, Derouin F, Rouveix B, Pocidalo JJ. In vivo assessment of antimicrobial agents against T. gondii:
quantification of parasites in blood, lungs and brain of infected mice. Antimicrob Agents Chemother
25. Hughes TH, McNabb PC, Makres TD, Feldman S. Efficacy of trimethoprim
and sulfamethoxazole in the prevention and treatment of Pneumocystis carinii
pneumonitis. Antimicrob Agents Chemother
26. Weber A, Opheim KE, Siber GR, Ericson JF, Smith AL. High-performance liquid chromatographic quantitation of trimethoprim
, sulfamethoxazole, and N4
-acetylsulfamethoxazole in body fluids. J Chromatogr
27. Remington JS. Trimethoprim
-sulfamethoxazole in murine toxoplasmosis. Antimicrob Agents Chemother
28. Allegra CJ, Kovacs JA, Drake JC, Swan JC, Chabner BA, Masur H. Activity of antifolates against Pneumocystis carinii
dihydrofolate reductase and identification of a potent new agent. J Exp Med
29. Walzer PD, Foy J, Steele P, et al. Activities of antifolate, antiviral and others in an immunosuppressed rat model of Pneumocystis carinii
pneumonia. Antimicrob Agents Chemother
30. Comley JC, Mullin RJ, Wolfe LA, Hanlon MH, Ferone R. Microculture screening assay for primary in vitro evaluation of drugs against Pneumocystis carinii. Antimicrob Agents Chemother