International Journal of Antimicrobial Agents 44 (2014) 514–519
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Evaluation of the in vitro activity of levornidazole, its metabolites and comparators against clinical anaerobic bacteria Jiali Hu a,b , Jing Zhang a,b,∗ , Shi Wu a,b , Demei Zhu a,b , Haihui Huang a,b , Yuancheng Chen a,b , Yang Yang a,b , Yingyuan Zhang a,b a b
Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China Key Laboratory of Clinical Pharmacology of Antibiotics, National Health and Family Planning Commission, Shanghai, China
a r t i c l e
i n f o
Article history: Received 12 June 2014 Accepted 29 July 2014 Keywords: Levornidazole Metabolite Anaerobe Minimum inhibitory concentration Minimum bactericidal concentration
a b s t r a c t This study evaluated the in vitro anti-anaerobic activity and spectrum of levornidazole, its metabolites and comparators against 375 clinical isolates of anaerobic bacteria, including Gram-negative bacilli (181 strains), Gram-negative cocci (11 strains), Gram-positive bacilli (139 strains) and Gram-positive cocci (44 strains), covering 34 species. Minimum inhibitory concentrations (MICs) of levornidazole, its five metabolites and three comparators against these anaerobic isolates were determined by the agar dilution method. Minimum bactericidal concentrations (MBCs) of levornidazole and metronidazole were measured against 22 strains of Bacteroides fragilis. Levornidazole showed good activity against B. fragilis, other Bacteroides spp., Clostridium difficile, Clostridium perfringens and Peptostreptococcus magnus, evidenced by MIC90 values of 0.5, 1, 0.25, 2 and 1 mg/L, respectively. The activity of levornidazole and the comparators was poor for Veillonella spp. Generally, levornidazole displayed activity similar to or slightly higher than that of metronidazole, ornidazole and dextrornidazole against anaerobic Gram-negative bacilli, Grampositive bacilli and Gram-positive cocci, especially B. fragilis. Favourable anti-anaerobic activity was also seen with levornidazole metabolites M1 and M4 but not M2, M3 or M5. For the 22 clinical B. fragilis strains, MBC50 and MBC90 values of levornidazole were 2 mg/L and 4 mg/L, respectively. Both MBC50 /MIC50 and MBC90 /MIC90 ratios of levornidazole were 4, similar to those of metronidazole. Levornidazole is an important anti-anaerobic option in clinical settings in terms of its potent and broad-spectrum in vitro activity, bactericidal property, and the anti-anaerobic activity of its metabolites M1 and M4. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Levornidazole, the levo isomer of ornidazole, is a thirdgeneration nitroimidazole derivative newly developed after metronidazole, tinidazole and ornidazole. This novel antibiotic has shown good anti-anaerobic and antiprotozoal activity [1]. Levornidazole was approved by the China Food and Drug Administration in August 2009. The ornidazole products used in clinical settings are mostly racemic compounds composed of equal amounts of the l-isomer and d-isomer. In general, the adverse reactions of ornidazole are mainly stomach discomfort, dizziness, somnolence and other adverse reactions of the nervous system [2]. Dextrornidazole is the major component of ornidazole contributing to toxicity of the central nervous system [3]. Levornidazole is similar to or slightly better than racemic ornidazole in terms of
∗ Corresponding author at: Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai 200040, China. Tel.: +86 21 5288 8190; fax: +86 21 6248 4347. E-mail addresses: zhangj [email protected], zhangj [email protected] (J. Zhang).
pharmacokinetic properties [4]. Early in vitro pharmacodynamic studies also demonstrated that levornidazole has a comparable antimicrobial spectrum and more potent antimicrobial activity compared with ornidazole [5]. However, only a small sample of strains was tested in those studies without identifying each strain to specific species. Therefore, it is important to further investigate the in vitro antibacterial activity of levornidazole. Studies have shown that five phase I metabolites are derived from ornidazole in animals and human liver. Both 1-chloro-3(2-hydroxymethyl-5-nitro-1-imidazolyl)-2-propanol (M1) and 2methyl-5-nitroimidazole (M2) are the oxidative products of ornidazole, whilst 3-(2-methyl-5-nitro-1-imidazolyl)-1,2-propanediol (M4) is produced by hydrolysis of the chloride in the side chain of the imidazole ring. N-(3-chloro-2-hydroxypropyl) acetamide (M3) and acetamide (M5) are generated by cleavage of the imidazole ring following hydrolysis of ornidazole [6]. Nanjing Sanhome Pharmaceutical Co., Ltd. (Nanjing, China) has developed the five metabolites of ornidazole (Table 1). The metabolites and metabolic pathway of levornidazole are similar to ornidazole.
http://dx.doi.org/10.1016/j.ijantimicag.2014.07.027 0924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
J. Hu et al. / International Journal of Antimicrobial Agents 44 (2014) 514–519
515
Table 1 Levornidazole and its five metabolites. Drug
Structural formula
Molecular formula
Molecular weight (g/mol)
Levornidazole
C7 H10 ClN3 O3
220
M1
C7 H10 ClN3 O4
236
M2
C4 H5 N3 O2
127
M3
C5 H10 ClNO2
152
M4
C7 H11 N3 O4
201
M5
C2 H5 NO
This study aimed to compare the in vitro antibacterial activities of levornidazole, metronidazole, ornidazole, dextrornidazole, and the five metabolites (M1, M2, M3, M4 and M5) of levornidazole to further characterise the antibacterial activity and spectrum of levornidazole. The results will provide useful data for future clinical use and further research of levornidazole.
59
antibiotic-associated diarrhoea. The 76 strains of Bacteroides spp. and 45 strains of C. perfringens were isolated from patients with colonisation. Quality control (QC) strains included Bacteroides fragilis ATCC 25285, C. difficile ATCC 70057 and Prevotella melaninogenica ATCC 25845. All QC and reference strains were characterised by morphology as well as physiological and biochemical tests before they were used in this study.
2. Materials and methods 2.1. Bacterial strains A total of 375 strains of anaerobes were collected from patients who were treated in Huashan Hospital (Shanghai, China) during the period January 2006 to February 2013. These strains were mainly isolated from blood, faeces, secretions and other specimens and included Gram-negative bacilli (181 strains), Gram-positive bacilli (139 strains), Gram-positive cocci (44 strains) and Gram-negative cocci (11 strains), covering 34 species (Table 2). Overall, 200 of the strains were isolated from faeces, including 72 strains of Clostridium difficile, 76 strains of Bacteroides spp., 45 strains of Clostridium perfringens and 7 strains of other Clostridium spp. The 72 strains of C. difficile and 7 strains of other Clostridium spp. were considered to be from infections, mainly isolated from patients with
2.2. Culture media Brucella broth and Brucella agar were from Oxoid USA Inc. (Columbia, MD). Laked sheep blood (5% v/v) was provided by Shanghai Zhudi Germ-free Animal Blood Supplier (Shanghai, China). Haemin (5 mg/L) and vitamin K1 (1 mg/L) were products of Sigma Chemical Co. (St. Louis, MO).
2.3. Antimicrobial agents Levornidazole, ornidazole and dextrornidazole as well as levornidazole metabolites M1, M3, M4 and M5 were the products of Nanjing Sanhome Pharmaceutical Co. Ltd. Metronidazole and levornidazole metabolite M2 were provided by the Department of
516
J. Hu et al. / International Journal of Antimicrobial Agents 44 (2014) 514–519
Table 2 Comparative in vitro activity of levornidazole against 375 strains of anaerobic bacteria. Organisms/compound
Bacteroides fragilis (46) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Bacteroides thetaiotaomicron (28) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Bacteroides ovatus (27) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Bacteroides uniformis (18) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Other Bacteroides spp. (39) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Prevotella spp. (23) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Clostridium difficile (72) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Clostridium perfringens (45) Levornidazole Ornidazole
Susceptibility (%)a
MIC (mg/L) Range
MIC50
MIC90
0.25–16 0.25–32 0.25–16 0.5–32 0.25–16 0.25–64 16 to >128 >128 >128
0.5 0.5 0.5 1 0.5 1 64 >128 >128
0.5 1 1 2 1 1 64 >128 >128
0.25–2 0.5–4 0.5–2 0.5–2 0.5–2 0.5–4 32 to >128 >128 >128
1 1 1 1 0.5 1 64 >128 >128
1 2 1 2 1 2 64 >128 >128
0.06–2 0.06–2 0.125–1 0.25–2 0.125–1 0.125–2 16 to >128 64 to >128 >128
0.5 1 1 2 1 1 16 >128 >128
1 2 1 2 1 2 16 >128 >128
0.125–2 0.25–2 0.25–2 0.25–4 0.25–4 0.25–4 32 to >128 >128 >128
1 1 1 2 1 1 64 >128 >128
2 2 2 2 1 2 64 >128 >128
0.125–2 0.25–2 0.25–2 0.5–2 0.25–2 0.25–4 32 to >128 64 to >128 >128
1 1 1 1 1 1 64 >128 >128
1 2 1 2 1 2 64 >128 >128
0.06–64 0.06–64 0.06–64 0.06 to >64 0.125–16 0.125 to >64 16 to >128 64 to >128 >128
1 1 0.5 1 1 1 64 >128 >128
2 2 2 2 1 2 >128 >128 >128
0.06–1 0.06–1 0.06–0.5 0.06–1 0.06–1 0.125–1 4–128 64 to >128 >128
0.125 0.125 0.25 0.25 0.25 1 16 >128 >128
0.25 0.25 0.5 0.25 0.25 1 32 >128 >128
2 1
2 2
0.5–2 0.5–4
S
I
R
97.8
2.2
0
100
0
0
100
0
0
100
0
0
100
0
0
0
4.3
0
0
95.7
100
J. Hu et al. / International Journal of Antimicrobial Agents 44 (2014) 514–519
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Table 2 (Continued) Organisms/compound
Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Other Clostridium spp. (16) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Peptostreptococcus spp. (44) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Veillonella spp. (11) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Eubacterium and Bifidobacterium (6) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5
Susceptibility (%)a
MIC (mg/L) Range
MIC50
MIC90
S
I
R
0.5–4 0.25–2 0.125–2 1–4 16 to >128 >128 >128
1 1 1 2 128 >128 >128
2 2 2 4 >128 >128 >128
100
0
0
0.25–4 0.25–4 0.125–8 0.5–4 0.125–4 0.5–4 8 to >128 64 to >128 >128
1 1 1 1 0.5 1 64 >128 >128
2 2 2 2 2 4 >128 >128 >128
100
0
0
0.03–2 0.06–4 0.03–4 0.03–4 0.03–2 0.06–8 8 to >128 >128 >128
0.5 1 0.5 1 0.25 1 128 >128 >128
1 4 2 2 1 4 >128 >128 >128
100
0
0
2–64 4–64 4–64 4–64 1–64 4–64 32 to >128 64 to >128 >128
4 8 4 8 4 8 >128 >128 >128
32 32 32 32 16 32 >128 >128 >128
63.6
18.2
18.2
0.25–16 0.5–32 0.5–16 0.5–32 0.25–32 0.25–32 32 to >128 64 to >128 >128
0.5 1 0.5 1 1 1 64 >128 >128
16 32 16 32 32 32 >128 >128 >128
5b
1b
0
MIC, minimum inhibitory concentration; MIC50/90 , MIC that inhibits 50% and 90% of the bacterial isolates, respectively; S, susceptible; I, intermediate; R, resistant. a Clinical and Laboratory Standards Institute (CLSI) susceptibility breakpoint for metronidazole (≤8 mg/L) [8]. b The figure indicates the actual number of strains instead of percentage.
Technical Service, National Institutes for Food and Drug Control of China (Shanghai, China).
2.4. Determination of minimum inhibitory concentrations (MICs) MICs of antimicrobial agents were determined for the 375 strains of anaerobic isolates in accordance with the double dilution agar method as described by the Clinical and Laboratory Standards Institute (CLSI) [7]. An inoculum size of ca. 0.5 McFarland standard (1 × 108 CFU/mL) was inoculated with an A400 multipoint inoculator (Denley Instruments Ltd., Billingshurst, UK). The inoculated plates were dried at room temperature. The plates were then observed to measure the MIC after incubation under anaerobic conditions at 35 ◦ C for 48 h. QC strains were included in each run of tests. The test strains were cultured in parallel with controls both under anaerobic and oxytolerant conditions.
2.5. Determination of minimum bactericidal concentrations (MBCs) MBCs of levornidazole and metronidazole were determined by the microdilution method for 22 clinical B. fragilis strains as well as the reference strain B. fragilis ATCC 25285. The samples were also subjected to oxytolerant test. CFUs of the test strain were counted 48 h later. The MBC was defined as the concentration required to reduce the initial number of viable bacteria by ≥99.9%. 2.6. Data processing and interpretation of antimicrobial susceptibility testing results According to the CLSI breakpoints [8], the results of antibacterial susceptibility testing were considered valid only when the MIC of metronidazole for the QC strain B. fragilis ATCC 25285 was within the range of 0.25–1 mg/L as specified by the CLSI. SPSS for Windows
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Table 3 Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of levornidazole against 22 clinical strains of Bacteroides fragilis. Antimicrobial agent Levornidazole Metronidazole
MIC (mg/L)
MBC (mg/L)
Range
MIC50
MIC90
Range
MBC50
MBC90
0.25–2 0.25–2
0.5 0.5
1 1
1–4 1–8
2 2
4 4
MIC50/90 , MIC that inhibits 50% and 90% of the bacterial isolates, respectively; MBC50/90 , MBC for 50% and 90% of the isolates, respectively.
v.16.0. (SPSS Inc., Chicago, IL) was used to analyse the data, including the MIC range, MIC50 and MIC90 (MIC that inhibits 50% and 90% of the bacterial isolates, respectively) MBC range, and MBC50 and MBC90 (MBC for 50% and 90% of the isolates, respectively). 3. Results 3.1. Minimum inhibitory concentration results Levornidazole demonstrated good antibacterial activity against B. fragilis, other Bacteroides spp., C. difficile, C. perfringens and Peptostreptococcus magnus among the 375 strains of anaerobes. The MIC90 of levornidazole for the above-listed species was 0.5, 1, 0.25, 2 and 1 mg/L, respectively. Levornidazole showed comparable or slightly better activity against Gram-negative bacilli such as B. fragilis compared with metronidazole, ornidazole and dextrornidazole. The metabolites M1 and M4 of levornidazole also provided in vitro anti-anaerobic activity against the above-described anaerobes. However, the metabolites M2, M3 and M5 had hardly any anti-anaerobic activity (Table 2). Regarding B. fragilis, the MIC90 of levornidazole was 0.5 mg/L, suggesting that levornidazole was better than other drugs of the same class, especially better than dextrornidazole (MIC90 = 2 mg/L). Levornidazole had good activity against other Bacteroides spp. (MIC90 = 1 mg/L), similar to other comparators. Meanwhile, compared with Bacteroides spp., levornidazole showed poor activity against Prevotella spp. (MIC90 = 2 mg/L). Apart from one strain of B. fragilis (MIC = 16 mg/L) and one strain of Prevotella disiens (MIC = 64 mg/L), favourable anti-anaerobic activity was observed with levornidazole and its metabolites M1 and M4 against B. fragilis and other anaerobic Gram-negative bacilli, similar to metronidazole. Levornidazole demonstrated poor activity against anaerobic Gram-negative cocci. Among the 11 strains of Veillonella spp., 2 strains were resistant to metronidazole and 2 strains were metronidazole-intermediate. This finding should be further confirmed with a larger number of strains. Levornidazole and its metabolites M1 and M4 also displayed strong activity against C. difficile (levornidazole MIC90 = 0.25 mg/L) as well as favourable activity against C. perfringens (levornidazole MIC90 = 2 mg/L) of the Gram-positive bacilli. In addition, levornidazole and its metabolites M1 and M4 had good activity against the Gram-positive Peptostreptococcus (levornidazole MIC90 = 1 mg/L), which was similar to metronidazole, ornidazole and dextrornidazole (Table 2). The metabolites M2, M3 and M5 of levornidazole only showed weak or almost no activity against Gram-negative anaerobes. 3.2. Minimum bactericidal concentration results Both levornidazole and metronidazole had the same MIC50 (0.5 mg/L), MIC90 (1 mg/L), MBC50 (2 mg/L) and MBC90 (4 mg/L) against the 22 clinical strains of B. fragilis (Table 3). Both MBC50 /MIC50 and MBC90 /MIC90 ratios were 4. The results indicated that levornidazole and metronidazole had similar good bactericidal activity against B. fragilis.
4. Discussion In this study, the in vitro susceptibility of 375 clinical strains of anaerobes was tested with levornidazole and relevant comparators. The results indicated that the anti-anaerobic activity of levornidazole was comparable with or slightly better than metronidazole, ornidazole and dextrornidazole. Levornidazole showed slightly higher activity against B. fragilis than other drugs of the same class, especially higher than dextrornidazole. It is therefore necessary to test the activity of each ornidazole enantiomer separately [9,10]. Levornidazole also displayed a similar broad anti-anaerobic spectrum as ornidazole and metronidazole, covering Bacteroides spp. (including B. fragilis), Fusobacterium spp., Clostridium spp. (including C. perfringens), some Eubacterium spp. (E. lentum) and Peptostreptococcus spp. [11]. The in vitro pharmacodynamic studies of levornidazole against 72 strains of C. difficile showed slightly stronger antibacterial activity (MIC90 = 0.25 mg/L) than metronidazole. Currently, phase 1 clinical trials have been completed for levornidazole tablet. We can expect its use in the future just like metronidazole to treat anaerobic infections [12]. In the hypoxic and anaerobic environment, levornidazole can penetrate into the cells of susceptible micro-organisms and the nitro group is readily oxidised to an amino group, which makes the DNA spiral structure break or blocks its transcription and replication in vivo, and finally causes the death of bacteria. This study indicated that levornidazole had a favourable anti-anaerobic effect. In addition, the anti-anaerobic MIC50 and MIC90 values of the levornidazole metabolites M1 and M4 were similar to or slightly lower than the corresponding values of levornidazole and metronidazole. Both metabolites had in vitro anti-anaerobic activity, especially M1. Based on the structure, M1 was derived from the hydroxyl oxidation of levornidazole in the 2-methyl group. The activity of M1 was similar to or slightly stronger than levornidazole, suggesting hydroxyl displacement of the 2-methyl group had little effect on the activity of levornidazole. M4, the hydrolytic metabolite of levornidazole, was formed by hydrolysis of chlorine in the side chain. The activity of M4 was slightly lower than M1 and levornidazole, indicating that a chlorine atom in the side chain has an effect on the activity of levornidazole to some extent. The nitro group of M2 was hard to oxidise owing to complete destruction of side chains. The MIC90 value of M2 was generally 64 mg/L for most anaerobes. This suggests that the side chain (3-chloro-2-hydroxypropyl) may be essential for anti-anaerobic activity. Hydrolytic cleavage of the imidazole ring, presumably after reduction of the nitro group, gives rise to M3 and M5. Both M3 and M5 were completely inactivated. This proves that the basic skeleton of the imidazole ring is also an essential group for antianaerobic activity. The results therefore support that the side chain (3-chloro-2-hydroxypropyl) and the imidazole ring are probably the essential groups for the anti-anaerobic activity of levornidazole. The results of levornidazole clinical trials have also shown that levornidazole has similar efficacy to ornidazole in the treatment of intra-abdominal, oral and pelvic anaerobic infections. However,
J. Hu et al. / International Journal of Antimicrobial Agents 44 (2014) 514–519
levornidazole is associated with less adverse events than ornidazole [13–15]. Furthermore, the adverse events of ornidazole were significantly fewer than metronidazole [16,17]. More recent studies also demonstrated that the central nervous system toxicity of ornidazole was primarily associated with dextrornidazole, which can inhibit Na+ , K+ -ATPase, Ca2+ -ATPase and the key enzyme succinate dehydrogenase of the respiratory chain [18]. In summary, the anti-anaerobic activity of levornidazole is similar to or slightly higher than metronidazole, ornidazole and dextrornidazole. All these agents have good in vitro activity against most species of anaerobes. Anti-anaerobic activity is seen with levornidazole metabolites M1 and M4, but not M2, M3 or M5. Levornidazole and metronidazole have similar bactericidal activity against B. fragilis. As a novel nitroimidazole derivative, levornidazole is safer than ornidazole while maintaining the same efficacy, which provides a better option for clinicians to prevent and manage anaerobic infections. Funding: This work was supported by the New Drug Creation and Manufacturing Programme of the Ministry of Science and Technology of China [2012ZX09303004-001] and the New Drug Creation and Manufacturing Programme of the Ministry of Science and Technology of China for a phase 4 clinical study of levornidazole and sodium chloride injection [2012ZX09104101]. Competing interests: Nanjing Sanhome Pharmaceutical Co., Ltd. (Nanjing, China) provided the test materials used in this study. Ethical approval: Not required. References [1] Wang H, Hao K, Wang GJ. Determination of the resistant mutant-preventing concentrations of four antimicrobial agents including levornidazole and justification of clinical dosage. Prog Pharmaceut Sci 2012;6:277–81.
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[2] Zhao WZ, Jia DG, Lei ZB. Prevention and management of ornidazole-associated adverse reactions. Pharmaceut Clin Res 2011;1:72–5. [3] Sun JH, Wang ZQ, Gu XL, Mu R. Comparative study of l- and d-ornidazole in terms of central nervous system toxicity in mice. J Chin Pharm Univ 2008;4:343–7. [4] Zhang C, Teng ZJ, Li L. Application of levornidazole in preparation of antianaerobic drugs. Application number CN 1686117 A. Jiangsu, China; 2005, October. [5] Li HM, Mei YN, Xu D. In vitro antibacterial activity of ornidazole against 111 clinical strains of anaerobes. Acta Univer Med Nanjing (Nat Sci) 2008;11:1500–2. [6] Schwartz DE, Jordan JC, Vetter W, Oesterhelt G. Metabolic studies of ornidazole in the rat, in the dog and in man. Xenobiotica 1979;9:571–81. [7] Clinical and Laboratory Standards Institute. Methods for antimicrobial susceptibility testing of anaerobic bacteria; approved standard. Document M11-A8. 8th ed. Wayne, PA: CLSI; 2013. [8] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twenty-fourth informational supplement. Document M100-S24. Wayne, PA: CLSI; 2014. [9] Yin YJ, Zhang QM, Yuan Z. Separation of ornidazole enantiomers by highperformance liquid chromatography. Chin J New Drug 2010;13:1161–3. [10] Mu LL, Cheng ZN, Guo X, Luo X, Yu P. Investigation of chiral inversion and pharmacokinetics of laevo-ornidazole by high-performance liquid chromatography. J Clin Pharm Ther 2013;38:31–5. [11] Löfmark S, Edlund C, Nord CE. Metronidazole is still the drug of choice for treatment of anaerobic infections. Clin Infect Dis 2010;50(Suppl. 1):16–23. [12] Xie AY, Cheng ZN, Ouyang DS, Guo X, Huang ZJ, Zhang GX, et al. Phase I clinical trial of tolerance study for levornidazole tablets. Chin J Clin Pharm Ther 2011;16:412–6. [13] Chen S, Zhang HD, Wang YC, Lou DH, Tao XX. Efficacy of levornidazole sodium chloride injection in the treatment of oral anaerobic infections. Jiangsu Med J 2011;6:714–5. [14] Hao YF, Wang Q, Yuan XJ, Wang LP, Lu ZK. Comparative clinical study of levornidazole, ornidazole, and tinidazole in treatment of pelvic inflammatory diseases. Chin J Human Sex 2012;8:38–9. [15] Shen GM. Clinical efficacy evaluation of levornidazole in treatment of intraabdominal anaerobic infections. Chin Med Eng 2012;10:96. [16] Zhang H. Cost effectiveness analysis of ornidazole and metronidazole in treatment of gingivitis. Strait Pharm J 2009;21:96–7. [17] Pan X. Clinical observation on the therapeutic effect of ornidazole and metronidazole in treatment of gingivitis. Strait Pharm J 2011;23:152–3. [18] Chen K, Sun JH, Wang ZQ, Ji H. The inhibitory effects of ornidazole enantiomer on central nervous system in mice. J Chin Pharm Univ 2012;3:271–4.
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Evaluation of the in vitro activity of levornidazole, its metabolites and comparators against clinical anaerobic bacteria Jiali Hu a,b , Jing Zhang a,b,∗ , Shi Wu a,b , Demei Zhu a,b , Haihui Huang a,b , Yuancheng Chen a,b , Yang Yang a,b , Yingyuan Zhang a,b a b
Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China Key Laboratory of Clinical Pharmacology of Antibiotics, National Health and Family Planning Commission, Shanghai, China
a r t i c l e
i n f o
Article history: Received 12 June 2014 Accepted 29 July 2014 Keywords: Levornidazole Metabolite Anaerobe Minimum inhibitory concentration Minimum bactericidal concentration
a b s t r a c t This study evaluated the in vitro anti-anaerobic activity and spectrum of levornidazole, its metabolites and comparators against 375 clinical isolates of anaerobic bacteria, including Gram-negative bacilli (181 strains), Gram-negative cocci (11 strains), Gram-positive bacilli (139 strains) and Gram-positive cocci (44 strains), covering 34 species. Minimum inhibitory concentrations (MICs) of levornidazole, its five metabolites and three comparators against these anaerobic isolates were determined by the agar dilution method. Minimum bactericidal concentrations (MBCs) of levornidazole and metronidazole were measured against 22 strains of Bacteroides fragilis. Levornidazole showed good activity against B. fragilis, other Bacteroides spp., Clostridium difficile, Clostridium perfringens and Peptostreptococcus magnus, evidenced by MIC90 values of 0.5, 1, 0.25, 2 and 1 mg/L, respectively. The activity of levornidazole and the comparators was poor for Veillonella spp. Generally, levornidazole displayed activity similar to or slightly higher than that of metronidazole, ornidazole and dextrornidazole against anaerobic Gram-negative bacilli, Grampositive bacilli and Gram-positive cocci, especially B. fragilis. Favourable anti-anaerobic activity was also seen with levornidazole metabolites M1 and M4 but not M2, M3 or M5. For the 22 clinical B. fragilis strains, MBC50 and MBC90 values of levornidazole were 2 mg/L and 4 mg/L, respectively. Both MBC50 /MIC50 and MBC90 /MIC90 ratios of levornidazole were 4, similar to those of metronidazole. Levornidazole is an important anti-anaerobic option in clinical settings in terms of its potent and broad-spectrum in vitro activity, bactericidal property, and the anti-anaerobic activity of its metabolites M1 and M4. © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Levornidazole, the levo isomer of ornidazole, is a thirdgeneration nitroimidazole derivative newly developed after metronidazole, tinidazole and ornidazole. This novel antibiotic has shown good anti-anaerobic and antiprotozoal activity [1]. Levornidazole was approved by the China Food and Drug Administration in August 2009. The ornidazole products used in clinical settings are mostly racemic compounds composed of equal amounts of the l-isomer and d-isomer. In general, the adverse reactions of ornidazole are mainly stomach discomfort, dizziness, somnolence and other adverse reactions of the nervous system [2]. Dextrornidazole is the major component of ornidazole contributing to toxicity of the central nervous system [3]. Levornidazole is similar to or slightly better than racemic ornidazole in terms of
∗ Corresponding author at: Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai 200040, China. Tel.: +86 21 5288 8190; fax: +86 21 6248 4347. E-mail addresses: zhangj [email protected], zhangj [email protected] (J. Zhang).
pharmacokinetic properties [4]. Early in vitro pharmacodynamic studies also demonstrated that levornidazole has a comparable antimicrobial spectrum and more potent antimicrobial activity compared with ornidazole [5]. However, only a small sample of strains was tested in those studies without identifying each strain to specific species. Therefore, it is important to further investigate the in vitro antibacterial activity of levornidazole. Studies have shown that five phase I metabolites are derived from ornidazole in animals and human liver. Both 1-chloro-3(2-hydroxymethyl-5-nitro-1-imidazolyl)-2-propanol (M1) and 2methyl-5-nitroimidazole (M2) are the oxidative products of ornidazole, whilst 3-(2-methyl-5-nitro-1-imidazolyl)-1,2-propanediol (M4) is produced by hydrolysis of the chloride in the side chain of the imidazole ring. N-(3-chloro-2-hydroxypropyl) acetamide (M3) and acetamide (M5) are generated by cleavage of the imidazole ring following hydrolysis of ornidazole [6]. Nanjing Sanhome Pharmaceutical Co., Ltd. (Nanjing, China) has developed the five metabolites of ornidazole (Table 1). The metabolites and metabolic pathway of levornidazole are similar to ornidazole.
http://dx.doi.org/10.1016/j.ijantimicag.2014.07.027 0924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
J. Hu et al. / International Journal of Antimicrobial Agents 44 (2014) 514–519
515
Table 1 Levornidazole and its five metabolites. Drug
Structural formula
Molecular formula
Molecular weight (g/mol)
Levornidazole
C7 H10 ClN3 O3
220
M1
C7 H10 ClN3 O4
236
M2
C4 H5 N3 O2
127
M3
C5 H10 ClNO2
152
M4
C7 H11 N3 O4
201
M5
C2 H5 NO
This study aimed to compare the in vitro antibacterial activities of levornidazole, metronidazole, ornidazole, dextrornidazole, and the five metabolites (M1, M2, M3, M4 and M5) of levornidazole to further characterise the antibacterial activity and spectrum of levornidazole. The results will provide useful data for future clinical use and further research of levornidazole.
59
antibiotic-associated diarrhoea. The 76 strains of Bacteroides spp. and 45 strains of C. perfringens were isolated from patients with colonisation. Quality control (QC) strains included Bacteroides fragilis ATCC 25285, C. difficile ATCC 70057 and Prevotella melaninogenica ATCC 25845. All QC and reference strains were characterised by morphology as well as physiological and biochemical tests before they were used in this study.
2. Materials and methods 2.1. Bacterial strains A total of 375 strains of anaerobes were collected from patients who were treated in Huashan Hospital (Shanghai, China) during the period January 2006 to February 2013. These strains were mainly isolated from blood, faeces, secretions and other specimens and included Gram-negative bacilli (181 strains), Gram-positive bacilli (139 strains), Gram-positive cocci (44 strains) and Gram-negative cocci (11 strains), covering 34 species (Table 2). Overall, 200 of the strains were isolated from faeces, including 72 strains of Clostridium difficile, 76 strains of Bacteroides spp., 45 strains of Clostridium perfringens and 7 strains of other Clostridium spp. The 72 strains of C. difficile and 7 strains of other Clostridium spp. were considered to be from infections, mainly isolated from patients with
2.2. Culture media Brucella broth and Brucella agar were from Oxoid USA Inc. (Columbia, MD). Laked sheep blood (5% v/v) was provided by Shanghai Zhudi Germ-free Animal Blood Supplier (Shanghai, China). Haemin (5 mg/L) and vitamin K1 (1 mg/L) were products of Sigma Chemical Co. (St. Louis, MO).
2.3. Antimicrobial agents Levornidazole, ornidazole and dextrornidazole as well as levornidazole metabolites M1, M3, M4 and M5 were the products of Nanjing Sanhome Pharmaceutical Co. Ltd. Metronidazole and levornidazole metabolite M2 were provided by the Department of
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Table 2 Comparative in vitro activity of levornidazole against 375 strains of anaerobic bacteria. Organisms/compound
Bacteroides fragilis (46) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Bacteroides thetaiotaomicron (28) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Bacteroides ovatus (27) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Bacteroides uniformis (18) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Other Bacteroides spp. (39) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Prevotella spp. (23) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Clostridium difficile (72) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Clostridium perfringens (45) Levornidazole Ornidazole
Susceptibility (%)a
MIC (mg/L) Range
MIC50
MIC90
0.25–16 0.25–32 0.25–16 0.5–32 0.25–16 0.25–64 16 to >128 >128 >128
0.5 0.5 0.5 1 0.5 1 64 >128 >128
0.5 1 1 2 1 1 64 >128 >128
0.25–2 0.5–4 0.5–2 0.5–2 0.5–2 0.5–4 32 to >128 >128 >128
1 1 1 1 0.5 1 64 >128 >128
1 2 1 2 1 2 64 >128 >128
0.06–2 0.06–2 0.125–1 0.25–2 0.125–1 0.125–2 16 to >128 64 to >128 >128
0.5 1 1 2 1 1 16 >128 >128
1 2 1 2 1 2 16 >128 >128
0.125–2 0.25–2 0.25–2 0.25–4 0.25–4 0.25–4 32 to >128 >128 >128
1 1 1 2 1 1 64 >128 >128
2 2 2 2 1 2 64 >128 >128
0.125–2 0.25–2 0.25–2 0.5–2 0.25–2 0.25–4 32 to >128 64 to >128 >128
1 1 1 1 1 1 64 >128 >128
1 2 1 2 1 2 64 >128 >128
0.06–64 0.06–64 0.06–64 0.06 to >64 0.125–16 0.125 to >64 16 to >128 64 to >128 >128
1 1 0.5 1 1 1 64 >128 >128
2 2 2 2 1 2 >128 >128 >128
0.06–1 0.06–1 0.06–0.5 0.06–1 0.06–1 0.125–1 4–128 64 to >128 >128
0.125 0.125 0.25 0.25 0.25 1 16 >128 >128
0.25 0.25 0.5 0.25 0.25 1 32 >128 >128
2 1
2 2
0.5–2 0.5–4
S
I
R
97.8
2.2
0
100
0
0
100
0
0
100
0
0
100
0
0
0
4.3
0
0
95.7
100
J. Hu et al. / International Journal of Antimicrobial Agents 44 (2014) 514–519
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Table 2 (Continued) Organisms/compound
Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Other Clostridium spp. (16) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Peptostreptococcus spp. (44) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Veillonella spp. (11) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5 Eubacterium and Bifidobacterium (6) Levornidazole Ornidazole Metronidazole Dextrornidazole M1 M4 M2 M3 M5
Susceptibility (%)a
MIC (mg/L) Range
MIC50
MIC90
S
I
R
0.5–4 0.25–2 0.125–2 1–4 16 to >128 >128 >128
1 1 1 2 128 >128 >128
2 2 2 4 >128 >128 >128
100
0
0
0.25–4 0.25–4 0.125–8 0.5–4 0.125–4 0.5–4 8 to >128 64 to >128 >128
1 1 1 1 0.5 1 64 >128 >128
2 2 2 2 2 4 >128 >128 >128
100
0
0
0.03–2 0.06–4 0.03–4 0.03–4 0.03–2 0.06–8 8 to >128 >128 >128
0.5 1 0.5 1 0.25 1 128 >128 >128
1 4 2 2 1 4 >128 >128 >128
100
0
0
2–64 4–64 4–64 4–64 1–64 4–64 32 to >128 64 to >128 >128
4 8 4 8 4 8 >128 >128 >128
32 32 32 32 16 32 >128 >128 >128
63.6
18.2
18.2
0.25–16 0.5–32 0.5–16 0.5–32 0.25–32 0.25–32 32 to >128 64 to >128 >128
0.5 1 0.5 1 1 1 64 >128 >128
16 32 16 32 32 32 >128 >128 >128
5b
1b
0
MIC, minimum inhibitory concentration; MIC50/90 , MIC that inhibits 50% and 90% of the bacterial isolates, respectively; S, susceptible; I, intermediate; R, resistant. a Clinical and Laboratory Standards Institute (CLSI) susceptibility breakpoint for metronidazole (≤8 mg/L) [8]. b The figure indicates the actual number of strains instead of percentage.
Technical Service, National Institutes for Food and Drug Control of China (Shanghai, China).
2.4. Determination of minimum inhibitory concentrations (MICs) MICs of antimicrobial agents were determined for the 375 strains of anaerobic isolates in accordance with the double dilution agar method as described by the Clinical and Laboratory Standards Institute (CLSI) [7]. An inoculum size of ca. 0.5 McFarland standard (1 × 108 CFU/mL) was inoculated with an A400 multipoint inoculator (Denley Instruments Ltd., Billingshurst, UK). The inoculated plates were dried at room temperature. The plates were then observed to measure the MIC after incubation under anaerobic conditions at 35 ◦ C for 48 h. QC strains were included in each run of tests. The test strains were cultured in parallel with controls both under anaerobic and oxytolerant conditions.
2.5. Determination of minimum bactericidal concentrations (MBCs) MBCs of levornidazole and metronidazole were determined by the microdilution method for 22 clinical B. fragilis strains as well as the reference strain B. fragilis ATCC 25285. The samples were also subjected to oxytolerant test. CFUs of the test strain were counted 48 h later. The MBC was defined as the concentration required to reduce the initial number of viable bacteria by ≥99.9%. 2.6. Data processing and interpretation of antimicrobial susceptibility testing results According to the CLSI breakpoints [8], the results of antibacterial susceptibility testing were considered valid only when the MIC of metronidazole for the QC strain B. fragilis ATCC 25285 was within the range of 0.25–1 mg/L as specified by the CLSI. SPSS for Windows
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Table 3 Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of levornidazole against 22 clinical strains of Bacteroides fragilis. Antimicrobial agent Levornidazole Metronidazole
MIC (mg/L)
MBC (mg/L)
Range
MIC50
MIC90
Range
MBC50
MBC90
0.25–2 0.25–2
0.5 0.5
1 1
1–4 1–8
2 2
4 4
MIC50/90 , MIC that inhibits 50% and 90% of the bacterial isolates, respectively; MBC50/90 , MBC for 50% and 90% of the isolates, respectively.
v.16.0. (SPSS Inc., Chicago, IL) was used to analyse the data, including the MIC range, MIC50 and MIC90 (MIC that inhibits 50% and 90% of the bacterial isolates, respectively) MBC range, and MBC50 and MBC90 (MBC for 50% and 90% of the isolates, respectively). 3. Results 3.1. Minimum inhibitory concentration results Levornidazole demonstrated good antibacterial activity against B. fragilis, other Bacteroides spp., C. difficile, C. perfringens and Peptostreptococcus magnus among the 375 strains of anaerobes. The MIC90 of levornidazole for the above-listed species was 0.5, 1, 0.25, 2 and 1 mg/L, respectively. Levornidazole showed comparable or slightly better activity against Gram-negative bacilli such as B. fragilis compared with metronidazole, ornidazole and dextrornidazole. The metabolites M1 and M4 of levornidazole also provided in vitro anti-anaerobic activity against the above-described anaerobes. However, the metabolites M2, M3 and M5 had hardly any anti-anaerobic activity (Table 2). Regarding B. fragilis, the MIC90 of levornidazole was 0.5 mg/L, suggesting that levornidazole was better than other drugs of the same class, especially better than dextrornidazole (MIC90 = 2 mg/L). Levornidazole had good activity against other Bacteroides spp. (MIC90 = 1 mg/L), similar to other comparators. Meanwhile, compared with Bacteroides spp., levornidazole showed poor activity against Prevotella spp. (MIC90 = 2 mg/L). Apart from one strain of B. fragilis (MIC = 16 mg/L) and one strain of Prevotella disiens (MIC = 64 mg/L), favourable anti-anaerobic activity was observed with levornidazole and its metabolites M1 and M4 against B. fragilis and other anaerobic Gram-negative bacilli, similar to metronidazole. Levornidazole demonstrated poor activity against anaerobic Gram-negative cocci. Among the 11 strains of Veillonella spp., 2 strains were resistant to metronidazole and 2 strains were metronidazole-intermediate. This finding should be further confirmed with a larger number of strains. Levornidazole and its metabolites M1 and M4 also displayed strong activity against C. difficile (levornidazole MIC90 = 0.25 mg/L) as well as favourable activity against C. perfringens (levornidazole MIC90 = 2 mg/L) of the Gram-positive bacilli. In addition, levornidazole and its metabolites M1 and M4 had good activity against the Gram-positive Peptostreptococcus (levornidazole MIC90 = 1 mg/L), which was similar to metronidazole, ornidazole and dextrornidazole (Table 2). The metabolites M2, M3 and M5 of levornidazole only showed weak or almost no activity against Gram-negative anaerobes. 3.2. Minimum bactericidal concentration results Both levornidazole and metronidazole had the same MIC50 (0.5 mg/L), MIC90 (1 mg/L), MBC50 (2 mg/L) and MBC90 (4 mg/L) against the 22 clinical strains of B. fragilis (Table 3). Both MBC50 /MIC50 and MBC90 /MIC90 ratios were 4. The results indicated that levornidazole and metronidazole had similar good bactericidal activity against B. fragilis.
4. Discussion In this study, the in vitro susceptibility of 375 clinical strains of anaerobes was tested with levornidazole and relevant comparators. The results indicated that the anti-anaerobic activity of levornidazole was comparable with or slightly better than metronidazole, ornidazole and dextrornidazole. Levornidazole showed slightly higher activity against B. fragilis than other drugs of the same class, especially higher than dextrornidazole. It is therefore necessary to test the activity of each ornidazole enantiomer separately [9,10]. Levornidazole also displayed a similar broad anti-anaerobic spectrum as ornidazole and metronidazole, covering Bacteroides spp. (including B. fragilis), Fusobacterium spp., Clostridium spp. (including C. perfringens), some Eubacterium spp. (E. lentum) and Peptostreptococcus spp. [11]. The in vitro pharmacodynamic studies of levornidazole against 72 strains of C. difficile showed slightly stronger antibacterial activity (MIC90 = 0.25 mg/L) than metronidazole. Currently, phase 1 clinical trials have been completed for levornidazole tablet. We can expect its use in the future just like metronidazole to treat anaerobic infections [12]. In the hypoxic and anaerobic environment, levornidazole can penetrate into the cells of susceptible micro-organisms and the nitro group is readily oxidised to an amino group, which makes the DNA spiral structure break or blocks its transcription and replication in vivo, and finally causes the death of bacteria. This study indicated that levornidazole had a favourable anti-anaerobic effect. In addition, the anti-anaerobic MIC50 and MIC90 values of the levornidazole metabolites M1 and M4 were similar to or slightly lower than the corresponding values of levornidazole and metronidazole. Both metabolites had in vitro anti-anaerobic activity, especially M1. Based on the structure, M1 was derived from the hydroxyl oxidation of levornidazole in the 2-methyl group. The activity of M1 was similar to or slightly stronger than levornidazole, suggesting hydroxyl displacement of the 2-methyl group had little effect on the activity of levornidazole. M4, the hydrolytic metabolite of levornidazole, was formed by hydrolysis of chlorine in the side chain. The activity of M4 was slightly lower than M1 and levornidazole, indicating that a chlorine atom in the side chain has an effect on the activity of levornidazole to some extent. The nitro group of M2 was hard to oxidise owing to complete destruction of side chains. The MIC90 value of M2 was generally 64 mg/L for most anaerobes. This suggests that the side chain (3-chloro-2-hydroxypropyl) may be essential for anti-anaerobic activity. Hydrolytic cleavage of the imidazole ring, presumably after reduction of the nitro group, gives rise to M3 and M5. Both M3 and M5 were completely inactivated. This proves that the basic skeleton of the imidazole ring is also an essential group for antianaerobic activity. The results therefore support that the side chain (3-chloro-2-hydroxypropyl) and the imidazole ring are probably the essential groups for the anti-anaerobic activity of levornidazole. The results of levornidazole clinical trials have also shown that levornidazole has similar efficacy to ornidazole in the treatment of intra-abdominal, oral and pelvic anaerobic infections. However,
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levornidazole is associated with less adverse events than ornidazole [13–15]. Furthermore, the adverse events of ornidazole were significantly fewer than metronidazole [16,17]. More recent studies also demonstrated that the central nervous system toxicity of ornidazole was primarily associated with dextrornidazole, which can inhibit Na+ , K+ -ATPase, Ca2+ -ATPase and the key enzyme succinate dehydrogenase of the respiratory chain [18]. In summary, the anti-anaerobic activity of levornidazole is similar to or slightly higher than metronidazole, ornidazole and dextrornidazole. All these agents have good in vitro activity against most species of anaerobes. Anti-anaerobic activity is seen with levornidazole metabolites M1 and M4, but not M2, M3 or M5. Levornidazole and metronidazole have similar bactericidal activity against B. fragilis. As a novel nitroimidazole derivative, levornidazole is safer than ornidazole while maintaining the same efficacy, which provides a better option for clinicians to prevent and manage anaerobic infections. Funding: This work was supported by the New Drug Creation and Manufacturing Programme of the Ministry of Science and Technology of China [2012ZX09303004-001] and the New Drug Creation and Manufacturing Programme of the Ministry of Science and Technology of China for a phase 4 clinical study of levornidazole and sodium chloride injection [2012ZX09104101]. Competing interests: Nanjing Sanhome Pharmaceutical Co., Ltd. (Nanjing, China) provided the test materials used in this study. Ethical approval: Not required. References [1] Wang H, Hao K, Wang GJ. Determination of the resistant mutant-preventing concentrations of four antimicrobial agents including levornidazole and justification of clinical dosage. Prog Pharmaceut Sci 2012;6:277–81.
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