In vivo antibacterial activity of nemonoxacin, a novel non-fluorinatedquinolone

Cong-Ran Li1†, Yi Li1†, Guo-Qing Li1, Xin-Yi Yang1, Wei-Xin Zhang1, Ren-Hui Lou1, Jing-Fang Liu1, Min Yuan1,Philip Huang2, Shan Cen 1, Li-Yan Yu1, Li-Xun Zhao1, Jian-Dong Jiang1‡ and Xue-Fu You1*‡

1Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;2TaiGen Biopharmaceuticals Co. Ltd, Beijing 100020, China

*Corresponding author. Tel: +86-10-63165290; Fax: +86-10-63017302; E-mail: xuefuyou@hotmail.com†C.-R. L. and Y. L. contributed equally to this work.

‡J.-D. J. and X.-F. Y. contributed equally to this work.

Received 30 March 2010; returned 3 June 2010; revised 26 July 2010; accepted 12 August 2010

Objectives: To evaluate the in vivo antibacterial efficacy of nemonoxacin, a novel C8-methoxy non-fluorinatedquinolone in murine systemic and local infection models.

Methods: The efficacy of nemonoxacin in systemic infections was evaluated in mouse peritonitis models usingisolates of methicillin-susceptible Staphylococcus aureus (MSSA, n¼1), methicillin-resistant S. aureus (MRSA,n¼1), methicillin- and levofloxacin-resistant Staphylococcus capitis (levofloxacin-resistant MRSC, n¼1),penicillin-intermediate Streptococcus pneumoniae (PISP, n¼1), penicillin-resistant S. pneumoniae (PRSP,n¼2), Enterococcus faecalis (n¼2, including 1 vancomycin-resistant Enterococcus, VRE) and Escherichia coli(n¼3). The local infections included mouse pulmonary infections caused by PRSP (n¼1), Klebsiella pneumoniae(n¼1) and mouse ascending urinary tract infection caused by E. coli (n¼1).

Results: In the mouse systemic infection model, nemonoxacin demonstrated potent activity against MSSA(ED50¼2.08 mg/kg), MRSA (ED50¼2.59 mg/kg), levofloxacin-resistant MRSC (ED50¼2.52 mg/kg), PISP(ED50¼5.47 mg/kg), PRSP (ED50¼3.68–5.28 mg/kg) and E. coli (ED50¼3.13–5.28 mg/kg), and moderateactivity towards E. faecalis infection (ED50¼8.48–15.16 mg/kg). The therapeutic efficacy of nemonoxacinwas significantly higher (P,0.01) than that of levofloxacin in infections caused by Gram-positive isolates(MSSA, MRSA, levofloxacin-resistant MRSC, PISP, PRSP and E. faecalis), but less potent than that of levofloxacinagainst E. coli infection (P,0.01). Nemonoxacin in vivo efficacy results with Gram-positive isolates (2- to 5-foldED50 advantage over levofloxacin) are consistent with the MIC data (4- to 16-fold MIC advantage of nemonox-acin over levofloxacin). In the mouse pulmonary infection model, nemonoxacin showed potent activity towardsPRSP (higher than levofloxacin) and K. pneumoniae (lower than levofloxacin) infections. In the mouse ascendingurinary tract infection model, nemonoxacin exhibited potent activity against E. coli infection (lower than levo-floxacin).

Conclusions: The results validated the potent efficacy of nemonoxacin in vivo. The higher efficacy of nemonox-acin than of levofloxacin towards infections caused by Gram-positive cocci (especially MRSA, levofloxacin-resistant MRSC, PRSP and VRE) warrants investigation of its clinical use.

Keywords: systemic infections, local infections, pulmonary infections, ascending urinary tract infections

IntroductionNemonoxacin (TG-873870) (TaiGen Biopharmaceuticals Co. Ltd,Beijing, China) is a novel C8-methoxy non-fluorinated quinolone.The C8-methoxy substituents have been associated with animproved spectrum of activity, including increased activityagainst Gram-positive cocci, and reduced mutant selection.1,2

The removal of the fluorine residue could reduce the incidenceof toxic side effects.3 In vitro activity studies of nemonoxacindemonstrated that it had higher activity than that of levofloxacinand moxifloxacin against Gram-positive cocci, includingmethicillin-resistant Staphylococcus aureus, Staphylococcus epi-dermidis, Streptococcus pneumoniae and Enterococcus faecalis,while its activity against Gram-negative bacilli was similar to

# The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.For Permissions, please e-mail: journals.permissions@oxfordjournals.org

J Antimicrob Chemother 2010; 65: 2411–2415doi:10.1093/jac/dkq341 Advance Access publication 21 September 2010

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that of levofloxacin and moxifloxacin.4,5 In this study, the effi-cacy of nemonoxacin was evaluated in vivo in mouse systemicand local infection models using levofloxacin as a comparator.

Materials and methods

Antimicrobial agentsNemonoxacin (TG-873870) in the form of malate salt (TG-875649)(C24H32N3O9.5, mol. wt 514.53, purity 99.2%) was obtained from TaiGenBiopharmaceuticals Co. Ltd (Beijing, China). Levofloxacin (purity 97.3%)was purchased from the National Pharmaceutical and Biological ProductsControl Institute (Beijing, China). The compounds were dissolved in sterilewater and the concentrations were calculated based on their activemoiety.

MicroorganismsS. aureus ATCC 29213 (methicillin-susceptible S. aureus, MSSA),S. pneumoniae ATCC 49619 (penicillin-intermediate S. pneumoniae,PISP), E. faecalis ATCC 29212 and Escherichia coli ATCC 25922 werestandard isolates from ATCC (Manassas, VA, USA). S. aureus 0705(MRSA), Staphylococcus capitis 0687 (methicillin- and levofloxacin-resistant S. capitis, levofloxacin-resistant MRSC), S. pneumoniae 0518(penicillin-resistant S. pneumoniae, PRSP), S. pneumoniae 0613 (PRSP),E. faecalis 4041 (vancomycin-resistant Enterococcus, VRE), Klebsiellapneumoniae 0607, E. coli 0635, E. coli 0638 and E. coli 0526 were clinicalisolates. Bacteria were stored at 2808C until use.

Laboratory animalsCD-1 ICR mice were purchased from Vital River Laboratories (Beijing,China) and acclimatized for 1 day prior to use. All animals were housedin cages (five animals of the same sex per cage) under constant temp-erature (228C) and humidity, and had free access to food and waterthroughout the study, except for the mouse ascending urinary tractinfection model during the water restriction period. All studies withanimals were approved by the Animal Research Committee of the Insti-tute of Medicinal Biotechnology (Beijing, China).

Mouse systemic infection modelThe in vivo efficacy of nemonoxacin against mouse systemic infectionsversus that of levofloxacin was evaluated with two strains of S. aureus,one strain of S. capitis, three strains of S. pneumoniae, two strains ofE. faecalis and three strains of E. coli (see Table 1). The experiment wascarried out according to a previously described method, with modifi-cations.6 For each isolate infection, CD-1 ICR mice (body weight, 18–22 g) were allocated randomly into 11 groups, with 5 groups (5 doses)for each compound and 1 control group (10 mice per group, 5 malesand 5 females). The mice were infected intraperitoneally with 0.5 mL ofa bacterial suspension in 5% mucin (100% minimum lethal dose). Nemo-noxacin and levofloxacin (saline for the control group) were administeredorally 15 min and 6 h after infection. The dose ranges of nemonoxacinwere 1.6–4.0 mg/kg for S. aureus and S. capitis infections, 2.4–10.0 mg/kg for S. pneumoniae infections, 5.0–22.6 mg/kg for E. faecalisinfections, and 1.6–10.0 mg/kg for E. coli infections. Deaths in eachgroup were recorded daily for 7 days, and the 50% effective dose(ED50) and 95% confidence limits were calculated by probit analysis.7

Mouse pulmonary infection modelThe therapeutic efficacies of nemonoxacin and levofloxacin in mousepulmonary infections caused by S. pneumoniae 0613 (PRSP) and

K. pneumoniae 0607 were evaluated with CD-1 ICR mice (body weight,18–22 g; 10 mice per group, 5 males and 5 females) according to pub-lished methods, with modifications.8,9 Four different doses of nemonoxacinor levofloxacin were used in the experiments for each isolate infection(see Table 2). For S. pneumoniae 0613 infection, the mice were first sub-jected to cyclophosphamide immunocompromise (150 mg/kg/day for3 days).10,11 Then, the mice were anaesthetized with ether and infectedintranasally using 50 mL of fresh bacterial suspension in saline (challengedose, 4.0×107 cfu/mouse). Nemonoxacin or levofloxacin was administeredorally 6, 12 and 24 h after infection. The mice were sacrificed 28 h afterinfection and the lungs were removed under sterile conditions. The lungswere homogenized in saline (0.1 g of tissue to a final volume of 1 mL),and viable colony counts were determined after blood agar plates werespread with 0.1 mL of properly diluted homogenates and incubated at358C for 48 h.

For K. pneumoniae 0607 infection, the mice were infected withoutimmunocompromise. Briefly, the mice were anaesthetized with etherand infected intranasally using 50 mL of bacterial suspension in saline(challenge dose, 1.0×107 cfu/mouse). Nemonoxacin or levofloxacin wasadministered orally 6, 12 and 24 h after infection. The mice weresacrificed 28 h after infection and the lungs were removed under sterileconditions. The lungs were homogenized in saline (0.1 g of tissue to afinal volume of 1 mL), and viable colony counts were determined afternutrient agar plates were spread with 0.1 mL of properly diluted hom-ogenates and incubated at 358C for 48 h. The lower detection limitwas 2 log cfu/g, which corresponded to the weakest dilution of lungtissue of 1021 that avoided significant drug carryover. The statisticalsignificance was determined using SPSS 12.0.

Mouse ascending urinary tract infection modelThe therapeutic efficacies of nemonoxacin and levofloxacin in mouseascending urinary tract infection caused by E. coli 0526 were evaluatedwith CD-1 ICR mice (body weight, 18–22 g; 10 mice per group, 5 malesand 5 females), as previously described with modifications.12,13 Fourdifferent doses of nemonoxacin or levofloxacin were used (see Table 3).The mice were subjected to water restriction for 24 h prior to infectionand 6 h after infection. Under pentobarbital sodium (70 mg/kg) anaes-thesia, the mice were subjected to bladder exposure by surgery andinfected subsequently by injection of 50 mL of bacterial suspension insaline (challenge dose, 3.0×107 cfu/mouse) into the bladders. Nemonox-acin or levofloxacin was administered orally 6, 12 and 24 h after infec-tion. The mice were sacrificed 28 h after infection and the kidneys wereremoved under sterile conditions. The kidneys were homogenized insaline (0.1 g of tissue to a final volume of 1 mL), and viable colonycounts were determined after nutrient agar plates were spread with0.1 mL of properly diluted homogenates and incubated at 358C for 48 h.The lower detection limit was 2 log cfu/g, which corresponded to theweakest dilution of kidney tissue of 1021 that avoided significant drugcarryover. The statistical significance was determined using SPSS 12.0.

Results

Mouse systemic infection model

The MICs, ED50s and 95% confidence limits of nemonoxacinand levofloxacin for the tested strains are shown in Table 1.Nemonoxacin showed potent and broad-spectrum in vivoactivity against both Gram-positive (S. aureus, S. capitis, S. pneu-moniae and E. faecalis) and Gram-negative (E. coli) isolates. TheED50s of nemonoxacin against S. aureus ATCC 29213 (MSSA),S. aureus 0705 (MRSA) and S. capitis 0687 (levofloxacin-resistantMRSC) were 2.08, 2.59 and 2.52 mg/kg, respectively, which weresignificantly lower (P,0.01) than the corresponding ED50s of

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levofloxacin (5.02, 8.45 and 4.32 mg/kg, respectively). The thera-peutic efficacies of nemonoxacin against S. pneumoniae ATCC49619 (PISP), S. pneumoniae 0518 (PRSP) and S. pneumoniae0613 (PRSP) infections (ED50s of 5.47, 3.68 and 5.28 mg/kg,respectively) were �3- to 5-fold higher (P,0.01) than those oflevofloxacin (ED50s of 19.14, 19.82 and 22.01 mg/kg, respect-ively). The activities of nemonoxacin against E. faecalis infection

were lower in comparison with its activities against S. aureus,S. capitis and S. pneumoniae infections. The ED50s of nemonoxa-cin against E. faecalis ATCC 29212 and E. faecalis 4041 (VRE)infections were 15.16 and 8.48 mg/kg, respectively, which weresignificantly lower (P,0.01) than those of levofloxacin (ED50sof 26.89 and 17.47 mg/kg, respectively). Nemonoxacin alsoshowed potent activity against Gram-negative isolates, even

Table 1. In vivo efficacies of nemonoxacin and the reference compound in mouse systemic infection

OrganismChallenge dose

(cfu/mouse)

Nemonoxacin Levofloxacin

P value of nemonoxacinversus levofloxacin

MICa

(mg/L)ED50 (95% confidence

limit) (mg/kg)MICa

(mg/L)ED50 (95% confidence

limit) (mg/kg)

S. aureus ATCC 29213 (MSSA) 2.1×105 0.03 2.08 (1.68–2.59) 0.125 5.02 (4.02–6.25) ,0.01S. aureus 0705 (MRSA) 2.5×105 0.03 2.59 (2.24–2.98) 0.25 8.45 (7.60–9.41) ,0.01S. capitis 0687

(levofloxacin-resistantMRSC)b

8.8×104 0.5 2.52 (2.17–2.93) 8 4.32 (3.96–4.72) ,0.01

S. pneumoniae ATCC 49619(PISP)

8.8×105 0.125 5.47 (4.55–6.57) 1 19.14 (16.98–21.57) ,0.01

S. pneumoniae 0518 (PRSP) 2.2×105 0.25 3.68 (3.11–4.36) 2 19.82 (17.57–22.37) ,0.01S. pneumoniae 0613 (PRSP) 1.8×105 0.25 5.28 (4.21–6.62) 2 22.01 (19.50–24.83) ,0.01E. faecalis ATCC 29212 3.3×106 0.03 15.16 (12.64–18.17) 0.5 26.89 (23.25–31.09) ,0.01E. faecalis 4041 (VRE) 1.1×108 0.06 8.48 (6.88–10.45) 1 17.47 (14.18–21.53) ,0.01E. coli ATCC 25922 6.0×105 0.03 3.13 (2.40–4.10) 0.015 0.68 (0.56–0.83) ,0.01E. coli 0635 1.5×105 0.06 3.38 (2.85–4.01) 0.015 0.97 (0.85–1.12) ,0.01E. coli 0638 1.0×105 0.06 5.28 (4.58–6.09) 0.015 0.84 (0.73–0.96) ,0.01

aMICs were determined by the agar dilution method according to CLSI recommendations.15

bLevofloxacin resistance was defined in accordance with the CLSI standard.16

Table 2. In vivo efficacies of nemonoxacin and the reference compound in mouse pulmonary infection

Organism Antibiotic MIC (mg/L) Dose (mg/kg) Log cfu/g (mean+SD, n¼10)

P value versus

control levofloxacin at the same dose

S. pneumoniae 0613 (PRSP) nemonoxacin 0.25 20 2.93+1.67 ,0.05 .0.0510 4.01+2.44 ,0.05 .0.05

5 4.81+2.14 ,0.05 .0.052.5 6.34+1.06 .0.05

levofloxacin 2 40 3.76+1.73 ,0.0520 4.34+1.96 ,0.0510 4.76+2.11 ,0.05

5 6.29+1.10 .0.05control 7.07+0.77

K. pneumoniae 0607 nemonoxacin 0.06 80 3.13+0.65 ,0.0540 3.63+0.54 ,0.0520 4.49+0.79 ,0.05 ,0.0510 5.88+0.79 .0.05 ,0.05

levofloxacin 0.03 20 3.02+0.33 ,0.0510 3.33+0.56 ,0.05

5 3.83+0.56 ,0.052.5 4.45+0.53 ,0.05

control 8.10+1.29

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though the efficacy was lower than that of levofloxacin. TheED50s of nemonoxacin against E. coli ATCC 25922, E. coli 0635and E. coli 0638 infections were 3.13, 3.38 and 5.28 mg/kg,respectively, which were 3- to 6-fold higher than those oflevofloxacin (P,0.01).

Mouse pulmonary infection model

The results of mouse pulmonary infections treated with nemo-noxacin and the reference compound (levofloxacin) are shownin Table 2. Both nemonoxacin and levofloxacin demonstratedpotent activities towards S. pneumoniae 0613 (PRSP) andK. pneumoniae 0607 infections, with most of the tested groupshaving significantly (P,0.05) decreased colony counts in com-parison with the control group. In comparison with levofloxacinat the same dose levels, nemonoxacin exhibited higher activityagainst S. pneumoniae 0613 infection [log cfu/g values of 2.93versus 4.34 at the 20 mg/kg dose level (P.0.05), 4.01 versus4.76 at the 10 mg/kg dose level (P.0.05) and 4.81 versus 6.29at the 5 mg/kg dose level (P.0.05) for nemonoxacin and levo-floxacin, respectively] and lower activity towards K. pneumoniae0607 infection [log cfu/g values of 4.49 versus 3.02 at the20 mg/kg dose level (P,0.05) and 5.88 versus 3.33 at the10 mg/kg dose level (P,0.05) for nemonoxacin and levofloxacin,respectively] (Table 2).

Mouse ascending urinary tract infection model

The results of mouse ascending urinary tract infection caused byE. coli 0526 are shown in Table 3. Nemonoxacin and levofloxacinshowed therapeutic efficacies, as all the tested groups of the twocompounds showed significant decreases (P,0.05) in kidneycolony counts in comparison with the control group. The efficacyof nemonoxacin was lower than that of levofloxacin, demon-strated by the higher colony counts of the nemonoxacingroups than those of the levofloxacin groups at the same dose

levels, i.e. log cfu/g values of 4.60 versus 3.47 at the 4 mg/kgdose level (P,0.05) and 4.85 versus 4.00 at the 2 mg/kg doselevel (P.0.05).

DiscussionThe in vivo activity of nemonoxacin was evaluated in this studyusing animal models of systemic and local infections. Theresults from the mouse systemic infection model (Table 1)mirrored the results of in vitro studies.4,5 Nemonoxacin demon-strated potent broad-spectrum antibacterial activity againstboth Gram-positive (S. aureus, S. capitis, S. pneumoniae andE. faecalis, higher than levofloxacin) and Gram-negative (E. coli,lower than levofloxacin) isolates. Nemonoxacin maintainedpotent antibacterial activity against the tested antibiotic-resistant isolates (i.e. MRSA, levofloxacin-resistant MRSC, PRSPand VRE, see Table 1), which is of great importance consideringthe problems these isolates have caused in the clinical setting.Of note is that nemonoxacin showed high activity towardslevofloxacin-resistant MRSC S. capitis 0687, which suggests thatnemonoxacin may be useful against infections caused bylevofloxacin-resistant isolates. The mouse local infectionmodels of pulmonary infection (Table 2) and ascending urinarytract infection (Table 3) further verified the aforementioned con-clusions. Nemonoxacin demonstrated potent efficacies in mousepulmonary infections caused by S. pneumoniae 0613 andK. pneumoniae 0607, and in mouse ascending urinary tractinfection caused by E. coli 0526.

The efficacy of nemonoxacin in mouse pulmonary infectioncaused by S. pneumoniae 0613 was more potent than that oflevofloxacin, although such differences were not significant. Ina prior study of nemonoxacin in mouse pulmonary infectioncaused by S. pneumoniae, the bacterial log killing effect of nemo-noxacin was two to six times more potent than that of moxiflox-acin at doses of 6.25–50 mg/kg.14 Taken together, these datasuggested that nemonoxacin was potentially at least as potentas the quinolones in use (levofloxacin, moxifloxacin) for bacterialpneumonia caused by S. pneumoniae.

In conclusion, this study demonstrated nemonoxacin to be apotent agent with broad-spectrum antibacterial activity and sig-nificant activity against antibiotic-resistant Gram-positive iso-lates in vivo. These results support further clinical studies usingnemonoxacin.

AcknowledgementsWe thank Ching-Hung Gene Hsu and David T. Chung at TaiGenBiopharmaceuticals Co. Ltd for reviewing the manuscript prior tosubmission.

FundingThis work was supported by the National Natural Science Foundation ofChina (30472058 and 30672502), the Beijing Natural Science Foundation(7062064) and the ‘11th Five-Year Plan’ of the Ministry of Sciences andTechnology, People’s Republic of China (2009ZX09303-005 and2008ZX09305-001).

Table 3. In vivo efficacies of nemonoxacin and the reference compoundin mouse ascending urinary tract infection caused by E. coli 0526

Dose (mg/kg)Log cfu/g

(mean+SD, n¼10)

P value versus

controllevofloxacin atthe same dose

Nemonoxacin (MIC 0.06 mg/L)16 3.53+0.61 ,0.058 3.59+0.89 ,0.054 4.60+0.75 ,0.05 ,0.052 4.85+0.69 ,0.05 .0.05

Levofloxacin (MIC 0.03 mg/L)4 3.47+0.76 ,0.052 4.00+0.72 ,0.051 4.86+1.21 ,0.050.5 4.75+1.21 ,0.05

Control 6.49+1.25

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Transparency declarationsP. H. is currently employed by and has stocks and options in TaiGenBiopharmaceutials Co. Ltd. All other authors: none to declare.

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