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In vivo susceptibility of Mycobacterium leprae to sitafloxacin (DU-6859a), either singly or in combination with rifampicin analogues
International Journal of Antimicrobial Agents 21 (2003) 251 /255 www.isochem.org
In vivo susceptibility of Mycobacterium leprae to sitafloxacin (DU-6859a), either singly or in combination with rifampicin analogues Arvind M. Dhople a,*, Kenji Namba b a
Department of Biological Sciences, Florida Institute of Technology, 150 West University Boulevard, Melbourne, FL 32901-6975, USA b New Product Research Laboratories I, Daiichi Pharmaceutical Company, Ltd., Tokyo, Japan Received 14 May 2002; accepted 1 July 2002
Abstract The antimicrobial effects of sitafloxacin (DU-6859a) against Mycobacterium leprae , either singly or in combination with either rifampicin, rifabutin or KRM-1648, were studied using a mouse footpad assay technique and the results were compared with those obtained with ofloxacin. When used singly, the minimum concentrations of sitafloxacin and ofloxacin needed to inhibit completely the growth of M. leprae were 25 and 100 mg per kg body weight per day, respectively, and the effects were bactericidal. Both sitafloxacin and ofloxacin exhibited excellent synergistic effects when combined with either rifabutin or KRM-1648, but not with rifampicin. Thus, incorporation of sitafloxacin and rifabutin (or KRM-1648) in the multidrug regimen for treating leprosy patients is suggested. # 2002 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Mycobacterium leprae ; Sitafloxacin; Ofloxacin; Rifampicin; Rifabutin; KRM-1648; Mouse footpad
1. Introduction The most effective treatment for leprosy currently being used worldwide is the multidrug therapy recommended by the World Health Organization consisting of dapsone, clofazimine and rifampicin [1]. Even though the number of registered cases under treatment has declined from about 12 million in 1981 to less than 1 million in 1999, the number of new cases detected per year has been steadily increasing with 840 000 new cases detected in 1999 alone-approximately equal to the global prevalence rate [2]. Furthermore, the emergence of strains of M. leprae resistant to all three front-line drugs has been reported [3] and also the increasing incidence of relapsed cases several years after completing the multidrug therapy [4,5]. To prevent generation of drug-resistant strains of M. leprae , to enhance the
* Corresponding author. Tel.: /1-321-674-7253; fax: /1-321-6747238 E-mail address: [email protected] (A.M. Dhople).
efficacies of current regimens and to reduce the duration of therapy, new bactericidal agents with highly potent antileprosy activity are urgently needed. In recent years, reports of in vitro activities of new fluoroquinolones against mycobacteria have suggested a potential role for these compounds in the management of mycobacterial diseases, including tuberculosis [6 /8]. Using an in vitro system as well as a mouse footpad system, Dhople and co-workers have shown the antileprosy activity of ofloxacin and also its synergism when combined with rifabutin [9,10]. Furthermore, they have also demonstrated the superiority of levofloxacin over ofloxacin in inhibiting the in vitro growth of M. leprae [11]. Recently, a new fluoroquinolone, sitafloxacin (DU-6859a) has been shown to be more potent than ofloxacin against various Gram-positive organisms [12] and also against mycobacteria, including M. tuberculosis [13,14]. Also, Dhople and Namba have shown superiority of sitafloxacin over other currently available fluoroquinolones in inhibiting the in vitro growth of M. leprae (submitted for publication).
0924-8579/02/$ - see front matter # 2002 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 2 ) 0 0 3 5 1 - 5
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A.M. Dhople, K. Namba / International Journal of Antimicrobial Agents 21 (2003) 251 /255
The aim of the present study was, therefore, to compare the activity of sitafloxacin with ofloxacin against M. leprae in mouse footpads, and also to study its synergism when combined with rifampicin analogues.
The results were analyzed by Student’s t-test and Fisher’s exact probability calculations. Differences were considered significant at 95% level of confidence.
3. Results 2. Materials and methods 2.1. Antimicrobial agents Sitafloxacin, (-)-7[(7S )-amino-5-azaspiro{2,4}heptan5-yl]-8-chloro-6-fluoro[(1R ,2S )-2-fluoro-1-cyclopropyl]1,4-dihydro-4-oxo-3-quinolinecarboxylic acid sesquihydrate, also known as DU-6859a, was obtained from Daiichi Pharmaceutical Company, Ltd., Tokyo, Japan, while ofloxacin was obtained from R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ. Both the drugs were first dissolved in small volumes of 0.1 N NaOH and diluted appropriately to obtain the desired concentration to be fed by gavage. Rifabutin and KRM1648 were gifts from Pharmacia Adria Laboratories, Columbus, Ohio and Kaneka Corp., Osaka, Japan, respectively. Rifampicin was obtained commercially from Sigma Chemical Co., St. Louis, MO. Rifampicin analogues were mixed in powdered mouse food (Purina Mills, St. Louis, MO) at the concentrations indicated and the mice were fed continuously ad libitum. 2.2. Bacteria M. leprae were harvested from the livers of ninebanded armadillos (Dasypus novemcinctus ) that had previously been inoculated in this laboratory with human- or armadillo-derived M. leprae and the bacteria were stored at /76 8C. Purification of M. leprae cells was carried out using DNase and Percoll gradient [15]. Cell counts were determined microscopically by the pinhead method [16].
3.1. Susceptibility of M. leprae to sitafloxacin The results presented in Tables 1 and 2 represent mean values and standard deviations derived from six samples of each variable. Inoculating M. leprae into mouse footpads does not result into disseminated infection during the life span of the mice, but rather the multiplication of the organism is restricted in the footpads only. Thus, the effect of drugs is evaluated by the extent of multiplication of the organism in the footpads. In all the experiments, M. leprae multiplied in normal fashion in the footpads of untreated control mice, thus, indicating the viability of inoculated M. leprae (Tables 1 and 2). In the mice treated with sitafloxacin (Table 1), at a dose of 12.5 mg per kg body weight per day, the multiplication of M. leprae in footpads was similar to that observed in untreated control mice, though the multiplication was slower than in controls even after withdrawing the drug after 8 months. On the other hand, in mice treated with sitafloxacin at a dose of 25 mg per kg body weight per day and higher, there was complete suppression of growth of organisms during the first 8 months of treatment period as well as 3 months after withdrawing the drug showing that the action of sitafloxacin was bactericidal. Using ofloxacin under similar conditions, daily dose of 50 mg per kg body weight was bacteriostatic, while 100 mg per kg body weight was bactericidal. To obtain similar bactericidal effects with rifampicin analogues (rifampicin, rifabutin and KRM-1648), the concentrations of drugs mixed in powdered food, and fed ad libitum, were 0.004, 0.0001, and 0.002% (w/w) respectively.
2.3. Mouse footpad assays Inoculation of M. leprae into mouse (BALB/c, 6week-old females) footpads and subsequent harvests were carried out using the method of Shepard [17]; quantitation of M. leprae in footpad harvests used the pinhead method [16]. Both hind footpads were inoculated with 6 /103 M. leprae cells. At periodic intervals during treatment (4, 6 and 8 months post inoculations), three mice from each variable group were sacrificed, tissues from each of the two hind footpads of each mouse were ground in 0.01 M phosphate buffer, pH 7.0, and M. leprae cells from each suspension were enumerated by the pinhead method. At the end of 8 months of treatment, drugs were withdrawn from the mouse food and last batch of mice was sacrificed 3 months later.
3.2. Susceptibility of M. leprae to sitafloxacin in combination with rifampicin analogues Combining sitafloxacin, at a dose of 12.5 mg per kg body weight per day or lower, with even 0.001% rifampicin had no effect on the multiplication of M. leprae in footpads, though the bacterial yield with a dose 12.5 mg of sitafloxacin combined with rifampicin was significantly lower compared with that in the control group (Table 2). On the other hand, when the mice were fed with sitafloxacin at a daily dose of 12.5 mg per kg body weight (but not lower) in combination with 0.002% rifampicin, no multiplication of M. leprae was observed in the footpads even after withdrawing the drugs after 8 months, thus, indicating complete inhibi-
A.M. Dhople, K. Namba / International Journal of Antimicrobial Agents 21 (2003) 251 /255
253
Table 1 Effect of sitafloxacin, ofloxacin and rifampicin analogues on M. leprae in mouse footpads Concentrationa
Drug
M. leprae ( /106) harvested per footpad at months post inoculation Four
Control Sitafloxacin
12.5 25 50 100 25 50 100 150 0.002 0.004 0.00003 0.0001 0.001 0.002
Ofloxacin
Rifampicin Rifabutin KRM-1648 a b
Eightb
Six
1.289/0.17 0.859/0.11 0 0 0 1.329/0.20 0 0 0 1.119/0.13 0 1.049/0.11 0 0.679/0.11 0
2.249/0.28 1.319/0.15 0 0 0 2.399/0.32 0 0 0 2.269/0.25 0 2.379/0.30 0 0.989/0.10 0
Eleven
2.979/0.46 1.649/0.20 0 0 0 3.149/0.38 0 0 0 3.139/0.36 0 3.419/0.38 0 1.389/0.19 0
3.329/0.42 2.289/0.32 0 0 0 3.419/0.51 1.629/0.17 0 0 3.489/0.44 0 3.649/0.37 0 1.989/0.30 0
Fluoroquinolones, mg per kg body weight per day, by gavage; Rifampicin analogues, % (w/w) mixed in food, fed ad libitum. Treatment discontinued.
tion of growth of organisms with this combination. To achieve similar results with ofloxacin, the doses of both drugs had to be reduced by 50% as with the combination of sitafloxacin and rifampicin. To obtain similar bactericidal inhibition in the growth of M. leprae in footpads with a combination of sitafloxacin (or ofloxacin) and rifabutin, the dose of either of the fluoroquinolones was 25% of the their full dose when used singly, and of rifabutin was 33% of the full dose when used singly to
obtain complete inhibition. Similarly, for a combination of either sitafloxacin or ofloxacin and KRM-1648, the concentration of each drug was 25% of its full dose when given singly to achieve bactericidal inhibition. Complete inhibition with this combination was also obtained when daily dose of sitafloxacin was 12.5 per kg body weight (50% of full dose if used singly) and KRM-1648 at 0.00025% (12.5% of full dose when used singly) was mixed in powdered chow.
Table 2 Effect of sitafloxacin (or ofloxacin), in combination with rifampicin analogues, on the growth of M. leprae in mouse footpads Sitafloxacina
Ofloxacina
Rifampicinb
Rifabutinb
KRM-1648b
M. leprae ( /106) harvested per footpad at months post inoculation Four
/ 12.5 3.12 6.25 12.5 / / / 12.5 3.12 6.25 / / 6.25 12.5 3.12 / / / a b c
/ / / / / 50 25 50 / / / 50 25 / / / 50 25 50
/ 0.001 0.002 0.002 0.002 0.001 0.002 0.002 / / / / / / / / / / /
/ / / / / / / / 0.00001 0.00003 0.00003 0.00001 0.00003 / / / / / /
/ / / / / / / / / / / / / 0.00025 0.00025 0.0005 0.00025 0.0005 0.0005
mg per kg body weight per day, by gavage. % (w/w) mixed in powdered chow and fed ad libitum. Treatment discontinued.
1.289/0.17 0.729/0.08 1.239/0.15 1.119/0.12 0 0 1.019/0.10 0 0.929/0.11 1.299/0.18 0 0 0 1.249/0.14 0 1.329/0.15 0 0.429/0.06 0
Six 2.249/0.28 0.979/0.16 2.229/0.31 1.579/0.15 0 0 1.909/0.23 0 1.189/0.13 2.049/0.25 0 0 0 2.019/0.25 0 2.359/0.34 0 0 0
Eightc 2.979/0.46 1.089/0.11 3.149/0.31 1.749/0.21 0 0 2.749/0.33 0 1.419/0.18 2.739/0.30 0 0 0 2.839/0.31 0 3.389/0.42 0 0 0
Eleven 3.329/0.42 1.419/0.15 3.869/0.40 1.979/0.31 0 1.529/0.18 3.329/0.48 0 1.899/0.25 3.289/0.41 0 1.489/0.21 0 3.439/0.40 0 3.769/0.49 1.389/0.21 0 0
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4. Discussion Using the in vitro system, we had demonstrated earlier the superiority of sitafloxacin over ofloxacin in inhibiting the growth of M. leprae (submitted for publication). The results presented here confirm our earlier findings about sitafloxacin, either used singly or in combination with either rifabutin or KRM-1648. Due to the inability of investigators to grow M. leprae in vitro (outside of its natural host), and, thus, establishing an effective vaccine, the main thrust for any leprosy control program has to be effective chemotherapy [18]. There has recently been a rise in the discovery of newer fluoroquinolones effective against infections caused by mycobacteria, specifically against M. avium complex and M. tuberculosis . However, very few have been tested against M. leprae and only ofloxacin is being evaluated in leprosy patients. Recently, attempts had been made to treat multibacillary leprosy patients with combination therapy consisting of rifampicin and ofloxacin daily for 4 weeks [19,20]. However, in both the cases the rates of relapses were very high and, thus, such system is totally unacceptable. One of the aims and advantages of multidrug therapy in leprosy is that it allows treatment to be administered for a shorter duration, thereby, enhancing compliance and minimizing the emergence of resistant strains. Multidrug regimens are currently given for 2 years in case of multibacillary leprosy patients. The development of new regimens, comprising bactericidal drugs, which achieve cure more rapidly are, therefore, highly desirable. Dhople [21] has earlier demonstrated the bactericidal effects, and superiority of both rifabutin and KRM1648 over rifampicin, against M. leprae in the in vitro system as well as in the mouse footpad system. Thus, sitafloxacin, rifabutin and KRM-1648 have now been shown to be potent bactericidal drugs against M. leprae . The bactericidal effect of rifabutin and KRM-1648, like rifampicin, is a result of specific, selective and strong, but reversible binding to DNA-dependent RNA polymerase. This enzyme blockade rapidly inhibits binding to DNA and subsequent initiation of protein synthesis. As a fluoroquinolone, sitafloxacin acts by inhibiting DNA gyrase. Thus, the actions of sitafloxacin and rifampicin analogues are complementary to each other and combining sitafloxacin with either rifabutin or KRM-1648 would be an ideal formulation in a multidrug therapy regimen in leprosy since the treatment can be administered for a short period of time, thus, avoiding long term toxic effects. If used singly, the dose of sitafloxacin for complete bactericidal effects against M. leprae is 25% of that of ofloxacin. Furthermore, if combined with either rifabutin or KRM-1648,
the dose of sitafloxacin can further be reduced to 25% of that if used singly, e.g. 6.25 mg per kg body weight per day. Similarly, combining with sitafloxacin, the dose of rifabutin or KRM-1648 can also be reduced to 33/25% of the dose if either of them were used singly. During the entire duration of this study, mice treated with either a single drug or with a combination of two drugs did not exhibit any signs of toxic effects due to drugs; also, no deaths occurred in mice in any group. This suggests that these drugs in the concentrations used were tolerated well by mice. The evidence presented here suggests a strong synergistic activity between sitafloxacin and rifabutin or KRM-1648 against M. leprae in vivo, and, therefore, studies should be undertaken in humans to determine the usefulness of such combinations in the treatment of human leprosy.
Acknowledgements The authors wish to thank Daiichi Pharmaceutical Company, Ltd., Tokyo, Japan, for the generous supply of sitafloxacin.
References [1] World Health Organization. WHO Expert Committee on Leprosy, sixth report; Tech. Rep. Ser. 768, WHO, Geneva, 1988. [2] World Health Organization. Remaining challenges towards the elimination of leprosy, Indian J. Lepr. 2000; 72:33 /45. [3] Ji B. Drug resistance in leprosy. Lepr Rev 1985;56:265 /78. [4] Chen XS, Li WZ, Jiang C, Ye GY. Studies on risk of leprosy relapses in China: relapses after treatment with multidrug therapy. Int J Lepr 1999;63:379 /87. [5] Shaw IN, Ibenezer G, Rao GS, Natarajan MM, Balasundaram MM. Relapses as histoid leprosy after receiving multidrug therapy (MDT): a report of three cases. Int J Lepr 2000;68:272 /6. [6] Heifets JB, Lindholm-Levy PJ. Bacteriostatic and bactericidal activities of ciprofloxacin and ofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex. Tubercle 1987;68:267 /76. [7] Tsukumura M. In vitro antituberculosis activity of new antibacterial substance ofloxacin (DL-8280). Am Rev Resp Dis 1985;131:348 /51. [8] World Health Organization. Study group on chemotherapy of leprosy for control program. WHO Tech. Rep. Ser. 675, WHO, Geneva 1982. [9] Dhople AM, Ibanez MA, Gardner GD. In vitro synergistic activity between ofloxacin and ansamycins against Mycobacterium leprae . Arzneim-Forsch Drug Res 1993;43:384 /6. [10] Dhople AM, Ibanez MA. In vivo susceptibility of Mycobacterium leprae to ofloxacin either singly or in combination of rifampin and rifabutin. Arzneim-Forsch Drug Res 1994;44:563 /5. [11] Dhople AM, Ibanez MA. In vitro activity of levofloxacin, singly and in combination with rifamycin analogs, against Mycobacterium leprae . Antimicrob Agents Chemother 1995;39:2116 /9. [12] Sato K, Hoshino K, Tanaka M, Hayakawa I, Osada Y. Antimicrobial activity of DU-6859, a new potent fluoroquinolone,
A.M. Dhople, K. Namba / International Journal of Antimicrobial Agents 21 (2003) 251 /255 against clinical isolates. Antimicrob Agents Chemother 1992;36:1491 /8. [13] Sato H, Tomioka H, Sato K, Dekio S. In vitro and in vivo antimycobacterial activities of a new quinolone, DU 6859a. Antimicrob Agents Chemother 1994;38:2877 / 82. [14] Tomioka H, Sato K, Akaki T, Kajitani H, Kawahara S, Sakatani M. Comparative in vitro antimicrobial activities of the newly synthesized quinolone HSR-903, sitafloxacin (DU-6859a), gatifloxacin (AM-1155) and levofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex. Antimicrob Agents Chemother 1999;43:3001 /4. [15] Dhople AM, Green KJ, Osborne LJ. Limited in vitro multiplication of Mycobacterium leprae . Ann Inst Pasteur Microbiol 1988;139:213 /23.
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[16] Hanks JH, Chatterjee BR, Lechat MF. A guide to the counting of mycobacteria in clinical and experimental materials. Int J Lepr 1964;32:156 /67. [17] Sheppard CC. The experimental disease that follows the injection of human leprosy bacilli into footpads of mice. J Exp Med 1960;329:161 /8. [18] Ji B, Grosset JH. Recent advances in the chemotherapy of leprosy. Lepr Rev 1990;61:313 /29. [19] Ganapati R, Pai VV, Revankar CR. Relapse of multibacillary leprosy after rifampin and ofloxacin treatment for 28 days: a case report. Int J Lepr 1998;66:56 /7. [20] Ji B, Jamet P, Sow S. High relapse rate among lepromatous leprosy patients treated with rifampin and ofloxacin daily for 4 weeks. Antimicrob Agents Chemother 1997;41:1953 /6. [21] Dhople AM. Search for newer antileprosy drugs. Indian J Lepr 2000;72:5 /20.
In vivo susceptibility of Mycobacterium leprae to sitafloxacin (DU-6859a), either singly or in combination with rifampicin analogues Arvind M. Dhople a,*, Kenji Namba b a
Department of Biological Sciences, Florida Institute of Technology, 150 West University Boulevard, Melbourne, FL 32901-6975, USA b New Product Research Laboratories I, Daiichi Pharmaceutical Company, Ltd., Tokyo, Japan Received 14 May 2002; accepted 1 July 2002
Abstract The antimicrobial effects of sitafloxacin (DU-6859a) against Mycobacterium leprae , either singly or in combination with either rifampicin, rifabutin or KRM-1648, were studied using a mouse footpad assay technique and the results were compared with those obtained with ofloxacin. When used singly, the minimum concentrations of sitafloxacin and ofloxacin needed to inhibit completely the growth of M. leprae were 25 and 100 mg per kg body weight per day, respectively, and the effects were bactericidal. Both sitafloxacin and ofloxacin exhibited excellent synergistic effects when combined with either rifabutin or KRM-1648, but not with rifampicin. Thus, incorporation of sitafloxacin and rifabutin (or KRM-1648) in the multidrug regimen for treating leprosy patients is suggested. # 2002 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Mycobacterium leprae ; Sitafloxacin; Ofloxacin; Rifampicin; Rifabutin; KRM-1648; Mouse footpad
1. Introduction The most effective treatment for leprosy currently being used worldwide is the multidrug therapy recommended by the World Health Organization consisting of dapsone, clofazimine and rifampicin [1]. Even though the number of registered cases under treatment has declined from about 12 million in 1981 to less than 1 million in 1999, the number of new cases detected per year has been steadily increasing with 840 000 new cases detected in 1999 alone-approximately equal to the global prevalence rate [2]. Furthermore, the emergence of strains of M. leprae resistant to all three front-line drugs has been reported [3] and also the increasing incidence of relapsed cases several years after completing the multidrug therapy [4,5]. To prevent generation of drug-resistant strains of M. leprae , to enhance the
* Corresponding author. Tel.: /1-321-674-7253; fax: /1-321-6747238 E-mail address: [email protected] (A.M. Dhople).
efficacies of current regimens and to reduce the duration of therapy, new bactericidal agents with highly potent antileprosy activity are urgently needed. In recent years, reports of in vitro activities of new fluoroquinolones against mycobacteria have suggested a potential role for these compounds in the management of mycobacterial diseases, including tuberculosis [6 /8]. Using an in vitro system as well as a mouse footpad system, Dhople and co-workers have shown the antileprosy activity of ofloxacin and also its synergism when combined with rifabutin [9,10]. Furthermore, they have also demonstrated the superiority of levofloxacin over ofloxacin in inhibiting the in vitro growth of M. leprae [11]. Recently, a new fluoroquinolone, sitafloxacin (DU-6859a) has been shown to be more potent than ofloxacin against various Gram-positive organisms [12] and also against mycobacteria, including M. tuberculosis [13,14]. Also, Dhople and Namba have shown superiority of sitafloxacin over other currently available fluoroquinolones in inhibiting the in vitro growth of M. leprae (submitted for publication).
0924-8579/02/$ - see front matter # 2002 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 0 2 ) 0 0 3 5 1 - 5
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A.M. Dhople, K. Namba / International Journal of Antimicrobial Agents 21 (2003) 251 /255
The aim of the present study was, therefore, to compare the activity of sitafloxacin with ofloxacin against M. leprae in mouse footpads, and also to study its synergism when combined with rifampicin analogues.
The results were analyzed by Student’s t-test and Fisher’s exact probability calculations. Differences were considered significant at 95% level of confidence.
3. Results 2. Materials and methods 2.1. Antimicrobial agents Sitafloxacin, (-)-7[(7S )-amino-5-azaspiro{2,4}heptan5-yl]-8-chloro-6-fluoro[(1R ,2S )-2-fluoro-1-cyclopropyl]1,4-dihydro-4-oxo-3-quinolinecarboxylic acid sesquihydrate, also known as DU-6859a, was obtained from Daiichi Pharmaceutical Company, Ltd., Tokyo, Japan, while ofloxacin was obtained from R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ. Both the drugs were first dissolved in small volumes of 0.1 N NaOH and diluted appropriately to obtain the desired concentration to be fed by gavage. Rifabutin and KRM1648 were gifts from Pharmacia Adria Laboratories, Columbus, Ohio and Kaneka Corp., Osaka, Japan, respectively. Rifampicin was obtained commercially from Sigma Chemical Co., St. Louis, MO. Rifampicin analogues were mixed in powdered mouse food (Purina Mills, St. Louis, MO) at the concentrations indicated and the mice were fed continuously ad libitum. 2.2. Bacteria M. leprae were harvested from the livers of ninebanded armadillos (Dasypus novemcinctus ) that had previously been inoculated in this laboratory with human- or armadillo-derived M. leprae and the bacteria were stored at /76 8C. Purification of M. leprae cells was carried out using DNase and Percoll gradient [15]. Cell counts were determined microscopically by the pinhead method [16].
3.1. Susceptibility of M. leprae to sitafloxacin The results presented in Tables 1 and 2 represent mean values and standard deviations derived from six samples of each variable. Inoculating M. leprae into mouse footpads does not result into disseminated infection during the life span of the mice, but rather the multiplication of the organism is restricted in the footpads only. Thus, the effect of drugs is evaluated by the extent of multiplication of the organism in the footpads. In all the experiments, M. leprae multiplied in normal fashion in the footpads of untreated control mice, thus, indicating the viability of inoculated M. leprae (Tables 1 and 2). In the mice treated with sitafloxacin (Table 1), at a dose of 12.5 mg per kg body weight per day, the multiplication of M. leprae in footpads was similar to that observed in untreated control mice, though the multiplication was slower than in controls even after withdrawing the drug after 8 months. On the other hand, in mice treated with sitafloxacin at a dose of 25 mg per kg body weight per day and higher, there was complete suppression of growth of organisms during the first 8 months of treatment period as well as 3 months after withdrawing the drug showing that the action of sitafloxacin was bactericidal. Using ofloxacin under similar conditions, daily dose of 50 mg per kg body weight was bacteriostatic, while 100 mg per kg body weight was bactericidal. To obtain similar bactericidal effects with rifampicin analogues (rifampicin, rifabutin and KRM-1648), the concentrations of drugs mixed in powdered food, and fed ad libitum, were 0.004, 0.0001, and 0.002% (w/w) respectively.
2.3. Mouse footpad assays Inoculation of M. leprae into mouse (BALB/c, 6week-old females) footpads and subsequent harvests were carried out using the method of Shepard [17]; quantitation of M. leprae in footpad harvests used the pinhead method [16]. Both hind footpads were inoculated with 6 /103 M. leprae cells. At periodic intervals during treatment (4, 6 and 8 months post inoculations), three mice from each variable group were sacrificed, tissues from each of the two hind footpads of each mouse were ground in 0.01 M phosphate buffer, pH 7.0, and M. leprae cells from each suspension were enumerated by the pinhead method. At the end of 8 months of treatment, drugs were withdrawn from the mouse food and last batch of mice was sacrificed 3 months later.
3.2. Susceptibility of M. leprae to sitafloxacin in combination with rifampicin analogues Combining sitafloxacin, at a dose of 12.5 mg per kg body weight per day or lower, with even 0.001% rifampicin had no effect on the multiplication of M. leprae in footpads, though the bacterial yield with a dose 12.5 mg of sitafloxacin combined with rifampicin was significantly lower compared with that in the control group (Table 2). On the other hand, when the mice were fed with sitafloxacin at a daily dose of 12.5 mg per kg body weight (but not lower) in combination with 0.002% rifampicin, no multiplication of M. leprae was observed in the footpads even after withdrawing the drugs after 8 months, thus, indicating complete inhibi-
A.M. Dhople, K. Namba / International Journal of Antimicrobial Agents 21 (2003) 251 /255
253
Table 1 Effect of sitafloxacin, ofloxacin and rifampicin analogues on M. leprae in mouse footpads Concentrationa
Drug
M. leprae ( /106) harvested per footpad at months post inoculation Four
Control Sitafloxacin
12.5 25 50 100 25 50 100 150 0.002 0.004 0.00003 0.0001 0.001 0.002
Ofloxacin
Rifampicin Rifabutin KRM-1648 a b
Eightb
Six
1.289/0.17 0.859/0.11 0 0 0 1.329/0.20 0 0 0 1.119/0.13 0 1.049/0.11 0 0.679/0.11 0
2.249/0.28 1.319/0.15 0 0 0 2.399/0.32 0 0 0 2.269/0.25 0 2.379/0.30 0 0.989/0.10 0
Eleven
2.979/0.46 1.649/0.20 0 0 0 3.149/0.38 0 0 0 3.139/0.36 0 3.419/0.38 0 1.389/0.19 0
3.329/0.42 2.289/0.32 0 0 0 3.419/0.51 1.629/0.17 0 0 3.489/0.44 0 3.649/0.37 0 1.989/0.30 0
Fluoroquinolones, mg per kg body weight per day, by gavage; Rifampicin analogues, % (w/w) mixed in food, fed ad libitum. Treatment discontinued.
tion of growth of organisms with this combination. To achieve similar results with ofloxacin, the doses of both drugs had to be reduced by 50% as with the combination of sitafloxacin and rifampicin. To obtain similar bactericidal inhibition in the growth of M. leprae in footpads with a combination of sitafloxacin (or ofloxacin) and rifabutin, the dose of either of the fluoroquinolones was 25% of the their full dose when used singly, and of rifabutin was 33% of the full dose when used singly to
obtain complete inhibition. Similarly, for a combination of either sitafloxacin or ofloxacin and KRM-1648, the concentration of each drug was 25% of its full dose when given singly to achieve bactericidal inhibition. Complete inhibition with this combination was also obtained when daily dose of sitafloxacin was 12.5 per kg body weight (50% of full dose if used singly) and KRM-1648 at 0.00025% (12.5% of full dose when used singly) was mixed in powdered chow.
Table 2 Effect of sitafloxacin (or ofloxacin), in combination with rifampicin analogues, on the growth of M. leprae in mouse footpads Sitafloxacina
Ofloxacina
Rifampicinb
Rifabutinb
KRM-1648b
M. leprae ( /106) harvested per footpad at months post inoculation Four
/ 12.5 3.12 6.25 12.5 / / / 12.5 3.12 6.25 / / 6.25 12.5 3.12 / / / a b c
/ / / / / 50 25 50 / / / 50 25 / / / 50 25 50
/ 0.001 0.002 0.002 0.002 0.001 0.002 0.002 / / / / / / / / / / /
/ / / / / / / / 0.00001 0.00003 0.00003 0.00001 0.00003 / / / / / /
/ / / / / / / / / / / / / 0.00025 0.00025 0.0005 0.00025 0.0005 0.0005
mg per kg body weight per day, by gavage. % (w/w) mixed in powdered chow and fed ad libitum. Treatment discontinued.
1.289/0.17 0.729/0.08 1.239/0.15 1.119/0.12 0 0 1.019/0.10 0 0.929/0.11 1.299/0.18 0 0 0 1.249/0.14 0 1.329/0.15 0 0.429/0.06 0
Six 2.249/0.28 0.979/0.16 2.229/0.31 1.579/0.15 0 0 1.909/0.23 0 1.189/0.13 2.049/0.25 0 0 0 2.019/0.25 0 2.359/0.34 0 0 0
Eightc 2.979/0.46 1.089/0.11 3.149/0.31 1.749/0.21 0 0 2.749/0.33 0 1.419/0.18 2.739/0.30 0 0 0 2.839/0.31 0 3.389/0.42 0 0 0
Eleven 3.329/0.42 1.419/0.15 3.869/0.40 1.979/0.31 0 1.529/0.18 3.329/0.48 0 1.899/0.25 3.289/0.41 0 1.489/0.21 0 3.439/0.40 0 3.769/0.49 1.389/0.21 0 0
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4. Discussion Using the in vitro system, we had demonstrated earlier the superiority of sitafloxacin over ofloxacin in inhibiting the growth of M. leprae (submitted for publication). The results presented here confirm our earlier findings about sitafloxacin, either used singly or in combination with either rifabutin or KRM-1648. Due to the inability of investigators to grow M. leprae in vitro (outside of its natural host), and, thus, establishing an effective vaccine, the main thrust for any leprosy control program has to be effective chemotherapy [18]. There has recently been a rise in the discovery of newer fluoroquinolones effective against infections caused by mycobacteria, specifically against M. avium complex and M. tuberculosis . However, very few have been tested against M. leprae and only ofloxacin is being evaluated in leprosy patients. Recently, attempts had been made to treat multibacillary leprosy patients with combination therapy consisting of rifampicin and ofloxacin daily for 4 weeks [19,20]. However, in both the cases the rates of relapses were very high and, thus, such system is totally unacceptable. One of the aims and advantages of multidrug therapy in leprosy is that it allows treatment to be administered for a shorter duration, thereby, enhancing compliance and minimizing the emergence of resistant strains. Multidrug regimens are currently given for 2 years in case of multibacillary leprosy patients. The development of new regimens, comprising bactericidal drugs, which achieve cure more rapidly are, therefore, highly desirable. Dhople [21] has earlier demonstrated the bactericidal effects, and superiority of both rifabutin and KRM1648 over rifampicin, against M. leprae in the in vitro system as well as in the mouse footpad system. Thus, sitafloxacin, rifabutin and KRM-1648 have now been shown to be potent bactericidal drugs against M. leprae . The bactericidal effect of rifabutin and KRM-1648, like rifampicin, is a result of specific, selective and strong, but reversible binding to DNA-dependent RNA polymerase. This enzyme blockade rapidly inhibits binding to DNA and subsequent initiation of protein synthesis. As a fluoroquinolone, sitafloxacin acts by inhibiting DNA gyrase. Thus, the actions of sitafloxacin and rifampicin analogues are complementary to each other and combining sitafloxacin with either rifabutin or KRM-1648 would be an ideal formulation in a multidrug therapy regimen in leprosy since the treatment can be administered for a short period of time, thus, avoiding long term toxic effects. If used singly, the dose of sitafloxacin for complete bactericidal effects against M. leprae is 25% of that of ofloxacin. Furthermore, if combined with either rifabutin or KRM-1648,
the dose of sitafloxacin can further be reduced to 25% of that if used singly, e.g. 6.25 mg per kg body weight per day. Similarly, combining with sitafloxacin, the dose of rifabutin or KRM-1648 can also be reduced to 33/25% of the dose if either of them were used singly. During the entire duration of this study, mice treated with either a single drug or with a combination of two drugs did not exhibit any signs of toxic effects due to drugs; also, no deaths occurred in mice in any group. This suggests that these drugs in the concentrations used were tolerated well by mice. The evidence presented here suggests a strong synergistic activity between sitafloxacin and rifabutin or KRM-1648 against M. leprae in vivo, and, therefore, studies should be undertaken in humans to determine the usefulness of such combinations in the treatment of human leprosy.
Acknowledgements The authors wish to thank Daiichi Pharmaceutical Company, Ltd., Tokyo, Japan, for the generous supply of sitafloxacin.
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