Comparative in vitro and in vivo activity of fleroxacin and ofloxacin against various mycobacteria

Comparative in vitro and in vivo activity of fleroxacin and ofloxacin against various mycobacteria

Tu bercle Tube&e (1991) 72. 176-180 0 Longman Group UK L.td 1991 Comparative in vifro and in viva activity of fleroxacin and ofloxacin against variou...

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Tu bercle Tube&e (1991) 72. 176-180 0 Longman Group UK L.td 1991

Comparative in vifro and in viva activity of fleroxacin and ofloxacin against various mycobacteria H. TOMIOKA, K. SAT0 and H. SAITO Department of Microbiology and Immunology, Shimane Medical University, Shimane, Japan

Summary - In vitro antimicrobial activity of fleroxacin (6,8- difluoro-1-(2-fluoroethyl)-l , 4-dihydro-7- (4-methyl-l -piperazinyl) -4-oxo-3-quinolinecarboxylic acid) and ofloxacin against representative pathogenic mycobacteria was evaluated by the agar dilution method, using 7Hll agar medium. Fleroxacin showed appreciable antimicrobial activity against Anycokcterium tuberculosis (M ICee=6.25 mg/l), M. kansasii (MICg0=3.13 mg/l), and M. fortuitum (MICW=6.25 mg/l), whereas M. ma&urn, M. scrofulaceum, M. avium, M. intracellulare, and M. chelonae were highly resistant to the agent. The activity of fleroxacin was comparable to that of ofloxacin. Fleroxacin showed antimicrobial activity against M. intracellulare phagocytosed in murine peritoneal macrophages at a concentration of 10 mg/l in the culture medium, but its activity was considerably lower than that of ofloxacin. On the other hand, the therapeutic activity of fleroxacin against M. fortuitum infection induced in mice was higher than that of ofloxacin. Neither fleroxacin nor ofloxacin was efficacious against M. intracellulare infection. Fleroxacin significantly depressed the growth of M. leprae in the mouse footpad.

Introduction

than M. tuberculosis, especially M.avium-intracellulare complex, arc generally resistant to all standard

Fleroxacin is a new quinolone with the chemical structure of 6,8difluoro-l- (2-fluoroethyl) -1,4dihydro-7- (4-methyl-l-piperazinyl) -4-0x0-3-quinolinecarboxylic acid. It shows potent activity against the isolates of the family Enrerobacteriaceae, Neisseria spp., and Haemophilus infiuenzae. It is also active against Staphylococcus spp., Pseudomonas aeruginosa, Branhamella catarrhalis, methicillin-resistant S. aureus and gentamicin-resistant P. aeruginosa. In general, the efficacy of fleroxacin is roughly comparable to that of norfloxacin and ofloxacin, but it is slightly less effective than ciprofloxacin [I]. Recently, it was reported that ofloxacin has some activity against Mycobacten’um iuberculosis and M. fortuitum infection [2-51. However, acid-fast bacilli other

antituberculosis drugs and many antimicrobial agents [6, 71. Therefore, the development of an effective drug against these mycobacteria is desirable. In this study, in vitro activity of fleroxacin against various mycobacteria was compared with that of ofloxacin, and the chemotherapeutic activity of fleroxacin was studied in mice infected with M. fortuitum, M. intracellulare and M. leprae.

Correspondence to: Dr Hajime Saitq Department of Microbiology and Immunology, Shimane Medical University, Izumo 693, Shimane. Japan

Materials and methods Drugs Flemxacin

and ofloxacin were donated by Kyorin Pharmaceutical Co., Ltd, Tokyo, Japan, and Daiichi Pharmaceutical Co., Ltd. Tokyo, respectively.

176

ACIWITY

177

OF FLEXOXACIN

Bacteria

suspended in 7H9 broth to yield about 106 cfu/ml. The susceptibility test was carried out by the two-fold agar dilution method, using 7Hll agar medium on which 5 ul of the test bacterial suspension were spotted. The MICs of the drugs were determined at 7 (rapid growers) or 14 (slow growers) days of cultivation at 37°C (33°C for M. marinum and M. chelonae subsp. chelonae) in a CO2 (5% COr95% humidified air) incubator.

Inc., Shizuoka, Japan, were injected intraperitoneally with 1 mg of zymosan A (Sigma Chemical Co., MO., USA) 4 days before cells were harvested in Hanks’ balanced salt solution (HBSS, pH 7.2) supplemented with 1% fetal bovine serum (FBS)(M.A. Bioproducts, Walkersville, Md., USA), centrifuged at 15Og for 5 min. washed with 1% PBS-HBSS, and then resuspended in 10% PBS-RPM1 1640 medium (Nissui Pharmaceutical Co., Tokyo)(S x [email protected]/ml). The cell suspensions (1 ml) were poured into 24-cell well culture dishes (Coming Glass Works, Corning, NY, USA). After incubation at 37-C for 2 h in a CR incubator, the wells were rinsed with 1% PBS-HBSS to remove non-adherent cells. The resultant macrophage monolayer was overlaid with 1 ml of 10% PBS-RPM1 1640 medium containing 5.5 x 106 cfu/ml of M. intracellulare N-260, incubated at 37°C for 1 h and washed with 1% PBS-HBSS to remove the non-phagocytosed bacteria. The macrophages were further incubated in 1 ml of 10% PBS-RPM1 1640 medium, in the presence or absence (control) of 10 mg/l of fleroxacin or ofloxacin at 37’C for up to 5 days in a CO;! incubator. The medium with or without the drugs was changed twice daily. The macrophages were then lysed by distilled water followed by sonication with Handy Sonic (Model UR-2OP; Tomy Seiko Co., Tokyo) for 20 s. The cfu in the cell lysate was counted on a 7Hll agar plate [8]. In some cases, the macrophages were stained with Giemsa solution, the number of macrophages was counted microscopically, and then the cfu per macrophage was calculated.

Preparation of macrophages and antimicrobial activity

Experimental infections in mice

All the strains of M. tuberculosis, M. avium, and M. intracellulare were fresh isolates from clinical specimens, and M. kansasii, M. marinum, M. scrofulaceum, M. fort&urn, M. chelonae subsp. abscessus, while those of M. chelonae subsp. chelonae were stocked in our laboratory. Strains of M. avium and M. intracellulare producing smooth and transparent colonies were used throughout the experiment. Both M. avium and M. intracellulare were identified by the DNAprobe test in our laboratory, using Gen-Probe Rapid Diagnostic System (Gen-Probe Inc., San Diego, CA, USA). M. fortuitum 18367, M. intracellulare N-260 and M. leprae Thai 53 were used for the experimental murine infection. Determination of MICs The organisms were grown in 7H9 broth (Difco Laboratories, Mich., USA) at 37°C (33°C for M. marinum and M. chelonae subsp. chelonae) for 3-7 days and

Peritoneal cells from female BALB/c mice (8-10 weeks old), which were purchased from Japan SLC

Table 1 Susceptibility

M. fortuitum

18367 and M. intracellulare N-260 species were grown in 7H9 broth at 37’C for 3-5

of various mycobacteria to fleroxacin and ofloxacin NlU?lbt??

Species

of

strain3

MICs (mgll) Fleroxacin SO%1

90462

oj7oxacin 50%

90%

M. tubercuimir M. kansasii M. marinum M. scrofulocewn M. avium M. intracellulare M. forlukwn M. chelonae (chelonae) M. chelonae (abscessw)

25 19 10 19 18 31 20 20 15

3.13 3.13 12.5 12.5 50 50 3.13 25 z-100

6.25 3.13 50 25 >loO 100 6.25 100 >lOO

1.56 1.56 12.5 12.5 2S 50 1.56 12.5 >I00

3.13 6.25 2s SO 100 100 3.13 50 >lOO

l MIC for SO% of the strains. 2 MIC for 90% of the strains.

178

TQMIOKA AND OTHERS

days and the bacterial suspensions were preparedby diluting organisms with the same medium so as to yield appropriate cfus of the organisms. A suspension of M. leprue Thai 53 was prepared from the infected footpads of female BALB/c (nu/nu) mice [9]. M. fortuitum infection. Five-week-old female ddY mice were infected intravenously (i.v.) with 9.2 x 106 of M. fortuitum. Fleroxacin (1 mg) or ofloxacin (1 mg) in distilled water (0.1 ml) was given by gavage (oesophageal intubation), daily, 6 times a week, from 24 h up to 4 weeks after the infection. Untreated mice were used as controls. The mice were killed 24 h after the last administration of the drugs. The kidneys were removed and lesions were observed macroscopically. The cfus in the kidneys were counted on 7Hll agar plate, as described previously [83.

Table 2 Antimicrobial activities of fleroxacin and ofloxacin against M. intracellulare phagocytosed in murine peritoneal macrophage-sl. Time after

infeclion &VI

0 3 5

Results Susceptibility of mycobacteria

Table 1 shows the susceptibilities of various mycobacteria to Reroxacin and ofloxacin. The MI% of fleroxacin against the test strains of M. tuberculosis, M. kunsasii, and M. fortuitum was 6.25, 3.13, and 6.25 mg/l, respectively. The other species showed a high resistance to fleroxacin. The activity of fleroxacin was comparable to that of oIloxacin. Antimicrobial activity against M. intracellulare phagocytoseii in macrophages

Table 2 shows the antimicrobial activities of the drugs against M. intracellulare phagocytosed in murine peritoneal macrophages. A significant inhibition of the growth of the intracellular organisms was noted in the presence of 10 mg/l of fleroxacin in the culture medium. Fleroxacin was less active than ofloxacin in retarding the growth of organisms in macrophages. Activity against murine experimental infections

cfulmacrophage Control

Fleroxacin

opoxocin

0.7 f 0.1 6.4 f 0.1 20.4 f 3.7

24 f 0.1 2 10.6 f 1.4

1.3 f 0.1 2 3.9 f 0.9 3

1 Mean f SE (n = 3). The IylIC values of fleroxacin and ofloxacin against M. tiracelhlare N-260 strain were determined as to be 100 and 50 mg/l, respebvely. 2 Significantly different from control (P < 0.01). 3 Significantly different from control (P < 0.05).

M. intracellulare inftdon. Five-week-old female BALB/c mice were infected i.v. with 1.2 x 107 of M. intrucellulure. Fleroxacin (1 mg) or ofloxacin (1 mg) in saline (0.1 ml) was given subcutaneously (sc) for 8 weeks, in the same frequency as described above. The lungs and spleen were removed and lung lesions were observed macroscopically, and then the cfus in the organs were counted on 7Hll agar plate [81. M. leprae infection. Five-week-old female BALB/c atliymic nude mice were infected with 1 x 106 of M. leprae injected SCinto the left hind footpad. Fleroxacin (3 mg) in distilled water (0.1 ml) was given to mice by gavage, once daily, 6 times per week, for up to 50 days from day 31 to day 80. On day 250, the mice were killed and the number of acid-fast bacilli in the infected footpad was enumerated by microscopy.

Table 3 shows the incidence of gross renal lesions in M. fortuitum-infected mice treated with or without drugs. Both fleroxacin and ofloxacin suppressed the development of renal lesions in mice at 2 and 4 weeks after infection. Table 4 shows the viable number of organisms in the kidneys of M. fortuitum-infected mice during the course of infection. The number of organisms in the kidneys was reduced by the administration of fleroxacin or ofloxacin and elimination of the organisms in fleroxacin-treated mice was greater than in ofloxacintreated mice. In mice infected with M. intrucellulure, neither fleroxacin nor ofloxacin reduced the number of viable organisms in the lungs and spleen (Table 5). As shown in Table 6, when nude mice infected with M. leprue in the hind footpad were given fleroxacin, the growth of the organisms at the site of infection was significantly reduced. Discussion It was reported that in vitro antibacterial activity of fleroxacin for many species is roughly comparable to that of ofloxacin [ 11,but its antimycobacterial activity has not been reported. In the present study, it was found that fleroxacin was highly active against M. tuberculosis, M. kansasii, and M. fortuitum, among the 8 species mycobacteria tested, and its activity was almost similar to that of otloxacin. However,

180

TOMIOKA

Table 6 Antileprosy Regbnen

activity

DCSe

of fleroxacin Lag-acid-faFt imdilfootpad~

(ms) None Fleroxacin

3

5.25 f 0.38 4.13 f 0.20 2

1 Mean f SE (n = 5). In repeated experiments the number of acid-fast bacilli detected in the tissue of footpad at day 1 after inoculation (lti bacilli/footpad), by the microscopic method used here, was smaller than 5 x 103 (below detectable level). 2 Significantly different from untreated mice (P < 0.05).

lungs and kidneys were 2.4, 2.9 and 6.8 mg/l or g, respectively. These concentrations of fleroxacin were about 2-3 times higher than those of ofloxacin @ersonal observation; internal report from Kyorin Pharmaceutical Co., Ltd). These results suggest that the therapeutic efficacy of fleroxacin may be greater than that of ofloxacin. In fact, in the present study, flemxacin exhibited a therapeutic activity greater than that of ofloxacin against M. fortuitum infection. Similar results have been reported in murine infection due to Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosu [ 11. We previously reported that ofloxacin exhibits bactericidal activity against M. feprue in mice, both in vitro and in vivo [93. In the present study, fleroxacin also caused a marked decrease in the growth of M. leprue in the footpads of mice. Recent studies have demonstrated a high resistance of M. leprue to DDS (diaminodiphenyl sulfone), which is the main drug used in the treatment of leprosy [ 10,111. Thus, fleroxacin may prove to be a potent new antileprosy drug.

AND OTHTas

References 1. Hirai K, Aoyama H, Hosoka M et al. In vitro and in vivo antibacterial activity of AM-833, a new quinolate derivative. Atibnicrob Age& Ckmotkr 1986; 29: 1059-1066. 2. Saito H, Sate K, Tom&a H, Watanabe T. In vitro and in vivu activities of norlloxacin, ofloxacin and ciprofloxacin against various mywbacteria. Kekkakn 1987; 62: 287-294. 3. Saito H, Sate K. Watanabe T, Tomioka H. In vitro and in vivo suscepibiities of M~ohacteriwn foruitum to ofloxacin (DL 8280). In: Ishigami J. ed. Recent advances in chemotherapy. Tokyo: University of Tokyo Press. 1985; p 3 19. 4. Tsukamura M. In vitro antituberculosis activity of a new antibacterial substance of ofloxacin (DL 8280). Am Rev Respir Dir 1985; 131: 348-351. 5. Tsukamura M, Nakamura E. Yoshii S, Amano H. Therapeutic effect of a new antibaaerial substance ofloxacin (DL 8280) on$onary tuberculosis. Am Rev Respir Dir 1985; 131: 6.

I.

8.

9.

10. 11.

Kuze F, Kurasawa T, Bando K. Lee Y, Maekawa N. In vitro and in vivo susceptibility of atypical mycobacteria to various drugs. Rev Infect Dir 1981; 3: 885-897. Woodley CL, Kilbum JO. In vitro susceptibility of Mycobacteriwn aviwn complex and Mycobacteriwn tuberculosis strains to a Spiro-piperidyl r&my&. Am Rev Respir Dir 1982; 126: 586-587. Saito H, Sate K, Tomioka H. Comparative in vifro and in vivo adivity of rifabutin and rifampicin against Mycobucrerium aviwn complex. Tubercle 198% 69: 187-192. Saito H, Tanioka H. Nagashima K. In vitro and in vivo activities of ofloxacin against Mycobacleriwn leprae infection induced in mice. Int J L.epr 1986; 54: 560-562. Pearson JMH. The problem of dapsone-resistant leprosy. Inl J Lepr 1981; 49: 417420. World Health Organization Group. Chemotherapy of leprosy for control programmes., WHO Tech Rep Ser Geneva: WHO 1982, p 675.

ACI’NITY

179

OF FLEROXAW

Table 3 Renal lesions in M. firttitum-infected

Regimen

No. of mice with renal lesions Week 2

Dose (msl

NOIle Fleroxacin ofloxacin

mice with or without lleroxacin or ofloxacin

1 1

Week 4

-

l+

2+

3+

0 0 0

ii

1 0 0

4 0 0

5

0 1 1

I+

2+

3+

1 4 4

3 0 0

1 0 0

no lesion; l+. discrete small lesions covering c 25% of surface; 2+, confluent lesions covering 25-5045 of surface; 3+, confluent ZCms covering > 5W of surface.

Table 4 Effects of fleroxacin and ofloxacin on the behaviour of M.

forfuhun in the kidneys during the course of infection

Log cjidkidneys~ Regimen

Dose (mhd

None Fleroxacin olloxacin

Day 1

Week 2

Week 4

5.23 f 0.06

5.67 f 0.19 3.47 f 0.522 4.61 f 0.362

5.34 f 0.25 222 f 0.183 3.41 f 0.592

1 1

1 Mean f SE (n = 5). 2 Significantly different from control (P < 0.05). 3 Significantly different fran control (P < 0.01).

Table 5 Effects of fleroxacin and ofloxacin 011the behaviour of M. intracellulare in the lungs and spleen during the wurse of infection Log cfulorgan’ Organ

h8s

Spleen

Regimen

None Fleroxacin Ofloxacin None Fleroxaciu Ofloxacin

Dose fmd

Day 1

Week 4

1 1 1 1

4.81 k 0.08 6.16 + 0.06 -

5.21 5.22 5.23 6.39 6.39 6.30

f f f f f f

Week 8 0.04 0.04 0.06 0.06 0.06 0.08

6.92 6.95 6.81 7.14 7.31 7.19

f f f f f f

0.06 0.05 0.17 0.05 0.02 0.09

1 Mean f SE (n = 5).

the 8 species mycobacteria tested, and its activity was almost similar to that of ofloxacin. However, M. avium and M. intracullare, which are resistant to most antituberculous or antimicrobial agents [6, 71, were resistant to fleroxacin and ofloxacin. Similarly, fleroxacin failed to exhibit any appreciable therapeutic activity against M. intracelhlare infection. We suggest several reasons why these drugs could reduce the growth of the organisms inside macrophages even when added at the concentration (10 m&I) lower than their MIC values (see Table 2, footnote). First,

it is possible that the organisms phagocytosed by macrophages (located in phagolysosomes) are rendered much more susceptible to these antimicrobial agents than extracellular ones because the former are damaged by the microbicidal mechanisms of the phagocytic cells. Secondly, these agents could be concentrated in phagolysosome vesicles to levels in excess of their MIC values. Thirdly, it may be also possible that some synergistic antimicrobial effect may be produced by interaction of these quinolones with certain substances contained in lysosomes.