- Email: [email protected]
Comparison of antimycobacterial activity of grepafloxacin against Mycobacterium avium with that of levofloxacin: accumulation of grepafloxacin in human macrophages
J Infect Chemother (2001) 7:16–21
© Japanese Society of Chemotherapy and the Japanese Association for Infectious Diseases 2001
ORIGINAL ARTICLE Masao Hirota · Tokie Totsu · Fusayo Adachi Kazuo Kamikawa · Junichi Watanabe · Shiro Kanegasaki Koh Nakata
Comparison of antimycobacterial activity of grepafloxacin against Mycobacterium avium with that of levofloxacin: accumulation of grepafloxacin in human macrophages
Received: July 4, 2000 / Accepted: October 10, 2000
Abstract The bactericidal activity of two new quinolones, grepafloxacin and levofloxacin, against five strains of Mycobacterium avium was investigated in vitro. The minimum inhibitory concentrations (MICs) of these two quinolones, determined by the broth microdilution method, were comparable for all strains tested. In contrast, grepafloxacin suppressed the intracellular growth of all the strains in monocyte-derived macrophages more strongly than levofloxacin, when the cells infected with these strains were incubated for 7 days in the presence of various concentrations of the two new quinolones. To find the reason for the strengthened intracellular killing activity of grepafloxacin, we determined the ratio of the concentration of the new quinolones in the cells to that in the medium (C/M concentration ratio). The C/M concentration ratio of grepafloxacin was increased to 34.7 by 7 days, whereas that of levofloxacin at 7 days was only 12.3. These data suggested that a higher level of intraphagocytic accumulation of grepafloxacin endows it with greater mycobactericidal activity.
Introduction
T. Totsu · F. Adachi · J. Watanabe · S. Kanegasaki The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
Mycobacterium avium infection has increased in the past decade because of the epidemic of AIDS. In AIDS patients, the phagocytosed organism can survive in macrophages, causing disseminated infection in the lungs, liver, spleen, bone marrow, lymph nodes, and blood.1,2 The prognosis for M. avium-infected AIDS patients is very poor, owing, in part, to the resistance of this organism to available antimicrobial agents, including antituberculosis drugs.3 Furthermore, severe toxicity is associated with many of these agents when they are used at clinically useful levels.4–5 In addition, the intraphagocytic localization of M. avium protects the organism from the action of certain antimicrobial agents.6 Recent studies suggest that the protection of intracellular organisms from antimicrobial activity probably reflects the failure of these antimicrobial agents to enter macrophages.7–9 Therapy with antimicrobial agents that can penetrate phagocytes and exhibit intracellular antimicrobial activity is oviously needed in this setting. New quinolones, which have been extensively developed in recent years, have potent, broadspectrum antibacterial activity with high efficacy. Because the new quinolones show high tissue and intracellular distribution,10,11 they are expected to be useful for the treatment of infections caused by M. avium. In this study, we compared the efficiency of intracellular killing by two new quinolones, grepafloxacin (GPFX) and levofloxacin (LVFX), in monocyte-derived macrophages. Our data showed that GPFX accumulated in macrophages at a concentration three times higher than that of LVFX, and GPFX suppressed the intraphagocytic growth of M. avium strains more efficiently than LVFX.
K. Kamikawa Department of Respiratory Group Therapeutic Application Development, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan
Materials and methods
Key words Macrophages · Grepafloxacin · Antimycobacterial activity · Mycobacterium avium
M. Hirota Department of Drug Metabolism, Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan
K. Nakata ( ) Department of Respiratory Diseases, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo, Tokyo 162-8655, Japan Tel. 181-3-3202-7181 (ext.2873); Fax 181-3-3207-1038 e-mail: [email protected]
Antimicrobial agents and reagents GPFX (Table 1) was produced by Otsuka Pharmaceutical (Tokyo, Japan). LVFX (Table 1) was purified from the
17 Table 1. Physicochemical characteristics of grepafloxacin and levofloxacin Compound
Solubility in water (mg/ml)
Dissociation constants pKa1; pKa2
Grepafloxacin
36.0
7.1; 8.8a
Levofloxacin
25.8a
5.5; 8.0a
a
Values are cited from Taira et al. (1993)18
commercially available tablets. The purity of prepared LVFX was 99.98%. These antimicrobial agents were dissolved in dimethyl sulfoxide (DMSO) and diluted with RPMI (Nissui Tokyo, Japan) /10% fetal calf serum (FCS) medium before use. We purchased 7H9 broth, with albumin, dextrose, and catalase (ADC) enrichment; and 7H10 agar, with oleic acid, albumin, dextrose, and catalase (OADC) enrichment, from Difco Laboratories (Detroit, MI, USA). Recombinant human (rh) granulocytemacrophage colony stimulating factor (GM-CSF; specific activity, 2.25 3 108 U/mg) was purchased from R&D Systems (Minneapolis, MN, USA). M. avium strains and culture The 14-2, 25-3, and 36-1 strains of M. avium were serially isolated from the blood of the same patient at different times. Strain 14-2 was sensitive to clarithromycin, while strains 25-3 and 36-1 were moderately and strongly resistant, respectively, to this agent. Strain MS-6 was isolated from the blood of another patient, and was strongly resistant to clarithromycin. Cells in blood samples were lysed, using sterile distilled water, and pellets were obtained by centrifugation at 1000 g for 10 min. The pellets were cultured in 7H9 broth with ADC enrichment at 37°C for 3–6 weeks. Each bacterial suspension was then diluted and plated onto 7H10 agar medium. The plates were incubated for 8 weeks at 37°C under 5% CO2. Bacterial cultures from a single colony were grown in 7H9 broth to their exponential phase (to an optical density [OD]650 of 0.15, corresponding to approximately 108 CFU/ml). The isolated strains were identified as M. avium, using DNA-DNA hybridization (DDH) of mycobacterium (Kyokuto, Tokyo, Japan) and by detecting the insertion sequence, IS1245, using the polymerase chain reaction.12 M. avium type strain, ATCC 25291, was obtained from the American Type Culture Collection (Manassas, VA, USA).
Determination of minimum inhibitory concentration (MICs) An aliquot of 20 µl of M. avium suspension (OD650 5 0.15) was added to each well of a 96-well microtiter plate containing 100 µl each of broth and solution of the new quinolone. A stock solution (16 µg/µl) of GPFX or LVFX was diluted serially with 7H9 broth. The plates were incubated at 37°C under 5% CO2 for 10 to 15 days, and the OD650 of each well was measured, using a microplate spectrophotometer (model 3550; Bio-Rad, Hercules, CA, USA). Each MIC was determined as the lowest concentration that completely inhibited the growth of each M. avium strain. Preparation of human monocytes Peripheral blood mononuclear cells (PBMC), obtained from healthy donors by Ficoll/Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation, were resuspended in RPMI 1640 medium (Nissui) supplemented with 10% heat-inactivated FCS. The PBMC were incubated for 15 min at 37°C in 250 ml tissue culture flasks (Falcon, Franklin Lakes, NJ, USA) coated with 10% human AB serum in phosphate-buffered saline (PBS). Non-adherent cells were removed by vigorous washing with PBS, and adherent cells were collected by gentle scraping, using a cell scraper (Sumitomo Bakelite, Tokyo, Japan), after incubation with 0.05% trypsin/0.53 mM ethylenediamine tetraacetic acid (EDTA) for 10 min at 37°C. At least 98% of the adherent cells were identified as monocytes by morphology and nonspecific esterase staining. The viability of cells was determined by trypan blue dye exclusion. Infection of monocytes/macrophages by M. avium strains The purified monocytes were suspended in RPMI 1640/ 10% FCS medium, supplemented with 10 ng/ml of recombi-
18
nant human granulocyte macrophage-colony stimulating factor (GM-CSF), and incubated at a density of 2 3 104 per well in 96-well tissue culture plates (Falcon) at 37°C under 5% CO2 in a humidified cell culture incubator. After 7 days, the monocyte-derived macrophages were exposed for 2 h to a single-cell suspension of each M. avium strain at a multiplicity of infection of 10 : 1. Extracellular bacteria were removed by washing three times with PBS. The macrophages infected with M. avium were further incubated for 7 days in the presence of various concentrations of the new quinolones (0–8 µg/ml). The final viability of the macrophages was ascertained by trypan blue dye exclusion to be more than 90%.
Evaluation of CFU The number of M. avium CFU in the macrophages was determined after lysis with 0.25% sodium dodecylsulfate, which was subsequently neutralized with 10% FCS in PBS. The lysate was diluted serially (104 to 106-fold) and plated in duplicate on 7H10 agar plates. The colonies were counted after 2 to 3 weeks of incubation at 37°C under 5% CO2. Triplicate wells were used for each experimental condition tested. Data values were expressed as means 6 SD and compared between groups by two-way analysis of variance with the aid of the SAS system, release 6.12 (SAS Institute, Nancy, NC, USA) for Macintosh computer. P values below 0.05 were defined as significant.
Measurement of the cellular/medium concentration ratio (C/M ratio) of the antimicrobial agents Two million monocytes were incubated with 10 ng/ml of recombinant GM-CSF in a 25-cm2 tissue culture flask (Falcon) for 7 days. The monocyte-derived macrophages were washed twice with PBS and further incubated with 0.8 µg/ml of GPFX or LVFX. After 1-day incubation and after 7-day incubation, the whole medium was recovered, centrifuged at 500 g for 10 min to remove non-adherent macrophages (less than 5%), immediately frozen, and stored at 280°C. The adherent macrophages were washed five times with 5 ml of silicone oil (DC550; specific gravity, 1.07; William F. Nye, New Bedford, MA, USA). The remaining medium around the macrophages was completely removed by this procedure. The flask with macrophages was immediately placed on dry ice for 10 min and then kept at 280°C. The average number of adherent macrophages recovered from the culture flasks was 1.71 3 106 (range, 1.63–1.80 3 106). The concentrations of GPFX or LVFX in the macrophages and the medium were determined using high-performance liquid chromatography (HPLC; HPLC column, TSK gel ODS-80Ts, 4.6 mm 3 150 mm [Tosoh, Tokyo, Japan]) according to the procedure described by Akiyama et al.13 with slight modifications. Briefly, macrophages were removed from the flask using a cell scraper (Falcon) and suspended in 0.1 ml of methanol. After sonication, the suspension was centrifuged at 21 600 g for 5 min,
and mixed with an equal volume of 1-cyclopropyl-6,8difluoro-1,4-dihydro-5-ethyl-7-(4-methyl-1-piperazinyl)4-oxo-3-quinolinecarboxylic acid (OPC-17203, Otsuka Pharmaceutical, Tokyo, Japan) (20 ng/ml) in methanol. For measurement of the concentration of GPFX or LVFX in the medium, the samples were mixed with OPC-17203 (2000 ng/ml) in methanol and extracted with chloroform for 10 min. The organic phase was evaporated to dryness at 40°C under a nitrogen stream. The residue was dissolved in the mobile phase (CH3CN [20 mM] : Na2SO4 : H3PO4 [1400 : 3600 : 4]). The pretreated solutions were injected to the HPLC column at 40°C with a flow rate of 0.8 ml/min. GPFX or LVFX was eluted with the mobile phase, and the detection of GPFX or LVFX on HPLC was performed at an excitation wavelength of 285 nm and fluorescence wavelength of 448 nm. The C/M ratio was determinating by calculating the cell volume of macrophages, estimated by the method of Krombach et al.14 The data values were expressed as means 6 SDs and compared between groups using the t-test, with the aid of the SAS system, release 6.12 (SAS Institute) for Windows. P values below 0.05 were defined as significant.
Results Antimycobacterial effect of new quinolones on extracellular and intracellular growth of M. avium strains The effect of GPFX or LVFX on the growth of M. avium strains in 7H9 broth was studied. As shown in Table 2, the MICs of GPFX and LVFX for the five M. avium strains were comparable, ranging from 0.25 to 1.0 µg/ml. On the other hand, GPFX suppressed the intracellular growth of all strains in the monocyte-derived macrophages more efficiently than LVFX when the cells were infected with these strains and incubated for 7 days in the presence of various concentrations of these antibiotics. As shown in Fig. 1, the number of M. avium CFU decreased with increased concentrations of GPFX. In all strains tested, the percentages of CFU in macrophages treated with GPFX were significantly lower than those in macrophages treated with LVFX, in the concentration range of 0.125–5.0 µg/ml. More than 94.5% of intracellular M. avium were killed by 1.0 µg/ml of GPFX, whereas the same concentration of LVFX killed 47%–74% of intracellular M. avium. Table 2. MIC values of grepafloxacin and levofloxacin for Mycobacterium avium strains (µg/ml) Strains
Grepafloxacin
Levofloxacin
14-2 25-3 36-1 MS-6 ATCC25291
0.25 0.25 0.50 0.50 1.00
0.50 0.50 1.00 0.50 1.00
Values expressed are the means of three independent experiments MIC, Minimum inhibitory concentration
19 Fig. 1A–E. Colony formation of intracellular M. avium strains in macrophages incubated with grepafloxacin or levofloxacin for 7 days. Macrophages were incubated with 0, 0.125, 0.250, 0.500, 1.000, 2.000, 4.000, and 8.000 µg/ml of grepafloxacin or levofloxacin for 7 days. Open circles indicate percent CFU in macrophages treated with levofloxacin; closed circles indicate percent CFU in macrophages treated with grepafloxacin. Values on the horizontal axis show the concentrations of antimicrobial agents added to the culture medium. Values on the vertical axis are expressed as percentages of control CFU (without antimicrobial agents). Error bars indicate SDs of three independent experiments. *P , 0.05 versus levofloxacin; **P , 0.01 versus levofloxacin
Cellular/medium (C/M) concentration ratios of the new quinolones To find the reason for the effectiveness of GPFX in macrophages, we evaluated the concentrations of GPFX and LVFX in macrophages and in the medium. When monocyte-derived macrophages were incubated with 0.8 µg/ ml of GPFX or LVFX for 1 day and 7 days, GPFX was accumulated in macrophages at concentrations of 21.3 µg/ ml (296.5 pmol/106 cells) and 28.1 µg/ml (390.6 pmol/106 cells), respectively, whereas the amounts of LVFX were 6.8 µg/ml (94.1 pmol/106 cells) and 10.2 µg/ml (140.9 pmol/106 cells), respectively. Degradation of antibiotics in the medium was not observed, because the concentrations of both GPFX and LVFX in the medium did not change during the incubation. The C/M ratios of GPFX at 1-day and at 7-day incubation were calculated to be 27.7 and 34.7, respectively, values which were significantly higher than those for LVFX, of 8.9 on day 1 and 12.3 on day 7 (Fig. 2). We repeated measurement of the C/M ratio three times, using blood monocytes from three different donors, and we obtained similar results. Thus, GPFX accumulated markedly in macrophages, in accordance with its relatively potent intracellular antimycobacterial activity against M. avium.
Discussion We investigated the effects of GPFX and LVFX against M. avium in in-vitro model. Incubation of M. avium-infected human macrophages with GPFX or LVFX resulted in a concentration-dependent decrease in viable bacteria, as
Fig. 2. Cellular/medium (C/M) concentration ratios of grepafloxacin (GPFX) and levofloxacin (LVFX). After the incubation of macrophages with 0.8 µg/ml grepafloxacin or levofloxacin for 1 day or 7 days, the concentrations of grepafloxacin and levofloxacin in the cells were measured by HPLC. Open columns indicate the C/M ratios of cells incubated with each compound for 1 day. Closed columns indicate the C/M ratios of cells incubated with each compound for 7 days. Data values are expressed as means 6 SDs of three independent experiments. P , 0.0001 versus levofloxacin
compared with numbers of the bacteria in control cells without antimicrobial agents. GPFX was more effective against intracellular M. avium growth than LVFX over the concentration ranges tested, although the MIC of these new quinolones against M. avium was comparable. GPFX accumulated markedly in macrophages, with a C/M ratio of about 35 at 7 days’ incubation, a value which was three times higher than that of LVFX. Because new quinolones show preferential distribution in various tissues such as lung, skin, and intestine, and high
20
intracellular concentrations,10,11 they have been suggested to be useful for the treatment of infectious diseases caused by intracellular pathogens such as Clamydia sp.,15 Legionella sp.,16 and mycobacteria17 in recent years. This underlines the need for evaluation of the accumulation of the new quinolones in macrophages. The accumulation of various antimicrobial agents in phagocytes has been investigated. Relatively high C/M ratios (between 2 and 8) have been demonstrated for many new quinolones, with ofloxacin having the highest ratio, of 8.18 In this study, we showed that the C/M ratio of LVFX was similar to that of ofloxacin. In comparison with ofloxacin and LVFX, our data showed that GPFX had an extremely high C/M ratio, greater than 20. This C/M ratio was comparable to those of clindamycin7 and the macrolides,19 which are known to be high. The mechanism of the intracellular uptake of antimicrobial agents by phagocytes, reported in previous studies, is broadly classified into two categories: passive and active transport systems. Macrolides are typical antibiotics that are classified in the latter category, and ofloxacin was reported to have the same mechanism.20 On the other hand, ciprofloxacin21 and sparfloxacin22 were reported to be incorporated mostly by a passive mechanism. Recently, Taira et al.18 reported that the uptake of GPFX by polymorphonuclear cells was extremely high, with a C/M ratio of 60, and rapid. The uptake reached a plateau within the first 5 min of incubation. They demonstrated that the accumulation of GPFX in polymorphonuclear cells involved an active transport system. Murata et al.23 showed that GPFX was distributed into rat lung at a high concentration by a similar transport system. Thus, it is suggested that the high concentration of GPFX in macrophages is due to the active transport system. In this study, we showed that GPFX was distributed in macrophages at a higher concentration than LVFX when the macrophages were incubated with the same concentration of these new quinolones. The solubility of GPFX in octanol-Sourensen buffer (pH 7.4), an organic solvent system, is tenfold higher than that of LVFX.23 In general, hydrophobicity is beneficial in antimicrobial agents, enabling them pass through the lipid bilayer of the cell membrane. Therefore, this physicochemical property may allow the high concentration of GPFX in macrophages. GPFX displays potent antimicrobial bioactivity, with a broad spectrum.24,25 Although little is known about its antimycobacterial activity, our present data confirmed the efficacy of this drug in both the intracellular and extracellular killing of M. avium. After the oral administration of 400 mg of GPFX daily, the concentrations in the bronchoepithelial cells and alveolar lining fluid are 3.1-fold and 12.2-fold, respectively, that in the plasma.26 The selective accumulation of GPFX in the lungs, and the potent antimycobacterial activity in macrophages shown in this study suggest that this drug would be effective against M. avium infection in the lung. Acknowledgments The authors thank Dr. Hiroyuki Sasabe, Dr. Hitoshi Akiyama, and Dr. Gohachiro Miyamoto for valuable discussions.
References 1. Benson CA, Ellner JJ. Mycobacterium avium complex infection and AIDS: advances in theory and practice. Clin Infect Dis 1993; 17:7–20. 2. Inderlied CB, Kemper CA, Bermudez LEM. The Mycobacterium avium complex. Clin Microbiol Rev 1993;6:266–310. 3. Rastogi N, Frehel C, Ryter A, Ohayon H, Lesourd M. Multiple drug resistance in Mycobacterium avium: is the wall architecture responsible for the exclusion of antimicrobial agents? Antimicrob Agents Chemother 1981;20:666–77. 4. Higgins K. Potential toxicity of ciprofloxacin. Ophthalmology 1991;98:120–1. 5. Taneja J, Kaur D. Study on hepatotoxicity and other side effects of antituberculosis drugs. J Indian Med Assoc 1990;88:278–80. 6. Frehel C, Ryter A, Rastogi N, David H. The electron-transparent zone in phagocytized Mycobacterium avium and other mycobacteria: formation, persistence and role in bacterial survival. Annu Inst Pasteur Microbiol 1986;137B:239–57. 7. Onyeji CO, Nightingale CH, Nicolau DP, Quintiliani R. Efficacies of liposome encapsulated clarithromycin and ofloxacin against Mycobacterium avium M. intracellular complex in human macrophages. Antimicrob Agents Chemother 1994;38:523–7. 8. Pellegrin I, Maugein J, Lapeyre C, Barbeau P, Leng B, Pellegrin JL. Activity of rifabutin, clarithromycin, ethambutol, sparfloxacin and amikacin, alone and in combination, against Mycobacterium avium complex in human macrophages. J Antimicrob Chemother 1996;37:501–10. 9. Shiratsuchi H, Jacobs MR, Pearson AJ, Venkataprasad N, Klopman G, Ellner JJ. Comparison of the activity of fluoroquinolones against Mycobacterium avium in cell-free systems and a human monocytes in-vitro infection model. J Antimicrob Chemother 1996;37:491–500. 10. Pascual A, Garcia I, Ballesta S, Perea EJ. Uptake and intracellular activity of trovafloxacin in human phagocytes and tissue-cultured epithelial cells. Antimicrob Agents Chemother 1997;41:274– 7. 11. Ito T, Yano I, Masuda S, Hashimoto Y, Inui K. Distribution characteristics of levofloxacin and grepafloxacin in rat kidney. Pharmacol Res 1999;16:534–9. 12. Garzelli C, Lari N, Nguon B, Cavallini M, Pistello M, Falcone G. Comparison of three restriction endonucleases in IS 1245-based RFLP typing of Mycobacterium avium. J Med Microbiol 1997;46: 933–9. 13. Akiyama H, Abe Y, Koike M, Kyushiki K, Fujio N, Odomi M, et al. Pharmacokinetics of grepafloxacin (I) – absorption, distribution and excretion after oral administration of grepafloxacin in animals as determined by HPLC. Jpn J Chemother 1995;43:99–106. 14. Krombach F, Munzing S, Allmeling AM, Gerlach JT, Behr J, Dorger M. Cell size of alveolar macrophages: an interspecies comparison. Environm Health Perspect 1997;105:1261–3. 15. Nagayana A, Nakao T, Tean H. In vitro activities of ofloxacin and four other new quinoline carboxilic acids against Chlamydia trachomatis. Antimicrob Agents Chemother 1988;32:1735–7. 16. Havlichel D, Saravolatz L, Pohlod D. Effect of quinolones and other antimicrobial agents on cell-associated Legionella pneumophila. Antimicrob Agents Chemother 1987;31:1529–34. 17. Rastogi N, Blom-Potan MC. Intracellular bactericidal activity of ciprofloxacin and ofloxacin against Mycobacterium tuberculosis H37Rv multiplying in the J-774 macrophages cell line. Zentralbl Bakteriol 1990;273:195–9. 18. Taira K, Koga H, Kohno S. Accumulation of a newly developed fluoroquinolone, OPC-17116, by human polymorphonuclear leukocytes. Antimicrob Agents Chemother 1993;37:1877–81. 19. Ishiguro M, Koga H, Kohno S, Hayashi T, Yamaguchi K, Hirota M. Penetration of macrolides into human polymorphonuclear leukocytes. J Antimicrob Chemother 1989;24:719–29. 20. Pascual A, Garcia I, Perea EJ. Fluorometric measurement of ofloxacin uptake by human polymorphonuclear leukocytes. Antimicrob Agents Chemother 1989;33:653–6. 21. Easmon CSF, Crane JP. Uptake of ciprofloxacin by macrophages. J Clin Pathol 1985;38:442–4. 22. Garcia I, Pascual A, Guzman MC, Perea EJ. Uptake and intracellular activity of sparfloxacin in human polymorphonuclear
21 leukocytes and tissue culture cells. Antimicrob Agents Chemother 1992;36:1053–6. 23. Murata M, Tamai I, Sai Y, Nagata O, Kato H, Tsuji A. Carriermediated lung distribution of HSR-903, a new quinolone antibacterial agent. J Pharmacol Exp Ther 1999;289:79–84. 24. Imada T, Miyazaki S, Nishida M, Yamaguchi K, Goto S. In vitro and in vivo antimicrobial activities of a new quinolone, OPC17116. Antimicrob Agents Chemother 1992;36:573–9.
25. Imada T, Goto S, Miyazaki S, Yamaguchi K, Kuwahara S. In vitro and in vivo antimicrobial effects of a new quinolone antimicrobial drug. Jpn J Chemother 1995;43:29–41. 26. Cook PJ, Andrews JM, Wise R, Honeybourne D, Moudgil H. Concentrations of OPC-17116, a new fluoroquinolome antibacterial, in serum and lung compartments. J Antimicrob Chemother 1995;35:317–26.
© Japanese Society of Chemotherapy and the Japanese Association for Infectious Diseases 2001
ORIGINAL ARTICLE Masao Hirota · Tokie Totsu · Fusayo Adachi Kazuo Kamikawa · Junichi Watanabe · Shiro Kanegasaki Koh Nakata
Comparison of antimycobacterial activity of grepafloxacin against Mycobacterium avium with that of levofloxacin: accumulation of grepafloxacin in human macrophages
Received: July 4, 2000 / Accepted: October 10, 2000
Abstract The bactericidal activity of two new quinolones, grepafloxacin and levofloxacin, against five strains of Mycobacterium avium was investigated in vitro. The minimum inhibitory concentrations (MICs) of these two quinolones, determined by the broth microdilution method, were comparable for all strains tested. In contrast, grepafloxacin suppressed the intracellular growth of all the strains in monocyte-derived macrophages more strongly than levofloxacin, when the cells infected with these strains were incubated for 7 days in the presence of various concentrations of the two new quinolones. To find the reason for the strengthened intracellular killing activity of grepafloxacin, we determined the ratio of the concentration of the new quinolones in the cells to that in the medium (C/M concentration ratio). The C/M concentration ratio of grepafloxacin was increased to 34.7 by 7 days, whereas that of levofloxacin at 7 days was only 12.3. These data suggested that a higher level of intraphagocytic accumulation of grepafloxacin endows it with greater mycobactericidal activity.
Introduction
T. Totsu · F. Adachi · J. Watanabe · S. Kanegasaki The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
Mycobacterium avium infection has increased in the past decade because of the epidemic of AIDS. In AIDS patients, the phagocytosed organism can survive in macrophages, causing disseminated infection in the lungs, liver, spleen, bone marrow, lymph nodes, and blood.1,2 The prognosis for M. avium-infected AIDS patients is very poor, owing, in part, to the resistance of this organism to available antimicrobial agents, including antituberculosis drugs.3 Furthermore, severe toxicity is associated with many of these agents when they are used at clinically useful levels.4–5 In addition, the intraphagocytic localization of M. avium protects the organism from the action of certain antimicrobial agents.6 Recent studies suggest that the protection of intracellular organisms from antimicrobial activity probably reflects the failure of these antimicrobial agents to enter macrophages.7–9 Therapy with antimicrobial agents that can penetrate phagocytes and exhibit intracellular antimicrobial activity is oviously needed in this setting. New quinolones, which have been extensively developed in recent years, have potent, broadspectrum antibacterial activity with high efficacy. Because the new quinolones show high tissue and intracellular distribution,10,11 they are expected to be useful for the treatment of infections caused by M. avium. In this study, we compared the efficiency of intracellular killing by two new quinolones, grepafloxacin (GPFX) and levofloxacin (LVFX), in monocyte-derived macrophages. Our data showed that GPFX accumulated in macrophages at a concentration three times higher than that of LVFX, and GPFX suppressed the intraphagocytic growth of M. avium strains more efficiently than LVFX.
K. Kamikawa Department of Respiratory Group Therapeutic Application Development, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan
Materials and methods
Key words Macrophages · Grepafloxacin · Antimycobacterial activity · Mycobacterium avium
M. Hirota Department of Drug Metabolism, Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan
K. Nakata ( ) Department of Respiratory Diseases, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo, Tokyo 162-8655, Japan Tel. 181-3-3202-7181 (ext.2873); Fax 181-3-3207-1038 e-mail: [email protected]
Antimicrobial agents and reagents GPFX (Table 1) was produced by Otsuka Pharmaceutical (Tokyo, Japan). LVFX (Table 1) was purified from the
17 Table 1. Physicochemical characteristics of grepafloxacin and levofloxacin Compound
Solubility in water (mg/ml)
Dissociation constants pKa1; pKa2
Grepafloxacin
36.0
7.1; 8.8a
Levofloxacin
25.8a
5.5; 8.0a
a
Values are cited from Taira et al. (1993)18
commercially available tablets. The purity of prepared LVFX was 99.98%. These antimicrobial agents were dissolved in dimethyl sulfoxide (DMSO) and diluted with RPMI (Nissui Tokyo, Japan) /10% fetal calf serum (FCS) medium before use. We purchased 7H9 broth, with albumin, dextrose, and catalase (ADC) enrichment; and 7H10 agar, with oleic acid, albumin, dextrose, and catalase (OADC) enrichment, from Difco Laboratories (Detroit, MI, USA). Recombinant human (rh) granulocytemacrophage colony stimulating factor (GM-CSF; specific activity, 2.25 3 108 U/mg) was purchased from R&D Systems (Minneapolis, MN, USA). M. avium strains and culture The 14-2, 25-3, and 36-1 strains of M. avium were serially isolated from the blood of the same patient at different times. Strain 14-2 was sensitive to clarithromycin, while strains 25-3 and 36-1 were moderately and strongly resistant, respectively, to this agent. Strain MS-6 was isolated from the blood of another patient, and was strongly resistant to clarithromycin. Cells in blood samples were lysed, using sterile distilled water, and pellets were obtained by centrifugation at 1000 g for 10 min. The pellets were cultured in 7H9 broth with ADC enrichment at 37°C for 3–6 weeks. Each bacterial suspension was then diluted and plated onto 7H10 agar medium. The plates were incubated for 8 weeks at 37°C under 5% CO2. Bacterial cultures from a single colony were grown in 7H9 broth to their exponential phase (to an optical density [OD]650 of 0.15, corresponding to approximately 108 CFU/ml). The isolated strains were identified as M. avium, using DNA-DNA hybridization (DDH) of mycobacterium (Kyokuto, Tokyo, Japan) and by detecting the insertion sequence, IS1245, using the polymerase chain reaction.12 M. avium type strain, ATCC 25291, was obtained from the American Type Culture Collection (Manassas, VA, USA).
Determination of minimum inhibitory concentration (MICs) An aliquot of 20 µl of M. avium suspension (OD650 5 0.15) was added to each well of a 96-well microtiter plate containing 100 µl each of broth and solution of the new quinolone. A stock solution (16 µg/µl) of GPFX or LVFX was diluted serially with 7H9 broth. The plates were incubated at 37°C under 5% CO2 for 10 to 15 days, and the OD650 of each well was measured, using a microplate spectrophotometer (model 3550; Bio-Rad, Hercules, CA, USA). Each MIC was determined as the lowest concentration that completely inhibited the growth of each M. avium strain. Preparation of human monocytes Peripheral blood mononuclear cells (PBMC), obtained from healthy donors by Ficoll/Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation, were resuspended in RPMI 1640 medium (Nissui) supplemented with 10% heat-inactivated FCS. The PBMC were incubated for 15 min at 37°C in 250 ml tissue culture flasks (Falcon, Franklin Lakes, NJ, USA) coated with 10% human AB serum in phosphate-buffered saline (PBS). Non-adherent cells were removed by vigorous washing with PBS, and adherent cells were collected by gentle scraping, using a cell scraper (Sumitomo Bakelite, Tokyo, Japan), after incubation with 0.05% trypsin/0.53 mM ethylenediamine tetraacetic acid (EDTA) for 10 min at 37°C. At least 98% of the adherent cells were identified as monocytes by morphology and nonspecific esterase staining. The viability of cells was determined by trypan blue dye exclusion. Infection of monocytes/macrophages by M. avium strains The purified monocytes were suspended in RPMI 1640/ 10% FCS medium, supplemented with 10 ng/ml of recombi-
18
nant human granulocyte macrophage-colony stimulating factor (GM-CSF), and incubated at a density of 2 3 104 per well in 96-well tissue culture plates (Falcon) at 37°C under 5% CO2 in a humidified cell culture incubator. After 7 days, the monocyte-derived macrophages were exposed for 2 h to a single-cell suspension of each M. avium strain at a multiplicity of infection of 10 : 1. Extracellular bacteria were removed by washing three times with PBS. The macrophages infected with M. avium were further incubated for 7 days in the presence of various concentrations of the new quinolones (0–8 µg/ml). The final viability of the macrophages was ascertained by trypan blue dye exclusion to be more than 90%.
Evaluation of CFU The number of M. avium CFU in the macrophages was determined after lysis with 0.25% sodium dodecylsulfate, which was subsequently neutralized with 10% FCS in PBS. The lysate was diluted serially (104 to 106-fold) and plated in duplicate on 7H10 agar plates. The colonies were counted after 2 to 3 weeks of incubation at 37°C under 5% CO2. Triplicate wells were used for each experimental condition tested. Data values were expressed as means 6 SD and compared between groups by two-way analysis of variance with the aid of the SAS system, release 6.12 (SAS Institute, Nancy, NC, USA) for Macintosh computer. P values below 0.05 were defined as significant.
Measurement of the cellular/medium concentration ratio (C/M ratio) of the antimicrobial agents Two million monocytes were incubated with 10 ng/ml of recombinant GM-CSF in a 25-cm2 tissue culture flask (Falcon) for 7 days. The monocyte-derived macrophages were washed twice with PBS and further incubated with 0.8 µg/ml of GPFX or LVFX. After 1-day incubation and after 7-day incubation, the whole medium was recovered, centrifuged at 500 g for 10 min to remove non-adherent macrophages (less than 5%), immediately frozen, and stored at 280°C. The adherent macrophages were washed five times with 5 ml of silicone oil (DC550; specific gravity, 1.07; William F. Nye, New Bedford, MA, USA). The remaining medium around the macrophages was completely removed by this procedure. The flask with macrophages was immediately placed on dry ice for 10 min and then kept at 280°C. The average number of adherent macrophages recovered from the culture flasks was 1.71 3 106 (range, 1.63–1.80 3 106). The concentrations of GPFX or LVFX in the macrophages and the medium were determined using high-performance liquid chromatography (HPLC; HPLC column, TSK gel ODS-80Ts, 4.6 mm 3 150 mm [Tosoh, Tokyo, Japan]) according to the procedure described by Akiyama et al.13 with slight modifications. Briefly, macrophages were removed from the flask using a cell scraper (Falcon) and suspended in 0.1 ml of methanol. After sonication, the suspension was centrifuged at 21 600 g for 5 min,
and mixed with an equal volume of 1-cyclopropyl-6,8difluoro-1,4-dihydro-5-ethyl-7-(4-methyl-1-piperazinyl)4-oxo-3-quinolinecarboxylic acid (OPC-17203, Otsuka Pharmaceutical, Tokyo, Japan) (20 ng/ml) in methanol. For measurement of the concentration of GPFX or LVFX in the medium, the samples were mixed with OPC-17203 (2000 ng/ml) in methanol and extracted with chloroform for 10 min. The organic phase was evaporated to dryness at 40°C under a nitrogen stream. The residue was dissolved in the mobile phase (CH3CN [20 mM] : Na2SO4 : H3PO4 [1400 : 3600 : 4]). The pretreated solutions were injected to the HPLC column at 40°C with a flow rate of 0.8 ml/min. GPFX or LVFX was eluted with the mobile phase, and the detection of GPFX or LVFX on HPLC was performed at an excitation wavelength of 285 nm and fluorescence wavelength of 448 nm. The C/M ratio was determinating by calculating the cell volume of macrophages, estimated by the method of Krombach et al.14 The data values were expressed as means 6 SDs and compared between groups using the t-test, with the aid of the SAS system, release 6.12 (SAS Institute) for Windows. P values below 0.05 were defined as significant.
Results Antimycobacterial effect of new quinolones on extracellular and intracellular growth of M. avium strains The effect of GPFX or LVFX on the growth of M. avium strains in 7H9 broth was studied. As shown in Table 2, the MICs of GPFX and LVFX for the five M. avium strains were comparable, ranging from 0.25 to 1.0 µg/ml. On the other hand, GPFX suppressed the intracellular growth of all strains in the monocyte-derived macrophages more efficiently than LVFX when the cells were infected with these strains and incubated for 7 days in the presence of various concentrations of these antibiotics. As shown in Fig. 1, the number of M. avium CFU decreased with increased concentrations of GPFX. In all strains tested, the percentages of CFU in macrophages treated with GPFX were significantly lower than those in macrophages treated with LVFX, in the concentration range of 0.125–5.0 µg/ml. More than 94.5% of intracellular M. avium were killed by 1.0 µg/ml of GPFX, whereas the same concentration of LVFX killed 47%–74% of intracellular M. avium. Table 2. MIC values of grepafloxacin and levofloxacin for Mycobacterium avium strains (µg/ml) Strains
Grepafloxacin
Levofloxacin
14-2 25-3 36-1 MS-6 ATCC25291
0.25 0.25 0.50 0.50 1.00
0.50 0.50 1.00 0.50 1.00
Values expressed are the means of three independent experiments MIC, Minimum inhibitory concentration
19 Fig. 1A–E. Colony formation of intracellular M. avium strains in macrophages incubated with grepafloxacin or levofloxacin for 7 days. Macrophages were incubated with 0, 0.125, 0.250, 0.500, 1.000, 2.000, 4.000, and 8.000 µg/ml of grepafloxacin or levofloxacin for 7 days. Open circles indicate percent CFU in macrophages treated with levofloxacin; closed circles indicate percent CFU in macrophages treated with grepafloxacin. Values on the horizontal axis show the concentrations of antimicrobial agents added to the culture medium. Values on the vertical axis are expressed as percentages of control CFU (without antimicrobial agents). Error bars indicate SDs of three independent experiments. *P , 0.05 versus levofloxacin; **P , 0.01 versus levofloxacin
Cellular/medium (C/M) concentration ratios of the new quinolones To find the reason for the effectiveness of GPFX in macrophages, we evaluated the concentrations of GPFX and LVFX in macrophages and in the medium. When monocyte-derived macrophages were incubated with 0.8 µg/ ml of GPFX or LVFX for 1 day and 7 days, GPFX was accumulated in macrophages at concentrations of 21.3 µg/ ml (296.5 pmol/106 cells) and 28.1 µg/ml (390.6 pmol/106 cells), respectively, whereas the amounts of LVFX were 6.8 µg/ml (94.1 pmol/106 cells) and 10.2 µg/ml (140.9 pmol/106 cells), respectively. Degradation of antibiotics in the medium was not observed, because the concentrations of both GPFX and LVFX in the medium did not change during the incubation. The C/M ratios of GPFX at 1-day and at 7-day incubation were calculated to be 27.7 and 34.7, respectively, values which were significantly higher than those for LVFX, of 8.9 on day 1 and 12.3 on day 7 (Fig. 2). We repeated measurement of the C/M ratio three times, using blood monocytes from three different donors, and we obtained similar results. Thus, GPFX accumulated markedly in macrophages, in accordance with its relatively potent intracellular antimycobacterial activity against M. avium.
Discussion We investigated the effects of GPFX and LVFX against M. avium in in-vitro model. Incubation of M. avium-infected human macrophages with GPFX or LVFX resulted in a concentration-dependent decrease in viable bacteria, as
Fig. 2. Cellular/medium (C/M) concentration ratios of grepafloxacin (GPFX) and levofloxacin (LVFX). After the incubation of macrophages with 0.8 µg/ml grepafloxacin or levofloxacin for 1 day or 7 days, the concentrations of grepafloxacin and levofloxacin in the cells were measured by HPLC. Open columns indicate the C/M ratios of cells incubated with each compound for 1 day. Closed columns indicate the C/M ratios of cells incubated with each compound for 7 days. Data values are expressed as means 6 SDs of three independent experiments. P , 0.0001 versus levofloxacin
compared with numbers of the bacteria in control cells without antimicrobial agents. GPFX was more effective against intracellular M. avium growth than LVFX over the concentration ranges tested, although the MIC of these new quinolones against M. avium was comparable. GPFX accumulated markedly in macrophages, with a C/M ratio of about 35 at 7 days’ incubation, a value which was three times higher than that of LVFX. Because new quinolones show preferential distribution in various tissues such as lung, skin, and intestine, and high
20
intracellular concentrations,10,11 they have been suggested to be useful for the treatment of infectious diseases caused by intracellular pathogens such as Clamydia sp.,15 Legionella sp.,16 and mycobacteria17 in recent years. This underlines the need for evaluation of the accumulation of the new quinolones in macrophages. The accumulation of various antimicrobial agents in phagocytes has been investigated. Relatively high C/M ratios (between 2 and 8) have been demonstrated for many new quinolones, with ofloxacin having the highest ratio, of 8.18 In this study, we showed that the C/M ratio of LVFX was similar to that of ofloxacin. In comparison with ofloxacin and LVFX, our data showed that GPFX had an extremely high C/M ratio, greater than 20. This C/M ratio was comparable to those of clindamycin7 and the macrolides,19 which are known to be high. The mechanism of the intracellular uptake of antimicrobial agents by phagocytes, reported in previous studies, is broadly classified into two categories: passive and active transport systems. Macrolides are typical antibiotics that are classified in the latter category, and ofloxacin was reported to have the same mechanism.20 On the other hand, ciprofloxacin21 and sparfloxacin22 were reported to be incorporated mostly by a passive mechanism. Recently, Taira et al.18 reported that the uptake of GPFX by polymorphonuclear cells was extremely high, with a C/M ratio of 60, and rapid. The uptake reached a plateau within the first 5 min of incubation. They demonstrated that the accumulation of GPFX in polymorphonuclear cells involved an active transport system. Murata et al.23 showed that GPFX was distributed into rat lung at a high concentration by a similar transport system. Thus, it is suggested that the high concentration of GPFX in macrophages is due to the active transport system. In this study, we showed that GPFX was distributed in macrophages at a higher concentration than LVFX when the macrophages were incubated with the same concentration of these new quinolones. The solubility of GPFX in octanol-Sourensen buffer (pH 7.4), an organic solvent system, is tenfold higher than that of LVFX.23 In general, hydrophobicity is beneficial in antimicrobial agents, enabling them pass through the lipid bilayer of the cell membrane. Therefore, this physicochemical property may allow the high concentration of GPFX in macrophages. GPFX displays potent antimicrobial bioactivity, with a broad spectrum.24,25 Although little is known about its antimycobacterial activity, our present data confirmed the efficacy of this drug in both the intracellular and extracellular killing of M. avium. After the oral administration of 400 mg of GPFX daily, the concentrations in the bronchoepithelial cells and alveolar lining fluid are 3.1-fold and 12.2-fold, respectively, that in the plasma.26 The selective accumulation of GPFX in the lungs, and the potent antimycobacterial activity in macrophages shown in this study suggest that this drug would be effective against M. avium infection in the lung. Acknowledgments The authors thank Dr. Hiroyuki Sasabe, Dr. Hitoshi Akiyama, and Dr. Gohachiro Miyamoto for valuable discussions.
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