VOl. 36, NO. 8
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1992, p. 1611-1613
0066-4804/92/081611-03$02.00/0
Copyright © 1992, American Society for Microbiology
Activity of Azithromycin against Mycobacterium avium Infection in Beige Mice M. H. CYNAMON* AND S. P. KLEMENS Veterans Affairs Medical Center and SUNY Health Science Center, Syracuse, New York 13210 Received 27 January 1992/Accepted 14 May 1992 The comparative activities of azithromycin and clarithromycin and the activities of azithromycin alone and in combination with other antimycobacterial agents were evaluated in the beige mouse model of disseminated Mycobacterium avium complex infection. Azithromycin was similar in activity to clarithromycin. Azithromycin plus clofazimine plus ethambutol reduced the number of splenic organisms more than azithromycin alone, while the combination was less active than azithromycin alone for bacteria in lungs. Rifabutin had activity active similar to that of azithromycin for organisms in spleens and lungs. Rifabutin plus azithromycin was more different not was significantly combination's the activity but than either agent alone for organisms in spleens, from that of rifabutin for organisms in lungs. The activity of azithromycin against several M. avium complex isolates was evaluated. The reduction of viable cell counts in spleens ranged from 1.7 to 0.8 log units. For the three isolates studied, there was little correlation between the' in vitro MIC and the in vivo activity.
Azithromycin (AZI) is an azalide antibiotic which differs from erythromycin chemically by a methyl-substituted nitrogen in the macrolide ring (11). AZI and clarithromycin (CLA) have in vitro activities against Mycobacterium avium complex (MAC) (6, 10). Both agents have been found to be active in the beige mouse model of disseminated MAC infection (4, 6) and in early clinical trials of disseminated MAC infection in patients with AIDS (3, 14). AZI achieves low peak levels in serum (approximately 1.5 ,ug/ml) (5) compared with an MIC for 90% of strains tested of 32 p,g/ml (n = 28) in vitro against MAC (10). High tissue concentrations of AZI are achieved particularly in spleens (45 p,g/g) and livers (73 ,ug/g) of rats given one oral dose of 50 mg/kg of body weight (5). The level of AZI in the lungs (18 p,g/ml) was much lower. The purpose of the present study was to compare the in vivo activity of AZI with that of CLA and to evaluate the activities of AZI alone and in combination with other antimycobacterial agents in the beige mouse model of disseminated MAC infection.
MATERIALS AND METHODS Drugs. AZI was provided by the Central Research Division, Pfizer Inc., Groton, Conn. Rifabutin (RBT) was provided by Adria Laboratories, Dublin, Ohio. CLA was provided by Abbott Laboratories, Abbott Park, Ill. Clofazimine (CFZ) was provided by CIBA-GEIGY Pharmaceuticals, Summit, N.J. Ciprofloxacin (CIP) was provided by Miles Pharmaceuticals, West Haven, Conn. Ethambutol (EMB) was purchased from Sigma Chemical Co., St. Louis, Mo. RBT and CFZ were dissolved in dimethyl sulfoxide and were subsequently diluted in distilled water prior to administration. AZI and CLA were dissolved in absolute ethanol and were subsequently diluted in distilled water. EMB and CIP were dissolved in distilled water. The final concentration of dimethyl sulfoxide or ethanol in drug preparations was 0.5%. Drug preparations were freshly made each morning prior to administration.
MAC isolates. The primary M. avium isolate used in these
*
Corresponding author.
studies was ATCC 49601, serotype 1, a clinical isolate from a patient with AIDS at SUNY Health Science Center, Syracuse, N.Y. This organism has been used previously in beige mouse studies in our laboratory (2, 8, 9). Two additional clinical isolates, F and G, were utilized. The MICs of AZI and CLA were determined with modified MuellerHinton broth, pH 7.4 (12), supplemented with 10% Middlebrook OADC (oleic acid-albumin-dextrose-catalase) enrichment (Difco Laboratories, Detroit, Mich.). MICs of the remaining antimicrobial agents were determined with modified Middlebrook 7H10 broth (7H10 agar formulation with agar and malachite green omitted), pH 6.6 (1), supplemented with 10% OADC enrichment. The MICs for ATCC 49601 are as follows: AZI, 8 ,ug/ml; CLA, 4 ,ug/ml; CFZ, 1 ,ug/ml; EMB, 4 ,ug/ml; RBT, 0.06 ,g/ml; and CIP, 8 ,ug/ml. The MIC of AZI for isolates F and G is 8 ,ug/ml. The morphology of the colonies on agar was of the transparent phenotype. The organisms were passaged through beige mice every 3 months to maintain virulence. Media. The organisms were grown in modified Middlebrook 7H10 broth with 10% Middlebrook OADC enrichment and 0.05% Tween 80 (13) on a rotary shaker at 37°C for 3 days. The culture suspension was diluted in 7H10 broth to yield 10 Klett units/ml (Klett-Summerson colorimeter; Klett Manufacturing, Brooklyn, N.Y.), or approximately 5 x 107 CFU/ml. The size of the inoculum was determined by titration and cell counts obtained from duplicate 7H10 agar plates (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 5% Middlebrook OADC enrichment. The plates were incubated at 37°C for 3 weeks prior to counting. Infection studies. Six-week-old beige mice (C57BL/6J-bgl/ bg) mice, bred at our facility, were infected intravenously through a caudal vein. Mice of the same sex were used in separate experiments. Each mouse received approximately 107 viable organisms suspended in 0.2 ml of 7H10 broth. In each experiment, a group of infected but untreated mice was sacrificed at the conclusion of therapy (late controls) and compared with treated groups of mice. In several studies, a group was sacrificed at the initiation of therapy (early controls). Generally, five mice per group were studied. Treatment was started 7 days after infection and was given orally by gavage for 10 days. Animals were sacrificed by 1611
1612
CYNAMON AND KLEMENS
ANTIMICROB. AGENTS CHEMOTHER. Early Control Late Control AZI 50 mg/kg AZI 100 mg/kg AZI 200 mg/kg CLA 200 mg/kg
8
z
9.a-
7-
z o,
oC 7
O Early Control * 0 * a O
01
Late Control AZI 200 mg/kg AZI + EMB AZI + CFZ AZI+CIP AZ + CFZ + EMB
6L.
0
UO-
(LZ1
5,
0-1
4.-
5
3.
SPLEEN
LIVER
-L
LUNG
FIG. 1. AZI dose response compared with CLA. Results are means for five mice per group. Error bars, standard deviations.
cervical dislocation 2 days after the last dose of drug. Spleens, livers, and lungs were aseptically removed and were ground in a tissue homogenizer. The number of viable organisms was determined by titration on 7H10 agar plates. Statistical evaluation. The viable cell counts were converted to logarithms, which were then evaluated with one- or two-variable analyses of variance. Statistically significant effects from the analyses of variance were further evaluated by the Tukey honestly significant difference tests (7) to make pairwise comparisons among means. RESULTS
Comparative activities of AZI and CLA (Fig. 1). AZI at 50, 100, and 200 mg/kg of body weight per day was compared with CLA at 200 mg/kg/day in female mice which had been infected with ATCC 49601 (2.8 x 107 organisms). AZI at each dose level and CLA reduced the numbers of organisms in spleens, livers, and lungs compared with the late controls (P < 0.01). AZI at 100 and 200 mg/kg and CLA at 200 mg/kg reduced the numbers of organisms in spleens compared with the early controls (P < 0.01). AZI at 100 mg/kg was not significantly different from AZI at 50 mg/kg. AZI at 200 mg/kg was more active than AZI at 100 mg/kg (P < 0.01). There was no significant difference between AZI at 100 mg/kg and CLA at 200 mg/kg. AZI at 200 mg/kg was more active than the latter agent (P < 0.05). In each treatment group, the number of bacteria in livers was lower than that in the early control group (P < 0.01). At 200 mg/kg, both AZI and CLA were more active than AZI at 50 mg/kg. AZI at 200 mg/kg was more active than AZI at 100 mg/kg or CLA at 200 mg/kg (P < 0.01). Only CLA reduced the number of bacteria in lungs compared with the early control group (P < 0.01). Each treatment resulted in cell counts in lungs that were lower than those in the late control group (P < 0.01). AZI at 200 mg/kg (P < 0.05) and CLA at 200 mg/kg (P < 0.01) were more active than AZI at 50 mg/kg. CLA at 200 mg/kg was more effective than AZI at 100 mg/kg. AZI at 200 mg/kg was not significantly different from AZI at 100 mg/kg or CLA at 200 mg/kg. Combinations of AZI with EMB, CFZ, and CIP (Fig. 2). AZI (200 mg/kg) was given daily alone or in combination with CIP (200 mg/kg), EMB (125 mg/kg), CFZ (20 mg/kg), or
LUNU
UrLrcc-N
FIG. 2. Activities of AZI alone and in combination with EMB, CFZ, and CIP.
EMB (125 mg/kg) plus CFZ (20 mg/kg) to male mice which had been infected with ATCC 49601 (2.2 x 107 organisms). An early control group had a lower number of organisms in lungs than the late controls (P < 0.01); however, no significant difference in the number of splenic organisms was found. Each of the AZI-containing regimens similarly reduced the numbers of organisms in spleens and lungs compared with the early and late control groups (P < 0.01). AZI+CFZ, AZI+EMB, and AZI+CIP had activities similar to that of AZI alone for bacteria in spleens. AZI+ EMB+CFZ was more active than AZI alone or the other combinations for bacteria in spleens (P < 0.01). AZI+EMB, AZI+CFZ, and AZI alone had similar activities for organisms in lungs. AZI+CIP and AZI+EMB+CFZ had comparable activities but less activity than the other regimens for bacteria in lungs (P < 0.05). Combination of AZI with RBT (Fig. 3). AZI at 200 mg/kg and RBT at 20 mg/kg both alone and in combination were given daily to male mice which had been infected with ATCC 49601 (3 x 107 organisms). AZI, RBT, and the combination reduced the numbers of organisms in spleens and lungs compared with untreated mice (P < 0.01). AZI and RBT had 11
-
a
-
*
* Late Control * AZI 200 mg/g RBT 20mgng O AZ + RBT
7 z
6
5
O c) n1
40
32-
SPLEEN FIG.
3.
LUNG
Activities of AZI and RBT alone and in combination.
AZITHROMYCIN ACTIVITY AGAINST M. AVIUM IN BEIGE MICE
VOL. 36, 1992
patients. Although our experiments do not address the issue of drug resistance, it is likely that combination therapy will suppress the emergence of resistant organisms. AZI+RBT and AZI+CFZ+EMB have enhanced activity in mice compared with the strongest single agent. It is not clear whether this would also be the case in humans with MAC infection. Further clinical evaluation of AZI in patients with disseminated MAC infection is in progress and should address the issues of efficacy, safety, and emergence of resistance.
z
0
n
1613
7-
IL
ACKNOWLEDGMENT This study was supported in part by a grant from the Central Research Division, Pfizer Inc.
0
-I-
ATCC
MAC
G
MAC F
49601
FIG. 4. Activity of AZI against several MAC isolates.
similar activities for bacteria in spleens and lungs. The combination was more active than either agent alone for organisms in spleens (P < 0.01). The combination was more active than AZI alone but was not significantly different from RBT alone for bacteria in lungs (P < 0.01). AZI against several MAC isolates (Fig. 4). AZI at 200 mg/kg given daily to male mice which had been infected with ATCC 49601 (2.6 x 107 organisms), female mice infected with isolate G (2.6 x 107 organisms), or male mice infected with isolate F (1.4 x 107 organisms). AZI was active against each of the isolates studied. AZI-treated mice had significantly fewer organisms in spleens (P < 0.01) than did their respective early controls. The reductions in the numbers of organisms ranged from 1.7 log units for ATCC 49601 to 0.8 log units for isolate G.
was
DISCUSSION
AZI has promising in vivo activity against the isolates of MAC studied. The activity of CLA at 200 mg/kg was similar to that of AZI at 200 mg/kg against ATCC 49601, CILA and AZI having in vitro MICs of 4 and 8 ,ug/ml, respectively. The combination of AZI+CFZ+EMB was more active than AZI alone or AZI in combination with either of the other agents. AZI and RBT had similar in vivo activities. AZI+RBT was more active than either agent alone for bacteria in spleens but was not significantly different from RBT alone for bacteria in lungs. AZI+RBT was similar in activity to AZI+CFZ+EMB with respect to organisms in spleens but was more active than AZI+CFZ+EMB against organisms in lungs. AZI was more active against ATCC 49601 than against isolate F or G. The reason(s) for the wide variation in the in vivo activity of this agent against isolates for which in vitro MICs are the same is not clear. A more extensive comparison of AZI with CLA would be useful to determine whether in vitro activity correlates with in vivo activity. Addition of a second or third agent to AZI might be most effective when AZI has modest activity against a particular MAC isolate. Multidrug therapy for mycobacterial infections is traditionally based on suppression of the emergence of resistant organisms. Since human immunodeficiency virus-infected patients with disseminated MAC infection usually have large populations of MAC organisms, it is likely that multidrug therapy will delay the emergence of resistance in this group of
REFERENCES 1. Cynamon, M. H. 1985. Comparative in vitro activities of MDL 473, rifampin, and ansamycin against Mycobactenum intracellulare. Antimicrob. Agents Chemother. 28:440-441. 2. Cynamon, M. H., C. E. Swenson, G. S. Palmer, and R. S. Ginsberg. 1989. Liposome-encapsulated-amikacin therapy of Mycobacterium avium complex infection in beige mice. Antimicrob. Agents Chemother. 33:1179-1183. 3. Dautzenberg, B., C. Truffot, S. Legris, M.-C. Meyohas, H. C. Berlie, A. Mercat, S. Cherret, and J. Grosset. 1991. Activity of clarithromycin against Mycobacterium avium infection in patients with the acquired immune deficiency syndrome. A controlled clinical trial. Am. Rev. Respir. Dis. 144:564-569. 4. Fernandes, P. B., D. J. Hardy, D. McDaniel, C. W. Hanson, and R. N. Swanson. 1989. In vitro and in vivo activities of clarithromycin against Mycobactenium avium. Antimicrob. Agents Chemother. 33:1531-1534. 5. Girard, A. E., D. Girard, A. R. English, T. D. Gootz, C. R. Cimochowski, J. A. Faiella, S. L. Haskell, and J. A. Retsema. 1987. Pharmacokinetic and in vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrob. Agents Chemother. 31:1948-1954. 6. Inderlied, C. B., P. T. Kolonoski, M. Wu, and L. S. Young. 1989. In vitro and in vivo activity of azithromycin (CP 62, 993) against the Mycobacterium avium complex. J. Infect. Dis. 159:994-997. 7. Kirk, R. E. 1986. Experimental design: procedures for the behavioral sciences. Brooks-Cole Publishing Co., Belmont, Calif. 8. Klemens, S. P., and M. H. Cynamon. 1991. In vivo activities of newer rifamycin analogs against Mycobactenum avium infection. Antimicrob. Agents Chemother. 35:2026-2030. 9. Klemens, S. P., M. H. Cynamon, C. E. Swenson, and R. S. Ginsberg. 1990. Liposome-encapsulated-gentamicin therapy of Mycobactenium avium complex infection in beige mice. Antimicrob. Agents Chemother. 34:967-970. 10. Naik, S., and R. Ruck. 1989. In vitro activities of several new macrolide antibiotics against Mycobacterium avium complex. Antimicrob. Agents Chemother. 33:1614-1616. 11. Retsema, J., A. Girard, W. Schelkly, M. Manousos, M. Anderson, G. Bright, R. Borovoy, L. Brennan, and R. Mason. 1987. Spectrum and mode of action of azithromycin (CP-62,993), a new 15-membered-ring macrolide with improved potency against gram-negative organisms. Antimicrob. Agents Chemother. 31:1939-1947. 12. Truffot-Pernot, C., B. Ji, and J. H. Grosset. 1991. Effect of pH on in vitro potency of clarithromycin against Mycobactenium avium complex, abstr. 672. Program Abstr. 31st Intersci. Conf. Antimicrob. Agents Chemother., Chicago, Ill. 13. Vestal, A. L. 1969. Procedures for the isolation and identification of mycobacteria. Public Health Service publication no. 1995, p. 113-115. Laboratory Division, National Communicable Disease Center, Atlanta. 14. Young, L. S., L. Wiviott, M. Wu, P. Kolonoski, R. Bolan, and C. B. Inderlied. 1991. Azithromycin for treatment of Mycobactenum avium-intracellulare complex infection in patients with AIDS. Lancet 338:1107-1109.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1992, p. 1611-1613
0066-4804/92/081611-03$02.00/0
Copyright © 1992, American Society for Microbiology
Activity of Azithromycin against Mycobacterium avium Infection in Beige Mice M. H. CYNAMON* AND S. P. KLEMENS Veterans Affairs Medical Center and SUNY Health Science Center, Syracuse, New York 13210 Received 27 January 1992/Accepted 14 May 1992 The comparative activities of azithromycin and clarithromycin and the activities of azithromycin alone and in combination with other antimycobacterial agents were evaluated in the beige mouse model of disseminated Mycobacterium avium complex infection. Azithromycin was similar in activity to clarithromycin. Azithromycin plus clofazimine plus ethambutol reduced the number of splenic organisms more than azithromycin alone, while the combination was less active than azithromycin alone for bacteria in lungs. Rifabutin had activity active similar to that of azithromycin for organisms in spleens and lungs. Rifabutin plus azithromycin was more different not was significantly combination's the activity but than either agent alone for organisms in spleens, from that of rifabutin for organisms in lungs. The activity of azithromycin against several M. avium complex isolates was evaluated. The reduction of viable cell counts in spleens ranged from 1.7 to 0.8 log units. For the three isolates studied, there was little correlation between the' in vitro MIC and the in vivo activity.
Azithromycin (AZI) is an azalide antibiotic which differs from erythromycin chemically by a methyl-substituted nitrogen in the macrolide ring (11). AZI and clarithromycin (CLA) have in vitro activities against Mycobacterium avium complex (MAC) (6, 10). Both agents have been found to be active in the beige mouse model of disseminated MAC infection (4, 6) and in early clinical trials of disseminated MAC infection in patients with AIDS (3, 14). AZI achieves low peak levels in serum (approximately 1.5 ,ug/ml) (5) compared with an MIC for 90% of strains tested of 32 p,g/ml (n = 28) in vitro against MAC (10). High tissue concentrations of AZI are achieved particularly in spleens (45 p,g/g) and livers (73 ,ug/g) of rats given one oral dose of 50 mg/kg of body weight (5). The level of AZI in the lungs (18 p,g/ml) was much lower. The purpose of the present study was to compare the in vivo activity of AZI with that of CLA and to evaluate the activities of AZI alone and in combination with other antimycobacterial agents in the beige mouse model of disseminated MAC infection.
MATERIALS AND METHODS Drugs. AZI was provided by the Central Research Division, Pfizer Inc., Groton, Conn. Rifabutin (RBT) was provided by Adria Laboratories, Dublin, Ohio. CLA was provided by Abbott Laboratories, Abbott Park, Ill. Clofazimine (CFZ) was provided by CIBA-GEIGY Pharmaceuticals, Summit, N.J. Ciprofloxacin (CIP) was provided by Miles Pharmaceuticals, West Haven, Conn. Ethambutol (EMB) was purchased from Sigma Chemical Co., St. Louis, Mo. RBT and CFZ were dissolved in dimethyl sulfoxide and were subsequently diluted in distilled water prior to administration. AZI and CLA were dissolved in absolute ethanol and were subsequently diluted in distilled water. EMB and CIP were dissolved in distilled water. The final concentration of dimethyl sulfoxide or ethanol in drug preparations was 0.5%. Drug preparations were freshly made each morning prior to administration.
MAC isolates. The primary M. avium isolate used in these
*
Corresponding author.
studies was ATCC 49601, serotype 1, a clinical isolate from a patient with AIDS at SUNY Health Science Center, Syracuse, N.Y. This organism has been used previously in beige mouse studies in our laboratory (2, 8, 9). Two additional clinical isolates, F and G, were utilized. The MICs of AZI and CLA were determined with modified MuellerHinton broth, pH 7.4 (12), supplemented with 10% Middlebrook OADC (oleic acid-albumin-dextrose-catalase) enrichment (Difco Laboratories, Detroit, Mich.). MICs of the remaining antimicrobial agents were determined with modified Middlebrook 7H10 broth (7H10 agar formulation with agar and malachite green omitted), pH 6.6 (1), supplemented with 10% OADC enrichment. The MICs for ATCC 49601 are as follows: AZI, 8 ,ug/ml; CLA, 4 ,ug/ml; CFZ, 1 ,ug/ml; EMB, 4 ,ug/ml; RBT, 0.06 ,g/ml; and CIP, 8 ,ug/ml. The MIC of AZI for isolates F and G is 8 ,ug/ml. The morphology of the colonies on agar was of the transparent phenotype. The organisms were passaged through beige mice every 3 months to maintain virulence. Media. The organisms were grown in modified Middlebrook 7H10 broth with 10% Middlebrook OADC enrichment and 0.05% Tween 80 (13) on a rotary shaker at 37°C for 3 days. The culture suspension was diluted in 7H10 broth to yield 10 Klett units/ml (Klett-Summerson colorimeter; Klett Manufacturing, Brooklyn, N.Y.), or approximately 5 x 107 CFU/ml. The size of the inoculum was determined by titration and cell counts obtained from duplicate 7H10 agar plates (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 5% Middlebrook OADC enrichment. The plates were incubated at 37°C for 3 weeks prior to counting. Infection studies. Six-week-old beige mice (C57BL/6J-bgl/ bg) mice, bred at our facility, were infected intravenously through a caudal vein. Mice of the same sex were used in separate experiments. Each mouse received approximately 107 viable organisms suspended in 0.2 ml of 7H10 broth. In each experiment, a group of infected but untreated mice was sacrificed at the conclusion of therapy (late controls) and compared with treated groups of mice. In several studies, a group was sacrificed at the initiation of therapy (early controls). Generally, five mice per group were studied. Treatment was started 7 days after infection and was given orally by gavage for 10 days. Animals were sacrificed by 1611
1612
CYNAMON AND KLEMENS
ANTIMICROB. AGENTS CHEMOTHER. Early Control Late Control AZI 50 mg/kg AZI 100 mg/kg AZI 200 mg/kg CLA 200 mg/kg
8
z
9.a-
7-
z o,
oC 7
O Early Control * 0 * a O
01
Late Control AZI 200 mg/kg AZI + EMB AZI + CFZ AZI+CIP AZ + CFZ + EMB
6L.
0
UO-
(LZ1
5,
0-1
4.-
5
3.
SPLEEN
LIVER
-L
LUNG
FIG. 1. AZI dose response compared with CLA. Results are means for five mice per group. Error bars, standard deviations.
cervical dislocation 2 days after the last dose of drug. Spleens, livers, and lungs were aseptically removed and were ground in a tissue homogenizer. The number of viable organisms was determined by titration on 7H10 agar plates. Statistical evaluation. The viable cell counts were converted to logarithms, which were then evaluated with one- or two-variable analyses of variance. Statistically significant effects from the analyses of variance were further evaluated by the Tukey honestly significant difference tests (7) to make pairwise comparisons among means. RESULTS
Comparative activities of AZI and CLA (Fig. 1). AZI at 50, 100, and 200 mg/kg of body weight per day was compared with CLA at 200 mg/kg/day in female mice which had been infected with ATCC 49601 (2.8 x 107 organisms). AZI at each dose level and CLA reduced the numbers of organisms in spleens, livers, and lungs compared with the late controls (P < 0.01). AZI at 100 and 200 mg/kg and CLA at 200 mg/kg reduced the numbers of organisms in spleens compared with the early controls (P < 0.01). AZI at 100 mg/kg was not significantly different from AZI at 50 mg/kg. AZI at 200 mg/kg was more active than AZI at 100 mg/kg (P < 0.01). There was no significant difference between AZI at 100 mg/kg and CLA at 200 mg/kg. AZI at 200 mg/kg was more active than the latter agent (P < 0.05). In each treatment group, the number of bacteria in livers was lower than that in the early control group (P < 0.01). At 200 mg/kg, both AZI and CLA were more active than AZI at 50 mg/kg. AZI at 200 mg/kg was more active than AZI at 100 mg/kg or CLA at 200 mg/kg (P < 0.01). Only CLA reduced the number of bacteria in lungs compared with the early control group (P < 0.01). Each treatment resulted in cell counts in lungs that were lower than those in the late control group (P < 0.01). AZI at 200 mg/kg (P < 0.05) and CLA at 200 mg/kg (P < 0.01) were more active than AZI at 50 mg/kg. CLA at 200 mg/kg was more effective than AZI at 100 mg/kg. AZI at 200 mg/kg was not significantly different from AZI at 100 mg/kg or CLA at 200 mg/kg. Combinations of AZI with EMB, CFZ, and CIP (Fig. 2). AZI (200 mg/kg) was given daily alone or in combination with CIP (200 mg/kg), EMB (125 mg/kg), CFZ (20 mg/kg), or
LUNU
UrLrcc-N
FIG. 2. Activities of AZI alone and in combination with EMB, CFZ, and CIP.
EMB (125 mg/kg) plus CFZ (20 mg/kg) to male mice which had been infected with ATCC 49601 (2.2 x 107 organisms). An early control group had a lower number of organisms in lungs than the late controls (P < 0.01); however, no significant difference in the number of splenic organisms was found. Each of the AZI-containing regimens similarly reduced the numbers of organisms in spleens and lungs compared with the early and late control groups (P < 0.01). AZI+CFZ, AZI+EMB, and AZI+CIP had activities similar to that of AZI alone for bacteria in spleens. AZI+ EMB+CFZ was more active than AZI alone or the other combinations for bacteria in spleens (P < 0.01). AZI+EMB, AZI+CFZ, and AZI alone had similar activities for organisms in lungs. AZI+CIP and AZI+EMB+CFZ had comparable activities but less activity than the other regimens for bacteria in lungs (P < 0.05). Combination of AZI with RBT (Fig. 3). AZI at 200 mg/kg and RBT at 20 mg/kg both alone and in combination were given daily to male mice which had been infected with ATCC 49601 (3 x 107 organisms). AZI, RBT, and the combination reduced the numbers of organisms in spleens and lungs compared with untreated mice (P < 0.01). AZI and RBT had 11
-
a
-
*
* Late Control * AZI 200 mg/g RBT 20mgng O AZ + RBT
7 z
6
5
O c) n1
40
32-
SPLEEN FIG.
3.
LUNG
Activities of AZI and RBT alone and in combination.
AZITHROMYCIN ACTIVITY AGAINST M. AVIUM IN BEIGE MICE
VOL. 36, 1992
patients. Although our experiments do not address the issue of drug resistance, it is likely that combination therapy will suppress the emergence of resistant organisms. AZI+RBT and AZI+CFZ+EMB have enhanced activity in mice compared with the strongest single agent. It is not clear whether this would also be the case in humans with MAC infection. Further clinical evaluation of AZI in patients with disseminated MAC infection is in progress and should address the issues of efficacy, safety, and emergence of resistance.
z
0
n
1613
7-
IL
ACKNOWLEDGMENT This study was supported in part by a grant from the Central Research Division, Pfizer Inc.
0
-I-
ATCC
MAC
G
MAC F
49601
FIG. 4. Activity of AZI against several MAC isolates.
similar activities for bacteria in spleens and lungs. The combination was more active than either agent alone for organisms in spleens (P < 0.01). The combination was more active than AZI alone but was not significantly different from RBT alone for bacteria in lungs (P < 0.01). AZI against several MAC isolates (Fig. 4). AZI at 200 mg/kg given daily to male mice which had been infected with ATCC 49601 (2.6 x 107 organisms), female mice infected with isolate G (2.6 x 107 organisms), or male mice infected with isolate F (1.4 x 107 organisms). AZI was active against each of the isolates studied. AZI-treated mice had significantly fewer organisms in spleens (P < 0.01) than did their respective early controls. The reductions in the numbers of organisms ranged from 1.7 log units for ATCC 49601 to 0.8 log units for isolate G.
was
DISCUSSION
AZI has promising in vivo activity against the isolates of MAC studied. The activity of CLA at 200 mg/kg was similar to that of AZI at 200 mg/kg against ATCC 49601, CILA and AZI having in vitro MICs of 4 and 8 ,ug/ml, respectively. The combination of AZI+CFZ+EMB was more active than AZI alone or AZI in combination with either of the other agents. AZI and RBT had similar in vivo activities. AZI+RBT was more active than either agent alone for bacteria in spleens but was not significantly different from RBT alone for bacteria in lungs. AZI+RBT was similar in activity to AZI+CFZ+EMB with respect to organisms in spleens but was more active than AZI+CFZ+EMB against organisms in lungs. AZI was more active against ATCC 49601 than against isolate F or G. The reason(s) for the wide variation in the in vivo activity of this agent against isolates for which in vitro MICs are the same is not clear. A more extensive comparison of AZI with CLA would be useful to determine whether in vitro activity correlates with in vivo activity. Addition of a second or third agent to AZI might be most effective when AZI has modest activity against a particular MAC isolate. Multidrug therapy for mycobacterial infections is traditionally based on suppression of the emergence of resistant organisms. Since human immunodeficiency virus-infected patients with disseminated MAC infection usually have large populations of MAC organisms, it is likely that multidrug therapy will delay the emergence of resistance in this group of
REFERENCES 1. Cynamon, M. H. 1985. Comparative in vitro activities of MDL 473, rifampin, and ansamycin against Mycobactenum intracellulare. Antimicrob. Agents Chemother. 28:440-441. 2. Cynamon, M. H., C. E. Swenson, G. S. Palmer, and R. S. Ginsberg. 1989. Liposome-encapsulated-amikacin therapy of Mycobacterium avium complex infection in beige mice. Antimicrob. Agents Chemother. 33:1179-1183. 3. Dautzenberg, B., C. Truffot, S. Legris, M.-C. Meyohas, H. C. Berlie, A. Mercat, S. Cherret, and J. Grosset. 1991. Activity of clarithromycin against Mycobacterium avium infection in patients with the acquired immune deficiency syndrome. A controlled clinical trial. Am. Rev. Respir. Dis. 144:564-569. 4. Fernandes, P. B., D. J. Hardy, D. McDaniel, C. W. Hanson, and R. N. Swanson. 1989. In vitro and in vivo activities of clarithromycin against Mycobactenium avium. Antimicrob. Agents Chemother. 33:1531-1534. 5. Girard, A. E., D. Girard, A. R. English, T. D. Gootz, C. R. Cimochowski, J. A. Faiella, S. L. Haskell, and J. A. Retsema. 1987. Pharmacokinetic and in vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrob. Agents Chemother. 31:1948-1954. 6. Inderlied, C. B., P. T. Kolonoski, M. Wu, and L. S. Young. 1989. In vitro and in vivo activity of azithromycin (CP 62, 993) against the Mycobacterium avium complex. J. Infect. Dis. 159:994-997. 7. Kirk, R. E. 1986. Experimental design: procedures for the behavioral sciences. Brooks-Cole Publishing Co., Belmont, Calif. 8. Klemens, S. P., and M. H. Cynamon. 1991. In vivo activities of newer rifamycin analogs against Mycobactenum avium infection. Antimicrob. Agents Chemother. 35:2026-2030. 9. Klemens, S. P., M. H. Cynamon, C. E. Swenson, and R. S. Ginsberg. 1990. Liposome-encapsulated-gentamicin therapy of Mycobactenium avium complex infection in beige mice. Antimicrob. Agents Chemother. 34:967-970. 10. Naik, S., and R. Ruck. 1989. In vitro activities of several new macrolide antibiotics against Mycobacterium avium complex. Antimicrob. Agents Chemother. 33:1614-1616. 11. Retsema, J., A. Girard, W. Schelkly, M. Manousos, M. Anderson, G. Bright, R. Borovoy, L. Brennan, and R. Mason. 1987. Spectrum and mode of action of azithromycin (CP-62,993), a new 15-membered-ring macrolide with improved potency against gram-negative organisms. Antimicrob. Agents Chemother. 31:1939-1947. 12. Truffot-Pernot, C., B. Ji, and J. H. Grosset. 1991. Effect of pH on in vitro potency of clarithromycin against Mycobactenium avium complex, abstr. 672. Program Abstr. 31st Intersci. Conf. Antimicrob. Agents Chemother., Chicago, Ill. 13. Vestal, A. L. 1969. Procedures for the isolation and identification of mycobacteria. Public Health Service publication no. 1995, p. 113-115. Laboratory Division, National Communicable Disease Center, Atlanta. 14. Young, L. S., L. Wiviott, M. Wu, P. Kolonoski, R. Bolan, and C. B. Inderlied. 1991. Azithromycin for treatment of Mycobactenum avium-intracellulare complex infection in patients with AIDS. Lancet 338:1107-1109.
