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In vitro activity of KRM-1648, either singly or in combination with ofloxacin, against Mycobacteriumulcerans
International Journal of Antimicrobial Agents 17 (2001) 57 – 61 www.ischemo.org
Original article
In vitro activity of KRM-1648, either singly or in combination with ofloxacin, against Mycobacterium ulcerans Arvind M. Dhople * Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL 32901, USA Received 27 April 2000; accepted 12 May 2000
Abstract The antimicrobial effect of a benzoxazinorifamycin, KRM-1648, either alone or in combination with ofloxacin, was evaluated in vitro against two type strains and six clinical isolates of Mycobacterium ulcerans. Growth of M. ulcerans was measured by plate counts and the BACTEC radiometric method. The minimal inhibitory concentration as well as minimal bactericidal concentration of KRM-1648 against M. ulcerans was between 0.012 and 0.025 mg/l, while corresponding values for rifampicin and rifabutin were in the range of 0.1–0.8 mg/l and 0.1–0.4 mg/l respectively. When combined with ofloxacin, KRM-1648 exhibited strong synergistic activity while only additive effects were observed with the combination of rifampicin (or rifabutin) and ofloxacin. These results suggest that KRM-1648 has a great potential in the treatment of M. ulcerans infection. © 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Mycobacterium ulcerans; Buruli ulcer; Benzoxazinorifamycin; KRM-1648, Rifampicin; Ofloxacin; Synergism
1. Introduction Infection with Mycobacterium ulcerans (M. ulcerans) causes a deforming disease, known as Buruli ulcer, occuring in swampy lowlands and river valleys in tropical or subtropical regions. The organism was first isolated by MacCallum and coworkers in 1948 [1] from six patients living in Bairnsdale, South Australia, and so the disease is also known as Bairnsdale ulcer [2]. M. ulcerans is the third major mycobacterial pathogen of man. It grows well on egg yolk-agar medium at 30– 33°C. The lesions often start as small subcutaneous nodules, gradually enlarge over days to weeks and lead to painless and chronic ulcers with characteristically undermined edges. After more than 50 years, the incidence of infection with M. ulcerans seems to be increasing sharply, particularly in West Africa [3,4] and so the World Health Organization has recently declared Buruli ulcer as an emerging public health problem [5]. * Tel.: +1-321-6747253; fax: 1-321-6747238. E-mail address: [email protected] (A.M. Dhople).
Despite the promising results in vitro [6] and in laboratory animals [7], the treatment of these ulcers has been disappointing. Surgery is widely regarded as the definitive treatment by the removal of necrotic tissue. Early pre-ulcerative lesions can be effectively treated with rifampicin alone or by heating at 40°C [8]. However, rifampicin is not usually effective against advanced ulcerative lesions. Also, antimycobacterial drugs are needed for postsurgical treatment to prevent relapses. A novel benzoxazinorifamycin, KRM-1648, has been shown to be more potent than rifampicin in inhibiting the growth of M. leprae in vitro and in mouse footpads [9,10]. It has also been demonstrated to be effective against M. tuberculosis [11,12] and M. a6ium complex [11]. Similarly, the antimycobacterial activities of ofloxacin against M. leprae [13,14], M. tuberculosis [15,16] and M. a6ium [16] are well established. Furthermore, Dhople and Ibanez have shown synergistic effects of KRM-1648, in combination with ofloxacin, against M. leprae [9,10]. We therefore devised a study to evaluate the effects of KRM-1648, either alone or in
0924-8579/01/$ - $20 © 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S0924-8579(00)00306-X
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A.M. Dhople / International Journal of Antimicrobial Agents 17 (2001) 57–61
combination with ofloxacin, on the in vitro growth of M. ulcerans.
2. Materials and methods
2.1. Antimicrobial agents Ofloxacin, obtained from R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ, USA, was first dissolved in small amount of 0.1 N NaOH and then diluted with distilled water. KRM-1648, [3%-hydroxy-5%(4-isobutyl-1-piperazinyl) benzoxazinorifamycin] was provided by Kaneka Corporation, Osaka, Japan; rifabutin, [1%,4-didehydro-1-deoxy-1,4-dihydro-5%-(2methylpropyl)-rifamycin was a gift of Pharmacia-Adria Laboratories, Dublin, OH, USA; rifampicin [3-(4methyl-1-piperazinyl-iminomethyl)-rifamycin was obtained commercially from Sigma Chemical Co., St. Louis, MO, USA. All rifampicin analogues were first dissolved in small volumes of methanol and then diluted with distilled water. All the diluted drug solutions were finally filter-sterilized with a 0.2 m Millipore membrane filter.
2.2. Organism Two type strains of M. ulcerans were used: ATCC 19423 and ATCC 35840. Six other M. ulcerans strains isolated from patients from six countries were also included in this study. These clinical isolates were obtained from Professor Portaels, Institute of Tropical Medicine, Antwerp, Belgium and were maintained on Lo¨wenstein-Jensen medium. Whenever needed, colonies from Lo¨wenstein-Jensen medium were subcultured using Middlebrook 7H9 broth containing OADC enrichment.
nostic Instrument System, Towson, MD, USA), 0.1 ml of this 7H12 broth culture was used undiluted to inoculate the above 7H12 broth vials containing drugs and palmitic acid, thus yielding 104 –105 CFU/ml [18]. Also, two drug-free vials per set of drug-containing vials were used as controls; one vial was inoculated in the same way as test vials and the other was inoculated with a 1:100 dilution of inoculum (1:100 control) to produce an initial concentration representing 1% of the bacterial population (102 –103 CFU/ml). The vials were incubated at 33°C in BACTEC 460B and the GI readings recorded every other day. The concentration of drug producing periodic GI increase and final GI reading lower than those in 1:100 control was considered to have inhibited more than 99% of bacterial population [19] and was therefore defined as MIC.
2.4. MBC determination For the determination of minimal bactericidal concentration (MBC) the method of Heifets was followed [20]. The 7H12 vials were inoculated with the organisms in the same was as described above for MIC determination, except that 1:100 control was eliminated. Beginning with an inoculum of 104 –105 CFU/ml, the cultures were allowed to incubate until growth was in exponential phase and the number of CFU/ml was 105 – 106, i.e. when GI readings were approximately 400. At this time, the drugs were added to achieve 1, 2, 4, 8 and 16 times the previously determined MIC. Samples were taken 14 days later, diluted appropriately depending on the GI readings and drug concentration (to prevent drug carry-over) and 0.5 ml of the dilutions were inoculated on 7H11 agar plates. Incubation was at 33°C for 28 days, after which colonies were counted and CFU/ml calculated. The MBC was the lowest concentration of drug that killed more than 99% of the bacterial population present when drugs were added.
2.3. MIC determination 2.5. Synergy studies Minimal inhibitory concentration (MIC) was defined as the lowest concentration of a drug that inhibited more than 99% of the bacterial population within 21 days of cultivation in 7H12 broth inoculated with 104 – 105 CFU/ml [17]. In the radiometric method adopted here, appropriate dilutions of the drugs were added individually in a volume of 0.1 ml to 7H12 broth vials containing [14C] palmitic acid (BBL, Sparks, MD, USA) to achieve the desired final concentrations. In preparing M. ulcerans suspension from each of the eight strains, a 7H12 vial was inoculated with 0.1 ml of a 1:2 dilution of a fresh 7H9 culture which equaled the optical density of a no. 1 McFarland standard. When growth in this vial reached a Growth Index (GI) reading of 400 – 500 in BACTEC 460B Instrument (Becton-Dickinson Diag-
Interactions between KRM-1648 (or rifampicin or rifabutin) and ofloxacin against M. ulcerans were investigated by combining each drug at concentrations which were lower than their respective MICs. The bactericidal effect of each combination was estimated by calculating the fractional inhibitory concentration (FIC), also known as the interaction index, following determination of the MIC of each agent alone and in combination, as described above. The FIC was calculated according to the following formula [21]: FIC=
MIC of A in presence of B MIC of A alone +
MIC of B in presence of A , MIC of B alone
A.M. Dhople / International Journal of Antimicrobial Agents 17 (2001) 57–61
where A and B are rifamycin analogue and ofloxacin respectively. Synergy was defined as a FIC5 0.5, an additive effect as a FIC of \0.5 and B 2.0 and antagonism as a FIC of ]2.0. For each sample, control as well as with drug, triplicate assays were performed in each case, and statistical significance was determined by Student’s t-test.
3. Results The effects of KRM-1648, and also of rifampicin, rifabutin and ofloxacin, incorporated singly into 7H12 medium, against M. ulcerans are presented in Table 1. In case of KRM-1648, the MIC against two type strains was 0.025 mg/l while the MIC for six clinical isolates varied between 0.012 and 0.025 mg/l. Furthermore, the MBC values for the type strains as well as for the isolates were the same as the MICs and thus, the MBC/MIC ratios for all the organisms were 1.0. The MICs and MBCs of both rifampicin and rifabutin were higher than those of KRM-1648. The MICs and MBCs of rifampicin were in the range of 0.1 – 0.4 and 0.2–0.8 mg/l respectively, thus giving the MBC/MIC ratio values of 1.0 for one of eight strains and 2.0 for seven of eight strains. In the case of rifabutin, the MBC/MIC ratio values were 1.0 (three of eight strains) and 2.0 (five of eight strains). The MIC values of ofloxacin ranged between 0.125 and 2.0 mg/l, with the MBC/MIC ratio values ranging from 1.0 to 4.0. The results obtained when each of the three rifamycin analogues was combined with ofloxacin, with each drug at concentrations lower than their respective MICs, are presented in Table 2. The combination of KRM-1648 and ofloxacin exhibited strong synergism; in seven of eight strains, the FIC was 0.5, while in one strain it was 0.75. On the other hand, no synergism was observed with any of the eight strains of M. ulcerans when either rifampicin or rifabutin was combined with ofloxacin: the FIC values ranged between 1.0 and 2.0.
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4. Discussion The data presented here clearly demonstrate the superiority of KRM-1648 over both rifampicin and rifabutin in inhibiting the in vitro growth of M. ulcerans. Such superiority of KRM-1648 over other rifampicin analogues was also demonstrated in the case of in vitro growth of M. leprae [9]. Similarly, the synergism exhibited with the combination of KRM-1648 and ofloxacin, but not with a combination of rifampicin and ofloxacin, is noteworthy. Pre-ulcerative and early ulcerative, but not advanced ulcers, can be effectively treated with rifampicin [8]. Several antituberculosis agents have been shown to be active against M. ulcerans in vitro but clinicians favour surgery over chemotherapy in initial management. The patients are treated with drugs when organisms are seen at the edges of resected lesions, or when disease has recurred after surgery [22]. One possible reason for antimycobacterial agents not being effective for the treatment of advanced ulcers might be because of low drug levels obtainable in tissues of patients with advanced ulcers. It has been demonstrated that in humans, the peak serum level of ofloxacin, after 400 mg/day oral dose, is 5.6 mg/l [23]. Even though it has not been evaluated in humans, peak plasma concentration of KRM-1649 in mice is 0.60 mg/l [24]. The results of present study demonstrate that this level of KRM1648 is inhibitory for the growth of M. ulcerans in vitro. Furthermore, the concentration of ofloxacin, when combined with KRM-1648 to achieve synergistic effects can also be obtained in sera of humans. Most of the M. ulcerans patients carry massive bacterial loads at some stage of the disease and this creates an ideal situation for the selection of drug resistant mutants. In such a situation, combined therapy, as in any other mycobacterial infection, will be highly advantageous. Thus, the combination of KRM-1648 and ofloxacin will have a great potential in clinical treatment of advanced ulcers. Recently, Portaels and
Table 1 In vitro activities of rifampicin analogues and ofloxacin used singly against Mycobacterium ulcerans a M. ulcerans
ATCC 19423 ATCC 35840 Clinical isolate 94-886 94-539 5143 842 8756 5155 a
MIC (mg/l)
MBC (mg/l)
MBC/MIC
1648
Rif
Rtb
Oflx.
1648
Rif
Rtb
Oflx.
0.025 0.025 0.012 0.012 0.025 0.012 0.025 0.025
0.4 0.4 0.4 0.1 0.2 0.2 0.4 0.2
0.2 0.2 0.4 0.1 0.1 0.2 0.2 0.1
0.5 0.5 1.0 2.0 0.5 0.125 2.0 1.0
0.025 0.025 0.012 0.012 0.025 0.012 0.025 0.025
0.8 0.8 0.4 0.2 0.4 0.4 0.8 0.4
0.4 0.4 0.4 0.2 0.2 0.4 0.2 0.1
1.0 1.0 2.0 4.0 1.0 0.5 2.0 2.0
1648 =KRM-1648; Rif =Rifampicin; Rtb = Rifabutin; Oflx. = Ofloxacin.
1648 1 1 1 1 1 1 1 1
Rif
Rtb
2 2 1 2 2 2 2 2
2 2 1 2 2 2 1 1
Oflx. 2 2 2 2 2 4 1 2
A.M. Dhople / International Journal of Antimicrobial Agents 17 (2001) 57–61
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Table 2 In vitro activities of rifampicin analogues in combination with ofloxacin against Mycobacterium ulcerans a MIC in combination M. ulcerans ATCC 19423 35840 Clinical isolate 94-886 94-539 5143 842 8756 5155 a
1648 0.006 0.006 0.003 0.003 0.006 0.003 0.006 0.006
MIC in combination
MIC in combination
Oflx.
FIC
Rif.
Oflx.
FIC
Rtb.
Oflx
FIC
0.125 0.125 0.25 0.5 0.125 0.06 0.5 0.25
0.5 0.5 0.5 0.5 0.5 0.75 0.5 0.5
0.2 0.2 0.2 0.1 0.1 0.2 0.2 0.1
0.25 0.25 0.5 1.0 0.25 0.125 1.0 0.5
1.0 1.0 1.0 1.5 1.0 2.0 1.0 1.0
0.1 0.1 0.2 0.05 0.1 0.1 0.1 0.05
0.25 0.25 0.5 1.0 0.25 0.125 1.0 0.5
1.0 1.0 1.0 1.0 1.5 1.5 1.0 1.0
1648 =KRM-1648; Rif =Rifampin; Rtb = Rifabutin; Oflx. = Ofloxacin; FIC = Fractional Inhibitory Concentration.
coworkers [25] showed the effect of clarithromycin on in vitro growth of large number of clinical isolates of M. ulcerans. It will be interesting to evaluate the effects of a combination of KRM-1648 and clarithromycin against M. ulcerans.
5. Conclusions We have found a new benzoxazinorifamycin, KRM1648, to be superior to rifampicin in inhibiting the growth of M. ulcerans in vitro. It is a bactericidal compound with an MBC/MIC ratio of 1.0. Furthermore, in combination with ofloxacin, it has demonstrated synergism. These results justify further studies with KRM-1648 in the mouse model and also in clinical trials.
Acknowledgements The author is grateful to Dr F. Portaels of Institute of Tropical Medicine, Antwerp, Belgium, for supplying the clinical isolates of M. ulcerans. Thanks are also due to Kaneka Corporation, Pharmacia-Adria Laboratories and R. W. Johnson Pharmaceutical Research Institute for the generous supply of KRM-1648, rifabutin and ofloxacin respectively.
References [1] MacCallum P, Tolhurst JC, Buckle G, Sissons HA. A new mycobacterial infection in Man. J Pathol Bacteriol 1948;60:93 – 122. [2] Vieth MGK, Johnson PDR, Flood PE, et al. A large localized outbreak of Mycobacterium ulcerans infection on a temperate southern Australian island. Epidemiol Infect 1997;119:313 – 8. [3] Meyers WM, Tignokpa N, Priuli GB, Portaels F. Mycobacterium ulcerans infection (Buruli ulcer); first reported patients in Togo. Br J Dermatol 1996;134:1116–21.
[4] Josse R, Guedenon A, Darie H, et al. Mycobacterium ulcerans cutaneous infections: Buruli ulcers. Med Trop (Mars) 1995;55:363 – 73. [5] World Health Organization website: http.www.who.int/gtbburuli/index.html [6] Schroeder KH. Investigation in the relationship of Mycobacterium ulcerans to Mycobacterium buruli. Amer Rev Resp Dis 1975;111:559 – 62. [7] Srivastava A, Singh NB, Gupta SK. Prospective treatment for Mycobacterium ulcerans infection in rat and mice through combined therapy with rifampin and septran. Curr Sci 1984;53:487– 8. [8] Meyers WM, Shelly WM, Connor DH. Heat treatment of Mycobacterium ulcerans infections without surgical incision. Amer J Trop Med Hyg 1974;23:924 – 9. [9] Dhople AM, Ibanez MA. The in vitro activities of novel benzoxazinorifamycins against Mycobacterium leprae. J Antimicro Chemother 1995;35:463 – 71. [10] Dhople AM, Ibanez MA. In vivo activities of novel benzoxazinorifamycins against Mycobacterium leprae. Indian J Lepr 1995;67:375 – 82. [11] Saito H, Tomioka H, Sato K, et al. In vitro antimycobacterial activities of newly synthesized benzoxazinorifamycins. Antimicro Agents Chemother 1991;35:542 – 7. [12] Klemens SP, Grossi MA, Cynamon MH. Activity of KRM1648, a new benzoxazinorifamycin, against Mycobacterium tuberculosis in a mouse model. Antimicro Agents Chemother 1994;38:2245 – 8. [13] 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. [14] In vivo susceptibility of Mycobacterium leprae to ofloxacin either singly or in combination with rifampicin and rifabutin ArzneimForsch/Drug Res 1994;44:563 – 565. [15] Mor N, Vanderkolk J, Heifets L. Inhibitory and bactericidal activities of levofloxacin against Mycobacterium tuberculosis in vitro and in human macrophages. Antimicro Agents Chemother 1994;38:1161 – 4. [16] Vacher S, Pellegrin JL, Leblanc F, et al. Comparative antimycobacterial activities of ofloxacin, ciprofloxacin and grepafloxacin. J Antimicro Chemother 1999;44:647 – 52. [17] Heifets LB, Lindholm-Levy PJ, Flory M. Comparison of bacteriostatic and bactericidal activity of isoniazid and ethionamide against Mycobacterium a6ium and Mycobacterium tuberculosis. Amer Rev Resp Dis 1991;143:268 – 70. [18] Heifets LB, Iseman MD, Cook JL, et al. Determination of in vitro susceptibility of Mycobacterium tuberculosis to cephalosporins by radiometric and conventional methods. An-
A.M. Dhople / International Journal of Antimicrobial Agents 17 (2001) 57–61 timicro Agents Chemother 1985;27:11–5. [19] Tarrand JJ, Groschel DH. Evaluation of the BACTEC radiometric method for detection of 1% resistant populations of M. tuberculosis. J Clin Micro 1985;21:941–6. [20] Heifets LB, Lindholm-Levy PJ. Bacteriostatic and bacteriocidal activity of ciprofloxacin and ofloxacin against Mycobacterium a6ium complex. Tubercle 1987;68:267–76. [21] Hallander HO, Dornbusch K, Gezilius L, et al. Synergism between aminoglycosides and cephalosporins with antipseudomonal activity; interaction index and killing curve method. Antomicto Agents Chemother 1982;22:743–52.
.
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[22] Johnson PDR, Stinear TP, Hayman JA. Mycobacterium ulcerans — a mini-review. J Med Micro 1999;48:511 – 3. [23] Saito H, Tomioka H, Sato K. In vitro antimycobacterial activity of the new quinolone OPC-17116. Chemother 1994;42:1–5. [24] Tomioka H, Saito H, Sato K, et al. Chemotherapeutic efficacy of a newly synthesized benzoxazinirifamycin, KRM-1648, against Mycobacterium a6ium complex infection induced in mice. Antimicro Agents Chemother 1992;36:387 – 93. [25] Portaels F, Traore H, De Ridder K, Meyers WM. In vitro susceptibility of Mycobacterium ulcerans to clarithromycin. Antimicro Agents Chemother 1998;42:2070 – 3.
Original article
In vitro activity of KRM-1648, either singly or in combination with ofloxacin, against Mycobacterium ulcerans Arvind M. Dhople * Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL 32901, USA Received 27 April 2000; accepted 12 May 2000
Abstract The antimicrobial effect of a benzoxazinorifamycin, KRM-1648, either alone or in combination with ofloxacin, was evaluated in vitro against two type strains and six clinical isolates of Mycobacterium ulcerans. Growth of M. ulcerans was measured by plate counts and the BACTEC radiometric method. The minimal inhibitory concentration as well as minimal bactericidal concentration of KRM-1648 against M. ulcerans was between 0.012 and 0.025 mg/l, while corresponding values for rifampicin and rifabutin were in the range of 0.1–0.8 mg/l and 0.1–0.4 mg/l respectively. When combined with ofloxacin, KRM-1648 exhibited strong synergistic activity while only additive effects were observed with the combination of rifampicin (or rifabutin) and ofloxacin. These results suggest that KRM-1648 has a great potential in the treatment of M. ulcerans infection. © 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Mycobacterium ulcerans; Buruli ulcer; Benzoxazinorifamycin; KRM-1648, Rifampicin; Ofloxacin; Synergism
1. Introduction Infection with Mycobacterium ulcerans (M. ulcerans) causes a deforming disease, known as Buruli ulcer, occuring in swampy lowlands and river valleys in tropical or subtropical regions. The organism was first isolated by MacCallum and coworkers in 1948 [1] from six patients living in Bairnsdale, South Australia, and so the disease is also known as Bairnsdale ulcer [2]. M. ulcerans is the third major mycobacterial pathogen of man. It grows well on egg yolk-agar medium at 30– 33°C. The lesions often start as small subcutaneous nodules, gradually enlarge over days to weeks and lead to painless and chronic ulcers with characteristically undermined edges. After more than 50 years, the incidence of infection with M. ulcerans seems to be increasing sharply, particularly in West Africa [3,4] and so the World Health Organization has recently declared Buruli ulcer as an emerging public health problem [5]. * Tel.: +1-321-6747253; fax: 1-321-6747238. E-mail address: [email protected] (A.M. Dhople).
Despite the promising results in vitro [6] and in laboratory animals [7], the treatment of these ulcers has been disappointing. Surgery is widely regarded as the definitive treatment by the removal of necrotic tissue. Early pre-ulcerative lesions can be effectively treated with rifampicin alone or by heating at 40°C [8]. However, rifampicin is not usually effective against advanced ulcerative lesions. Also, antimycobacterial drugs are needed for postsurgical treatment to prevent relapses. A novel benzoxazinorifamycin, KRM-1648, has been shown to be more potent than rifampicin in inhibiting the growth of M. leprae in vitro and in mouse footpads [9,10]. It has also been demonstrated to be effective against M. tuberculosis [11,12] and M. a6ium complex [11]. Similarly, the antimycobacterial activities of ofloxacin against M. leprae [13,14], M. tuberculosis [15,16] and M. a6ium [16] are well established. Furthermore, Dhople and Ibanez have shown synergistic effects of KRM-1648, in combination with ofloxacin, against M. leprae [9,10]. We therefore devised a study to evaluate the effects of KRM-1648, either alone or in
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A.M. Dhople / International Journal of Antimicrobial Agents 17 (2001) 57–61
combination with ofloxacin, on the in vitro growth of M. ulcerans.
2. Materials and methods
2.1. Antimicrobial agents Ofloxacin, obtained from R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ, USA, was first dissolved in small amount of 0.1 N NaOH and then diluted with distilled water. KRM-1648, [3%-hydroxy-5%(4-isobutyl-1-piperazinyl) benzoxazinorifamycin] was provided by Kaneka Corporation, Osaka, Japan; rifabutin, [1%,4-didehydro-1-deoxy-1,4-dihydro-5%-(2methylpropyl)-rifamycin was a gift of Pharmacia-Adria Laboratories, Dublin, OH, USA; rifampicin [3-(4methyl-1-piperazinyl-iminomethyl)-rifamycin was obtained commercially from Sigma Chemical Co., St. Louis, MO, USA. All rifampicin analogues were first dissolved in small volumes of methanol and then diluted with distilled water. All the diluted drug solutions were finally filter-sterilized with a 0.2 m Millipore membrane filter.
2.2. Organism Two type strains of M. ulcerans were used: ATCC 19423 and ATCC 35840. Six other M. ulcerans strains isolated from patients from six countries were also included in this study. These clinical isolates were obtained from Professor Portaels, Institute of Tropical Medicine, Antwerp, Belgium and were maintained on Lo¨wenstein-Jensen medium. Whenever needed, colonies from Lo¨wenstein-Jensen medium were subcultured using Middlebrook 7H9 broth containing OADC enrichment.
nostic Instrument System, Towson, MD, USA), 0.1 ml of this 7H12 broth culture was used undiluted to inoculate the above 7H12 broth vials containing drugs and palmitic acid, thus yielding 104 –105 CFU/ml [18]. Also, two drug-free vials per set of drug-containing vials were used as controls; one vial was inoculated in the same way as test vials and the other was inoculated with a 1:100 dilution of inoculum (1:100 control) to produce an initial concentration representing 1% of the bacterial population (102 –103 CFU/ml). The vials were incubated at 33°C in BACTEC 460B and the GI readings recorded every other day. The concentration of drug producing periodic GI increase and final GI reading lower than those in 1:100 control was considered to have inhibited more than 99% of bacterial population [19] and was therefore defined as MIC.
2.4. MBC determination For the determination of minimal bactericidal concentration (MBC) the method of Heifets was followed [20]. The 7H12 vials were inoculated with the organisms in the same was as described above for MIC determination, except that 1:100 control was eliminated. Beginning with an inoculum of 104 –105 CFU/ml, the cultures were allowed to incubate until growth was in exponential phase and the number of CFU/ml was 105 – 106, i.e. when GI readings were approximately 400. At this time, the drugs were added to achieve 1, 2, 4, 8 and 16 times the previously determined MIC. Samples were taken 14 days later, diluted appropriately depending on the GI readings and drug concentration (to prevent drug carry-over) and 0.5 ml of the dilutions were inoculated on 7H11 agar plates. Incubation was at 33°C for 28 days, after which colonies were counted and CFU/ml calculated. The MBC was the lowest concentration of drug that killed more than 99% of the bacterial population present when drugs were added.
2.3. MIC determination 2.5. Synergy studies Minimal inhibitory concentration (MIC) was defined as the lowest concentration of a drug that inhibited more than 99% of the bacterial population within 21 days of cultivation in 7H12 broth inoculated with 104 – 105 CFU/ml [17]. In the radiometric method adopted here, appropriate dilutions of the drugs were added individually in a volume of 0.1 ml to 7H12 broth vials containing [14C] palmitic acid (BBL, Sparks, MD, USA) to achieve the desired final concentrations. In preparing M. ulcerans suspension from each of the eight strains, a 7H12 vial was inoculated with 0.1 ml of a 1:2 dilution of a fresh 7H9 culture which equaled the optical density of a no. 1 McFarland standard. When growth in this vial reached a Growth Index (GI) reading of 400 – 500 in BACTEC 460B Instrument (Becton-Dickinson Diag-
Interactions between KRM-1648 (or rifampicin or rifabutin) and ofloxacin against M. ulcerans were investigated by combining each drug at concentrations which were lower than their respective MICs. The bactericidal effect of each combination was estimated by calculating the fractional inhibitory concentration (FIC), also known as the interaction index, following determination of the MIC of each agent alone and in combination, as described above. The FIC was calculated according to the following formula [21]: FIC=
MIC of A in presence of B MIC of A alone +
MIC of B in presence of A , MIC of B alone
A.M. Dhople / International Journal of Antimicrobial Agents 17 (2001) 57–61
where A and B are rifamycin analogue and ofloxacin respectively. Synergy was defined as a FIC5 0.5, an additive effect as a FIC of \0.5 and B 2.0 and antagonism as a FIC of ]2.0. For each sample, control as well as with drug, triplicate assays were performed in each case, and statistical significance was determined by Student’s t-test.
3. Results The effects of KRM-1648, and also of rifampicin, rifabutin and ofloxacin, incorporated singly into 7H12 medium, against M. ulcerans are presented in Table 1. In case of KRM-1648, the MIC against two type strains was 0.025 mg/l while the MIC for six clinical isolates varied between 0.012 and 0.025 mg/l. Furthermore, the MBC values for the type strains as well as for the isolates were the same as the MICs and thus, the MBC/MIC ratios for all the organisms were 1.0. The MICs and MBCs of both rifampicin and rifabutin were higher than those of KRM-1648. The MICs and MBCs of rifampicin were in the range of 0.1 – 0.4 and 0.2–0.8 mg/l respectively, thus giving the MBC/MIC ratio values of 1.0 for one of eight strains and 2.0 for seven of eight strains. In the case of rifabutin, the MBC/MIC ratio values were 1.0 (three of eight strains) and 2.0 (five of eight strains). The MIC values of ofloxacin ranged between 0.125 and 2.0 mg/l, with the MBC/MIC ratio values ranging from 1.0 to 4.0. The results obtained when each of the three rifamycin analogues was combined with ofloxacin, with each drug at concentrations lower than their respective MICs, are presented in Table 2. The combination of KRM-1648 and ofloxacin exhibited strong synergism; in seven of eight strains, the FIC was 0.5, while in one strain it was 0.75. On the other hand, no synergism was observed with any of the eight strains of M. ulcerans when either rifampicin or rifabutin was combined with ofloxacin: the FIC values ranged between 1.0 and 2.0.
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4. Discussion The data presented here clearly demonstrate the superiority of KRM-1648 over both rifampicin and rifabutin in inhibiting the in vitro growth of M. ulcerans. Such superiority of KRM-1648 over other rifampicin analogues was also demonstrated in the case of in vitro growth of M. leprae [9]. Similarly, the synergism exhibited with the combination of KRM-1648 and ofloxacin, but not with a combination of rifampicin and ofloxacin, is noteworthy. Pre-ulcerative and early ulcerative, but not advanced ulcers, can be effectively treated with rifampicin [8]. Several antituberculosis agents have been shown to be active against M. ulcerans in vitro but clinicians favour surgery over chemotherapy in initial management. The patients are treated with drugs when organisms are seen at the edges of resected lesions, or when disease has recurred after surgery [22]. One possible reason for antimycobacterial agents not being effective for the treatment of advanced ulcers might be because of low drug levels obtainable in tissues of patients with advanced ulcers. It has been demonstrated that in humans, the peak serum level of ofloxacin, after 400 mg/day oral dose, is 5.6 mg/l [23]. Even though it has not been evaluated in humans, peak plasma concentration of KRM-1649 in mice is 0.60 mg/l [24]. The results of present study demonstrate that this level of KRM1648 is inhibitory for the growth of M. ulcerans in vitro. Furthermore, the concentration of ofloxacin, when combined with KRM-1648 to achieve synergistic effects can also be obtained in sera of humans. Most of the M. ulcerans patients carry massive bacterial loads at some stage of the disease and this creates an ideal situation for the selection of drug resistant mutants. In such a situation, combined therapy, as in any other mycobacterial infection, will be highly advantageous. Thus, the combination of KRM-1648 and ofloxacin will have a great potential in clinical treatment of advanced ulcers. Recently, Portaels and
Table 1 In vitro activities of rifampicin analogues and ofloxacin used singly against Mycobacterium ulcerans a M. ulcerans
ATCC 19423 ATCC 35840 Clinical isolate 94-886 94-539 5143 842 8756 5155 a
MIC (mg/l)
MBC (mg/l)
MBC/MIC
1648
Rif
Rtb
Oflx.
1648
Rif
Rtb
Oflx.
0.025 0.025 0.012 0.012 0.025 0.012 0.025 0.025
0.4 0.4 0.4 0.1 0.2 0.2 0.4 0.2
0.2 0.2 0.4 0.1 0.1 0.2 0.2 0.1
0.5 0.5 1.0 2.0 0.5 0.125 2.0 1.0
0.025 0.025 0.012 0.012 0.025 0.012 0.025 0.025
0.8 0.8 0.4 0.2 0.4 0.4 0.8 0.4
0.4 0.4 0.4 0.2 0.2 0.4 0.2 0.1
1.0 1.0 2.0 4.0 1.0 0.5 2.0 2.0
1648 =KRM-1648; Rif =Rifampicin; Rtb = Rifabutin; Oflx. = Ofloxacin.
1648 1 1 1 1 1 1 1 1
Rif
Rtb
2 2 1 2 2 2 2 2
2 2 1 2 2 2 1 1
Oflx. 2 2 2 2 2 4 1 2
A.M. Dhople / International Journal of Antimicrobial Agents 17 (2001) 57–61
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Table 2 In vitro activities of rifampicin analogues in combination with ofloxacin against Mycobacterium ulcerans a MIC in combination M. ulcerans ATCC 19423 35840 Clinical isolate 94-886 94-539 5143 842 8756 5155 a
1648 0.006 0.006 0.003 0.003 0.006 0.003 0.006 0.006
MIC in combination
MIC in combination
Oflx.
FIC
Rif.
Oflx.
FIC
Rtb.
Oflx
FIC
0.125 0.125 0.25 0.5 0.125 0.06 0.5 0.25
0.5 0.5 0.5 0.5 0.5 0.75 0.5 0.5
0.2 0.2 0.2 0.1 0.1 0.2 0.2 0.1
0.25 0.25 0.5 1.0 0.25 0.125 1.0 0.5
1.0 1.0 1.0 1.5 1.0 2.0 1.0 1.0
0.1 0.1 0.2 0.05 0.1 0.1 0.1 0.05
0.25 0.25 0.5 1.0 0.25 0.125 1.0 0.5
1.0 1.0 1.0 1.0 1.5 1.5 1.0 1.0
1648 =KRM-1648; Rif =Rifampin; Rtb = Rifabutin; Oflx. = Ofloxacin; FIC = Fractional Inhibitory Concentration.
coworkers [25] showed the effect of clarithromycin on in vitro growth of large number of clinical isolates of M. ulcerans. It will be interesting to evaluate the effects of a combination of KRM-1648 and clarithromycin against M. ulcerans.
5. Conclusions We have found a new benzoxazinorifamycin, KRM1648, to be superior to rifampicin in inhibiting the growth of M. ulcerans in vitro. It is a bactericidal compound with an MBC/MIC ratio of 1.0. Furthermore, in combination with ofloxacin, it has demonstrated synergism. These results justify further studies with KRM-1648 in the mouse model and also in clinical trials.
Acknowledgements The author is grateful to Dr F. Portaels of Institute of Tropical Medicine, Antwerp, Belgium, for supplying the clinical isolates of M. ulcerans. Thanks are also due to Kaneka Corporation, Pharmacia-Adria Laboratories and R. W. Johnson Pharmaceutical Research Institute for the generous supply of KRM-1648, rifabutin and ofloxacin respectively.
References [1] MacCallum P, Tolhurst JC, Buckle G, Sissons HA. A new mycobacterial infection in Man. J Pathol Bacteriol 1948;60:93 – 122. [2] Vieth MGK, Johnson PDR, Flood PE, et al. A large localized outbreak of Mycobacterium ulcerans infection on a temperate southern Australian island. Epidemiol Infect 1997;119:313 – 8. [3] Meyers WM, Tignokpa N, Priuli GB, Portaels F. Mycobacterium ulcerans infection (Buruli ulcer); first reported patients in Togo. Br J Dermatol 1996;134:1116–21.
[4] Josse R, Guedenon A, Darie H, et al. Mycobacterium ulcerans cutaneous infections: Buruli ulcers. Med Trop (Mars) 1995;55:363 – 73. [5] World Health Organization website: http.www.who.int/gtbburuli/index.html [6] Schroeder KH. Investigation in the relationship of Mycobacterium ulcerans to Mycobacterium buruli. Amer Rev Resp Dis 1975;111:559 – 62. [7] Srivastava A, Singh NB, Gupta SK. Prospective treatment for Mycobacterium ulcerans infection in rat and mice through combined therapy with rifampin and septran. Curr Sci 1984;53:487– 8. [8] Meyers WM, Shelly WM, Connor DH. Heat treatment of Mycobacterium ulcerans infections without surgical incision. Amer J Trop Med Hyg 1974;23:924 – 9. [9] Dhople AM, Ibanez MA. The in vitro activities of novel benzoxazinorifamycins against Mycobacterium leprae. J Antimicro Chemother 1995;35:463 – 71. [10] Dhople AM, Ibanez MA. In vivo activities of novel benzoxazinorifamycins against Mycobacterium leprae. Indian J Lepr 1995;67:375 – 82. [11] Saito H, Tomioka H, Sato K, et al. In vitro antimycobacterial activities of newly synthesized benzoxazinorifamycins. Antimicro Agents Chemother 1991;35:542 – 7. [12] Klemens SP, Grossi MA, Cynamon MH. Activity of KRM1648, a new benzoxazinorifamycin, against Mycobacterium tuberculosis in a mouse model. Antimicro Agents Chemother 1994;38:2245 – 8. [13] 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. [14] In vivo susceptibility of Mycobacterium leprae to ofloxacin either singly or in combination with rifampicin and rifabutin ArzneimForsch/Drug Res 1994;44:563 – 565. [15] Mor N, Vanderkolk J, Heifets L. Inhibitory and bactericidal activities of levofloxacin against Mycobacterium tuberculosis in vitro and in human macrophages. Antimicro Agents Chemother 1994;38:1161 – 4. [16] Vacher S, Pellegrin JL, Leblanc F, et al. Comparative antimycobacterial activities of ofloxacin, ciprofloxacin and grepafloxacin. J Antimicro Chemother 1999;44:647 – 52. [17] Heifets LB, Lindholm-Levy PJ, Flory M. Comparison of bacteriostatic and bactericidal activity of isoniazid and ethionamide against Mycobacterium a6ium and Mycobacterium tuberculosis. Amer Rev Resp Dis 1991;143:268 – 70. [18] Heifets LB, Iseman MD, Cook JL, et al. Determination of in vitro susceptibility of Mycobacterium tuberculosis to cephalosporins by radiometric and conventional methods. An-
A.M. Dhople / International Journal of Antimicrobial Agents 17 (2001) 57–61 timicro Agents Chemother 1985;27:11–5. [19] Tarrand JJ, Groschel DH. Evaluation of the BACTEC radiometric method for detection of 1% resistant populations of M. tuberculosis. J Clin Micro 1985;21:941–6. [20] Heifets LB, Lindholm-Levy PJ. Bacteriostatic and bacteriocidal activity of ciprofloxacin and ofloxacin against Mycobacterium a6ium complex. Tubercle 1987;68:267–76. [21] Hallander HO, Dornbusch K, Gezilius L, et al. Synergism between aminoglycosides and cephalosporins with antipseudomonal activity; interaction index and killing curve method. Antomicto Agents Chemother 1982;22:743–52.
.
61
[22] Johnson PDR, Stinear TP, Hayman JA. Mycobacterium ulcerans — a mini-review. J Med Micro 1999;48:511 – 3. [23] Saito H, Tomioka H, Sato K. In vitro antimycobacterial activity of the new quinolone OPC-17116. Chemother 1994;42:1–5. [24] Tomioka H, Saito H, Sato K, et al. Chemotherapeutic efficacy of a newly synthesized benzoxazinirifamycin, KRM-1648, against Mycobacterium a6ium complex infection induced in mice. Antimicro Agents Chemother 1992;36:387 – 93. [25] Portaels F, Traore H, De Ridder K, Meyers WM. In vitro susceptibility of Mycobacterium ulcerans to clarithromycin. Antimicro Agents Chemother 1998;42:2070 – 3.