Synergistic Activity of Azithromycin and Pyrimethamine or

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1 ANTMCROBAL AGNTS AND CHMOTHRAPY, May 199, p /9/5997-5$./ Copyright 199, American Society for Microbiology Vol. 36, No. 5 Synergistic Activity of Azithromycin and Pyrimethamine or Sulfadiazine in Acute xperimental Toxoplasmosis FRANCS DROUN,1* RADy ALMADANY, FRAN(COS CHAU, BRNARD ROUVX, AND JAN JACQUS POCDALO Laboratoire de Parasitologie-Mycologie, Hopital Saint-Louis, 1, avenue Claude Vellefaux, Paris Cedex 1, 1 and NSRM U 13, Hopital Claude Bernard, Paris Cedex 19, France Received 4 October 1991/Accepted 1 February 199 The efficacy of azithromycin administered alone or combined with pyrimethamine or sulfadiazine was examined in a murine model of acute toxoplasmosis. Outbred Swiss mice acutely infected with tachyzoites of the virulent RH strain were treated for 1 days from day +1 postinfection. The efficacy of each regimen was assessed in terms of survival rates and sequential titration of parasites in blood, brain, and lungs by using a tissue culture method. Administration of azithromycin at 3, 15, or 75 mg/kg of body weight per day resulted in prolonged survival relative to that of untreated controls; sequential examination of parasite burden showed early eradiaction of Toxoplasma gondii from the lungs, whereas dissemination to the brain was not prevented. A remarkable synergistic effect was observed when azithromycin (15 mg/kgtday) was administered in combination with pyrimethamine or sulfadiazine at noncurative dosages, i.e., 1.5 and mg/kg/day, respectively. n mice treated with azithromycin plus sulfadiazine and azithromycin plus pyrimethamine, parasite burdens in blood and organs, relapses after cessation of therapy, and mortality were all markedly reduced relative to mice treated with any of the agents alone. These results show that azithromycin, which is remarkably active on pulmonary Toxoplasma infection, significantly potentiates the curative effect of sulfadiazine or pyrimethamine. Macrolides are inhibitory for Toxoplasma gondii tachyzoites in vitro, but a significant effect is only obtained at high concentrations (3, 4, 6). This suggests that a therapeutic effect will only be obtained in vivo if these concentrations are reached in body fluids or tissues. During the past few years, several new macrolide derivatives with greater oral biovailability and higher and more persistent levels in serum and tissues have been proposed (1); among these compounds, azithromycin is effective in the treatment of both acute and chronic toxoplasmosis when administered at 15 to 3 mg/kg of body weight per day to mice (1, ). This 15-membered ring macrolide is characterized by a very long elimination half-life in animals and humans and exceptional tissue distribution with accumulation in various type of cells (), yielding very high intracellular concentrations. These pharmacological considerations led us to reevaluate the activity of azithromycin in an experimental model of acute toxoplasmosis by sequential determination of the parasite burden in tissues. n addition, the combinations of azithromycin with pyrimethamine or sulfadiazine were investigated, since these two latter drugs show better diffusion into the brain and may complement the activity of azithromycin. MATRALS AND MTHODS Parasites. The RH strain of T. gondii was maintained in mice by passages every or 3 days; for each experiment, tachyzoites were collected from the peritoneal cavity of infected mice and resuspended in physiological saline. * Corresponding author. 997 n vivo experiments. n vivo studies were performed by using female Swiss Webster mice weighing 1 to grams (ffa Credo, Lyon, France) and infected intraperitoneally with 14 tachyzoites. The kinetics of the infection were determined by subculture of serial dilutions of blood, brain, and lung homogenates as previously described (11). From each blood and organ suspension, serial fourfold dilutions were prepared in the culture medium, and then 4 pl of each dilution was inoculated into duplicate wells of tissue culture plates. After 7 h of incubation at 37 C, cultures were fixed and examined for T. gondii by using an indirect immunofluorescence assay. The presence of parasitic foci was recorded in each well; the final titer was the last dilution which gave at least one parasitic focus. The number of parasites per gram or milliliter (parasite burden) was calculated as the reciprocal titer in tissue culture/volume (microliters) or weight (milligrams) x 1,. Azithromycin (Pfizer nc.), pyrimethamine (Sigma Chemical Co.), and sulfadiazine (Dohms Laboratories) were provided in powder form and prepared daily as liquid suspensions; after brief sonication, the homogeneized suspensions were administered to mice by tube feeding. xperimental design. The efficacy of azithromycin alone and in combination was examined in two sets of experiments. n each experiment mice were randomly allocated to separate groups. Thirty mice were not treated (controls), and 4 mice were used for each drug regimen. Treatments were administered for 1 days from day 1 after infection. Parasite burdens were determined after sacrifice at various intervals; five mice were used for each time point, and blood and organ samples were cultured. (i) Azithromycin alone. Azithromycin was administered at 75, 15, and 3 mg/kg/day. Mice were studied for 1 days after infection. Parasite burden was determined in blood and Downloaded from on April 9, 1 by guest

2 99 DROUN T AL. TABL 1. Activity of azithromycin administered for 1 days from day 1 postinfection % Survival' on following day Treatment after infection: Mean survival (mg/kg/day) (days) Controlb Azithromycin a Survival was estimated at the date of examination of parasitic burden in blood and tissues (Fig. 1). b Mean of two experiments. organs on days 4, 7, 1, 14, and 1 after infection in mice treated with 15 and 3 mg/kg/day, or on days 4, 7, 1, and 14 in mice treated with 75 mg/kg/day. (ii) Azithromycin combined with pyrimethamine or sulfadiazine. The efficacy of azithromycin combined with sulfadiazine or pyrimethamine administered at noncurative dosages was examined in two separate experiments. ach experiment comprised a group of untreated mice (controls) and a group of mice treated with azithromycin alone (15 mg/kg/ day). One group of mice was treated with sulfadiazine ( mg/kg/day), and another with the combination of azithromycin plus sulfadiazine. n a second experiment, one group of mice was treated with pyrimethamine (1.5 mg/kg/day) and another with azithromycin plus pyrimethamine. Mice were studied for 3 days after infection, and parasite burden was determined in the survivors in each group at days 4, 7, 1, 14, 1, and 3 after infection. Statistical analysis. Survival rates were estimated by using the Kaplan Meier product limit method. The parasite burden in blood, lungs, and brain was determined, and the mean value from five mice (± 1 standard error) was calculated from each time point. RSULTS Untreated mice. All the control mice died within 7 (range, 5 to 7) days. As in a previous study (11), parasite burdens were constantly higher in lung than in brain or blood; at day 4, the mean values of four experiments ( mice) were 6.31 ±.7 log units in lung, 3. ±.4 in brain, and.3 ± in blood. Azithromycin alone. Administration of azithromycin at 75, 15, and 3 mg/kg/day from day 1 after infection resulted in an increase in survival relative to control mice (Table 1). At the time of death, mice treated with azithromycin had neurological symptoms such as paralysis and clonic movements, whereas controls showed signs of pulmonary distress and intense weakness. The kinetics of infection in mice treated with azithromycin at 75, 15, and 3 mg/kg/day are presented in Fig. 1A, B, and C, respectively. n treated mice, parasitaemia was negative throughout the follow-up period. n mice treated with 3 mg/kg/day, parasite burden progressively increased in the brain from day 7 until day 1; lung involvement was only demonstrated at day 4 in one mouse and then remained undetectable. n mice treated with 15 mg/kg/day, a similar pattern, consisting of a progressive increase in parasite burden in the brain, was observed, contrasting with an early and persistent disappearance of lung parasites at day 7. n mice treated with 75 mg/kg/day, the disappearance of lung ANTMCROB. AGNTS CHMOTHR. parasites took longer; high parasite burdens were observed at day 4 and then decreased at day 7 to become negative at day 1. Azithromycin combined with sulfadiazine or pyrimethamine. n each of the two experiments, the mean time of death was, respectively, 5.1 and 5. days for control mice and 1 and 13 days for mice treated with azithromycin (15 mg/kg/day). n both experiments, the parasite burden in mice treated with azithromycin alone progressively increased in the brain until death, whereas it decreased in the lungs. n one experiment, parasites were present at a low level in the blood of one of five mice at day 4 and at day 1, and in the lung of one of five mice at day 1; in the other experiment, parasites were present in the lungs of two of the five mice at day 14. When sulfadiazine was administered alone at mg/kg/ day, 77% of the mice were alive at the end of the experiment (Table ). Parasite burdens were low in the lungs while mice were under therapy and then rapidly increased in the lungs and brain early after cessation of therapy and remained constant at day 1 and day 3 (Fig. B). When pyrimethamine was administered at 1.5 mg/kg/day, all the mice died within 3 days (mean survival, 1 days) (Table ). Parasite burdens increased between day 4 and day 7 and then decreased but did not become negative in lung and brain. After cessation of therapy, parasite burdens rapidly increased in blood and organs, with maximum values of log units in the brain and log units in the lungs at day 1 (Fig. C). (i) Combination of azithromycin and sulfadiazine. All the mice that received this combination were alive at day 3 (Table ). Throughout the follow-up period, parasites were undetectable in the lung and blood; the brains of only two mice were infected, one at day 7 and one at day 3, with low parasite counts (Fig. D). (ii) Combination of azithromycin and pyrimethamine. One mouse died at day 5, giving a final survival estimate of 93% at day 3 (Table ). Parasites were found at a low level at day 4 in the blood of one mouse and the brains of two mice, at day 14 and day 1 in the lungs of one mouse, and at day 3 in the brain and lungs of one mouse (Fig. ). DSCUSSON The results of this study confirm previous reports on the efficacy of azithromycin against murine toxoplasmosis when administered alone at a high dosage (1, ). Like these latter authors, we found the curative effect to be dose dependent, but complete protection was not obtained in our model, even at the dosage of 3 mg/kg/day. The kinetic study of parasite infection in blood and tissues showed that parasitic dissemination to the brain was not prevented. At the time of death, mice showed neurological symptoms, probably related to the high parasite burden in brain tissue. n contrast, parasites were not detectable in blood and were cleared from lungs. This specific efficacy was probably the main reason for the prolonged survival of treated mice, since pulmonary toxoplasmosis is the principal cause of death in this model of acute infection (11). This special effect on lung infection may be explained in part by the pharmacokinetics of azithromycin. This macrolide has a large tissue distribution and concentrates into alveolar macrophages with a prolonged uptake; this yields high intracellular concentrations which can be retained for long periods, even in the absence of extracellular antibiotic (). Since it has been shown that concentrations of at least 1 to,ug/ml are necessary to Downloaded from on April 9, 1 by guest

3 7,5O-... VOL. 36, A - - S.6 = ;.a4 SYNRGSTC ACTVTY OF AZTHROMYCN 999 B r "r O FG. 1. Kinetics of parasite burdens in blood (), lungs (-), and brain () in mice infected at day with 14 tachyzoites of the RH C strain. ach point represents the mean ± standard error of the mean for five mice. Shaded areas represent the period of administration of antimicrobial agents (day 1 to day 1 after infection). Mice were treated with azithromycin at 75 mg/kg/day (A), 15 mg/kg/day (B), and 3 mg/kg/day (C) for 1 days from day 1 after infection. n '-7_ centration in vitro (5), may only be partially effective in acute infections. The other main observation was the prolonged therapeutic effect of azithromycin in the lungs, since parasite burdens remained low even 15 days after cessation of therapy. On the basis of these results, azithromycin could act in synergy with 35 other compounds which individually fail to show prolonged efficacy. n a previous study using a similar experimental design we showed that the efficacy of pyrimethamine and sulfadiazine was limited to the period of administration, since relapses involving lungs and brain were constantly inhibit 9% of T. gondii growth in vitro (3, 5), parasites m; ay observed early after cessation of therapy (11). n the present only be eradicated in organs in which such concentratio] ns report, we confirm these observations and show that the can be maintained. This would be the case in the lung, whe re administration of azithromycin in addition to sulfadiazine or azithromycin concentrates remarkably (7), but not in t] he pyrimethamine reinforces the activity of each of the two brain, which it penetrates but does not concentrate in as wi ell drugs. n mice treated with azithromycin plus sulfadiazine as it does in other tissues. n a recent study by Araujo et al. and azithromycin plus pyrimethamine, parasite burdens in (1), concentrations of azithromycin in serum and tissue we re blood and organs, as well as mortality and the occurrence of determined in normal and T. gondii-infected mice that h; ad relapses after cessation of therapy, were markedly lower receive a daily dose of,ug of azithromycin for 1 da: ys than in mice treated with any of the agents alone. (i.e., a dose regimen comparable to that was used in oiur These results suggest that the search for new effective study); concentrations in brain tissue were 1,ug/ml in normrial combined therapy for toxoplasmosis should not be limited to mice and 1.7,ug/ml in T. gondii-infected mice. Such conce n- the evaluation of drug combinations with only complemen- tary pharmacologic effects on the parasite but should trations, which are comparable to the 5% inhibitory coon- also Downloaded from on April 9, 1 by guest TABL. n vivo activity of azithromycin, sulfadiazine, and pyrimethamine administered alone or in combination for 1 days from day 1 postinfection Treatment % Survivarl on following day after infection: Mean survival (mg/kg/day) (days) Controlb 1 5. Azithromycin (15) Pyrimethamine (1.5) Sulfadiazine () >3 Azithromycin + Sulfadiazine >3 Azithromycin + Pyrimethamine >3 a Survival was estimated at the date of examination of parasitic burden in blood and tissues (Fig. ). b Mean of two experiments.

4 1 DROUN T AL. ANTMCROB. AGNTS CHMOTHR. _. AB.7 A A.,, 7,, 6 B d CM a C 4.j _ io :5. 4 a. o.3 C -J a. U. U C; c take in account the specific effect of each compound in vivo. Azithromycin was previously found to have no synergistic effect in vitro with pyrimethamine (5) or sulfadiazine (unpublished observations). The remarkable in vivo synergy that we observed is probably related to a complementary effect of the two compounds on tissue infection. On the basis of these results, we consider that the combination of azithromycin plus sulfadiazine or azithromycin plus pyrimethamine should be evaluated in the clinical setting, particularly in patients with pulmonary toxoplasmosis. These combinations may also represent a possible alternative to the combination of pyrimethamine plus sulfadiazine in case of intolerance to one of these two compounds. The prolonged efficacy that we observed experimentally and 35 L FG.. Kinetics of parasite burdens in blood (), lungs (U), and brain () in mice infected at day with 14 tachyzoites of the RH strain. ach point represents the mean + standard error of the mean for five mice (B through ) or ten mice (A, mean of two experiments). Shaded areas represent the period of administration of antimicrobial agents (day 1 to day 1 after infection). Mice were treated with azithromycin at 15 mg/kg/day (A), sulfadiazine at mg/kg/day (B), pyrimethamine at 1.5 mg/kg/day (C), azithromycine combined with sulfadiazine (D), and azithromycin combined with pyrimethamine (). the fact that azithromycin was recently shown to be effective against Toxoplasma cysts (9) also support testing the efficacy, of these combinations for the primary and secondary pro- 35 phylaxis of toxoplasmosis. ACKNOWLDGMNTS This work was supported in part by a grant from the Agence Nationale pour la Recherche sur le SDA (ANRS). We thank A. Sulahian and D. Young for reviewing the manuscript. RFRNCS 1. Araujo, F., R. M. Shepard, and J. S. Remington n vivo activity of the macrolides antibiotic azithromycin, roxithromycin and spiramycin angainst Toxoplasma gondii. ur. J. Clin. Microbiol. nfect. Dis. 1: Araujo, F. G., D. R. Guptill, and J. S. Remington. 19. Azithromycin, a macrolide antibiotic with potent activity against Toxoplasma gondii. Antimicrob. Agents Chemother. 3: D 35 Downloaded from on April 9, 1 by guest

5 VOL. 36, Chamberland, S., H. A. Kirst, and W. L. Current Comparative activity of macrolides against Toxoplasma gondii demonstrating utility of an in vitro microassay. Antimicrob. Agents Chemother. 35: Chang, H. R., and J. C. F. Pechere. 19. n vitro effects of four macrolides (roxithromycin, spiramycin, azithromycin [CP- 6,993], and A-566) on Toxoplasma gondii. Antimicrob. Agents Chemother. 3: Derouin, F., and C. Chastang Activity in vitro against Toxoplasma gondii of azithromycin and clarithomycin alone and with pyrimethamine. J. Antimicrob. Chemother. 5: Derouin, F., J. Nalpas, and C. Chastang. 19. Mesure in vitro de l'effet inhibiteur de macrolides lincosamides et synergestines sur la croissance de Toxoplasma gondii. Pathol. Biol. 36: Girard, A.., D. Girard, A. R. nglish, T. D. Gootz, C. R. Cimochowski, J. A. Faiella, S. L. Haskell, and J. A. Retsema Pharmacokinetic and in vivo studies with azithromycin SYNRGSTC ACTVTY OF AZTHROMYCN 11 (CP-6,993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrob. Agents Chemother. 31: Gladue, R. P., G. M. Bright, R.. saacson, and M. F. Newborg n vitro and in vivo uptake of azithromycin (CP-6,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection. Antimicrob. Agents Chemother. 33: Huskinson-Mark, J., F. G. Araujo, and J. S. Remington valuation of the effect of drugs on the cyst form of Toaxoplasma gondii. J. nfect. Dis. 164: Kirst, H. A., and G. Sides New directions for macrolide antibiotics: pharmacokinetics and clinical efficacy. Antimicrob. Agents Chemother. 33: Piketty, C., F. Derouin, B. Rouveix, F. Chau, and J. J. Pocidalo n vivo assessment of antimicrobial agents against Toxoplasma gondii by quantification of parasites in the blood, lungs, and brain of infected mice. Antimicrob. Agents Chemother. 34: Downloaded from on April 9, 1 by guest