Nacer Lounis, Tom Gevers, Joke Van Den Berg, Luc Vranckx, and Koen Andries * Department of Antimicrobial Research, Tibotec BVBA, Johnson & Johnson,

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1 AAC Accepts, published online ahead of print on 8 September 2009 Antimicrob. Agents Chemother. doi: /aac Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. ATP synthase inhibition of Mycobacterium avium is not bactericidal Nacer Lounis, Tom Gevers, Joke Van Den Berg, Luc Vranckx, and Koen Andries * Department of Antimicrobial Research, Tibotec BVBA, Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Belgium. *Corresponding author: Koen Andries, DVM, PhD Tibotec BVBA, Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Belgium. Phone: Fax: kandries@its.jnj.com Key words: Mycobacterium avium, TMC207, R207910, experimental chemotherapy, diarylquinoline. 1

2 ABSTRACT The efficacy of ATP synthase inhibitor TMC207 was assessed in early and late Mycobacterium avium infections in mice. In contrast to what was earlier observed for M. tuberculosis, a bacteriostatic effect was obtained. In vitro, the MBC/MIC ratio was very high. The MBC is more relevant for assessment of PK/PD relationships than the MIC. Mycobacterium avium is a pathogen that causes disseminated disease in immunocompromised individuals and pulmonary disease in immunocompetent adults (15), is far less susceptible than Mycobacterium tuberculosis to most antimicrobial agents, and treatment options are very limited (7, 10). The most efficacious drugs are clarithromycin (CLA), the azalide antibiotic azithromycin and amikacin (AMK). They are generally part of a multidrug regimen including rifamycins and ethambutol (10) and need to be administered daily for up to 24 months (10). These regimen are expensive and poorly tolerated (2, 6). TMC207 (also known as R207910) is a diarylquinolone ATP synthase inhibitor with potent activity against M. tuberculosis (1, 3, 13). It has broad antimycobacterial activity, with MIC values against several clinical isolates of M. avium ranging from to 0.25 µg/ml (1, 8). Female C57Bl/6J mice aged 6-7 weeks (Janvier Breeding, France) were infected intraperitoneally with 0.5 ml of a bacterial suspension containing 2.3x10 7 CFU of M. avium 101. In a first group of 20 animals (Table 1), treatment started the day after infection (early infection model) and included a 2

3 negative control, a positive control (CLA) and two test groups (TMC207 or CLA+TMC). Mice were sacrificed after one month of treatment. A second group of 165 mice was kept untreated for one month (late infection model), and then treated with CLA alone, AMK alone, TMC207 alone, CLA+AMK, CLA+TMC, AMK+TMC or CLA+AMK+TMC for 4 months (Table 1). Five animals from each group were sacrificed at monthly intervals. All drugs were given 5 times weekly at the following doses: 25 mg/kg TMC207 orally, 200 mg/kg CLA orally, and 150 mg/kg AMIK subcutaneously. Treatment effects were assessed by CFU counts, determined by plating 3 serial 10-fold dilutions of homogenized spleen suspensions onto Löwenstein-Jensen plates. The Student t test with Bonferroni correction of the p value was used to analyse CFU counts. As four and seven groups were compared, p values were adjusted to and , respectively. In the early infection model, untreated control mice had 6.53±0.56 log10 CFU counts at day 0, increasing to 8.0±0.9 log10 CFU one month later (p=0.02). Monotherapy of CLA and TMC decreased CFU counts by 1.99 and 2.56 log10 compared to late controls (p=0.005 and p=0.002, respectively). The combination of TMC and CLA did not improve the activity of the individual compounds (p>0.05) (Table 2). When compared with the early controls, all regimens were bacteriostatic. In the late infection model, untreated control mice reached 8.0±0.9 log10 CFU counts one month after infection and remained stable over the next 4 months (Table 2). The regimens CLA alone, AMK alone, TMC alone, CLA+TMC, CLA+AMK, AMK+TMC and CLA+AMK+TMC did not show any activity after 1 month of treatment. After 2 months of treatment, all regimens 3

4 achieved a bacteriostatic activity. After three months of treatment the triple combination CLA+AMK+TMC achieved bactericidal activity (p=0.001) and was more active than CLA or TMC alone, but not more than AMK alone. After four months of treatment AMK alone, CLA+AMK, AMK+TMC and CLA+AMK+TMC, all achieved bactericidal activity. The activity of the TMC monotherapy was still bacteriostatic. The activity of the triple combination CLA+AMK+TMC was better than CLA alone or TMC alone but not better than AMK alone. The MIC of TMC207 against the M. avium strain used in the present study was µg/ml, consistent with earlier estimates (1, 8). Based on the impressive in vivo results obtained with M. tuberculosis which has an MIC of µg/ml (1), the weak in vivo activity of TMC207 against M. avium was disappointing. We decided to determine the MBC of TMC against M. avium and found it to be much higher than its MIC (128>>0.015 µg/ml). A bacteriostatic activity of TMC207 against M. avium was also confirmed in a killing study in which M. avium was exposed to 100 and 1000 times the MIC, which resulted in a 1 log10 CFU kill after 14 days of exposure. A M. tuberculosis sample exposed to the same concentrations of TMC207 was reduced by 5 log10, clearly illustrating that TMC207 has bactericidal activity against M. tuberculosis and bacteriostatic activity against M. avium. Our data reveal that the mechanism by which mycobacteria are inhibited in their growth (as reflected by the MIC) may be different from the mechanism by which some mycobacterial species are killed. It is currently not clear why some mycobacteria are not killed by TMC207, despite their growth being inhibited at low concentrations, while others do get killed, be it after a delay of a few days. 4

5 Many pharmacokinetic/pharmacodynamic parameters used to estimate efficacy of antimicrobial agents refer to the MIC value e.g. AUIC, AUC/MIC, Time>MIC and Cmax/MIC (14). For drugs with a high MBC/MIC ratio, parameters referring to the MBC (such as the AUBC or area under the bactericidal curve (14)) are probably more relevant. Based on the PK parameters of TMC207 on the one hand, and the MIC s for M. tuberculosis and M. avium on the other hand, one would not have been able to predict the dramatic difference in efficacy in the mouse model, using exactly the same dosing schedule as that used for M. tuberculosis (1). Very few compounds have shown bactericidal efficacy against MAC infections in the beige or the C57Bl/6J mouse models. Many drugs have been tested but with the exception of macrolides (4, 11, 16) and aminoglycosides (5, 9, 11, 12), none were bactericidal. AMK is the only antimicrobial tested which was able to increase the activity of CLA in the mouse model and to prevent the selection of resistant mutants of M. avium to CLA (12). Since AMK cannot be used for longer than 2 or 3 months for safety reasons (ototoxicity and nephrotoxicity), an alternative orally available compound is desirable. Provided that TMC207 has an acceptable safety profile, it could be used as a companion drug of CLA because of its bacteriostatic activity. The ability of TMC207 to prevent the selection of resistant mutants to CLA should be investigated in future studies. 5

6 REFERENCES 1. Andries, K., P. Verhasselt, J. Guillemont, H.W.H. Göhlmann, J.M. Neefs, H. Wrinkler, J.V. Gestel, P. Timmerman, M. Zhu, E. Lee, P. Williams, D. de Chaffoy, H. Huitric, S. Hoffner, E. Cambau, C. Truffot- Pernot, N. Lounis, and V. Jarlier A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. SCIENCE. 307: Benson, C.A., P.L. Williams, J.S. Currier, F. Holland, L.F. Mahon, R.R. MacGregor, C.B. Interlied, C. Flexner, J. Neidig, R. Chaisson, G. F. Notario, R. hafner, and the AIDS Clinical Trials Group 223 Protocol Team A prospective, randomized trial examining the efficacy and safety of clarithromycin in combination with ethambutol, rifabutin, or both for the treatment of disseminated Mycobacterium avium complex disease in persons with acquired immunodeficiency syndrome. Clin. Infect. Dis. 37: Diacon A.H., A. Pym, M. Grobusch, M. Patientia, R. Rustomjee, L. Page-Shipp, C. Pistorius, R. Krause, M. Bogoshi, G. Churyard, A. Venter, J. Allen, J.C. Palomino, T. De Marez, R.P.G. van Heeswijk, N. Lounis, P. Meyvisch, J. Verbeeck, W. Parys, K. De Beule, K. Andries, D.F. McNeeley. The investigational diarylquinoline TMC207 for multidrug-resistant TB. New England Journal of Medicine. N. Engl. J. Med. 360: Fernandes, P.B., D.J. Hardy, D. McDaniel, C.W. Hanson, and R.N. Swanson In vitro and in vivo activities of clarithromycin against Mycobacterium avium. Antimicrob. Agents Chemother. 33: Gangadharam, P.R.J., V.K. Perumal, N.R. Podapati, L. Kesavalu, and M. Iseman In vivo activity of amikacin alone or in combination with clofazimine or rifabutin or both against experimental Mycobacterium avium complex infections in beige mice. Antimicrob. Agents Chemother. 32:

7 6. Griffith, D.E., B.A. Brown-Elliott, S. Shepherd, J. McLarty, L. Griffith, and R.J. Wallace Jr Ethambutol ocular toxicity in treatment regimens for Mycobacterium avium complex lung disease. Am. J. Respir. Crit. Care Med. 172: Huang, J.H., P.N. Kao, V. Adi, and S.J. Ruoss Mycobacterium avium-intracellulare pulmonary infection in HIV-negative patients without preexisting lung disease: diagnostic and management limitations. Chest. 115: Huitric, E., P. Verhasselt, K. Andries, and S.E. Hoffner In vitro anti-mycobacterial spectrum of a diarylquinoline ATP synthase inhibitor. Antimicrob. Agents Chemother. Antimicrob. Agents Chemother. 51: Inderlied, C.B., PT. Kolonski, W. Martin, and L.S. Young Amikacin, ciprofloxacin, and imipenem treatment for disseminated Mycobacterium avium complex infection of beige mice. Antimicrob. Agents Chemother. 33: Iseman, M.D Medical management of pulmonary disease caused by Mycobacterium avium complex. Clin. Chest. Med. 23: Ji, B., N. Lounis, C. Truffot-Pernot, and J. Grosset Effectiveness of various antimicrobial agents against Mycobacterium avium Complex in the beige mouse model. 38: Lounis, N., B. Ji, C. Truffot-Pernot, and J. Grosset Selection of clarithromycin-resistant Mycobacterium avium Complex during combined therapy using the beige mouse model. Antimicrob. Agents Chemother. 39:

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9 Table 1: Experimental design Dates of sacrifices Treatment No groups of (mg/kg) mice D-28 D-27 D0 M1 M2 M3 M4 A. Control 30 inf B. CLA 5 inf. Trt C. TMC 5 Inf. Trt D. CLA+ TMC 5 Inf. Trt E. CLA 20 Inf. - Trt F. TMC 20 Inf. - Trt G. AMK 20 Inf. - Trt H. CLA+AMK 20 Inf. - Trt I. AMK+TMC 20 Inf. - Trt J. CLA+TMC 20 Inf. - Trt K. CLA+AMK+TMC 20 Inf. - Trt Total nb. mice CLA: clarithromycin 200 mg/kg; TMC: TMC mg/kg; AMK: amikacin 150 mg/kg; inf.: infection; Trt.: treatment. D: day, M1: 1 month; M2: 2 months; M3: 3 months; M4: 4 months. Downloaded from on December 17, 2018 by guest 9

10 Table 2: Mean CFU counts in the spleens in the M. avium infection models Groups Early infection modela Late infection modelb CFU counts (log10) CFU counts (log10) in the spleens in the spleens D-27 D 0 D 0 M 1 M 2 M 3 M 4 Controls 6.5± ± ± ± ± ± ±0.3 CLA ± ± ± ± ±0.5 TMC ± ± ± ± ±0.2 CLA +TMC ± ± ± ± ±1.7 AMK ± ± ± ±0.3 CLA+AMK ± ± ± ±1.3 AMK+TMC ± ± ± ±0.7 CLA+AMK+TMC ± ± ± ±0.5 a: mice were infected intraperitoneally and treated the next day for 28 days. b: mice were infected intraperitoneally and treated the a month later for 4 months. CLA: clarithromycin 200 mg/kg; TMC: TMC mg/kg; AMK: amikacin 150 mg/kg; inf.: infection; Trt.: treatment. D: day, M1: 1 month; M2: 2 months; M3: 3 months; M4: 4 months. Downloaded from on December 17, 2018 by guest 10