Topoisomerase IV; DNA gyrase
Bacterial DNA gyrase [1][2][3]
Bacterial topoisomerase IV [1][2][3]

Clinafloxacin shows efficacy against S. pneumonia in parC-gyrA mutants, with a minimum inhibitory concentration (MIC) of 1 μg/ml[2].
Clinafloxacin hydrochloride possesses antibacterial properties against S. aureus mutant strains with altered targets. It has MIC values of 0.016 µg/ml, 0.063 µg/ml, and 0.915 µg/ml against wild type S. aureus, gyrA mutant S. aureus, and gyrA mutant S. aureus, respectively[3].

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Against Staphylococcus aureus (including susceptible strains), Clinafloxacin (AM-1091; CI-960) exhibited potent concentration-dependent antibacterial activity, with MIC values ranging from 0.03 to 0.125 μg/mL. It showed higher affinity for DNA gyrase than topoisomerase IV in S. aureus [1]
- Against Proteus mirabilis (urinary tract infection isolates), the drug displayed antibacterial activity with MIC values of 0.06-0.25 μg/mL for ciprofloxacin-susceptible strains. For quinolone-resistant strains carrying gyrA or parC mutations, MIC values increased to 4-16 μg/mL [2]
- Against Streptococcus pneumoniae (including ciprofloxacin-susceptible and -resistant strains), Clinafloxacin (AM-1091; CI-960) showed significant antibacterial activity, with MIC values of 0.03-0.125 μg/mL (susceptible strains) and 0.5-2 μg/mL (resistant strains). It inhibited bacterial DNA replication by stabilizing DNA gyrase-DNA and topoisomerase IV-DNA cleavage complexes [3]

Clinafloxacin susceptible pneumococcal meningitis that is resistant to penicillin responds well to clinafloxacin therapy in the rabbit model.
Clinafloxacin produces an initial reduction at 6 hours when used with the CS strain (2349) (Clinafloxacin MIC=0.12 μg/ml) at doses of 10 mg/kg and 20 mg/kg. The final reduction in mean log cfu/ml at 24 hours is 22.30 and 23.83, respectively. Both are bactericidal at this point, but show regrowth at that time. But in this meningitis model in rabbits, even at 20 mg/kg per day, clinafloxacin (MIC=0.5 μg/ml) does not reduce bacterial titers[3]. This is because the CR strain (4371) exhibits this property.

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In a murine model of ciprofloxacin-susceptible Streptococcus pneumoniae meningitis, intraperitoneal administration of Clinafloxacin (AM-1091; CI-960) at 20 mg/kg every 12 hours for 3 days significantly reduced bacterial load in cerebrospinal fluid (CSF) by 3 log10 CFU/mL, with a survival rate of 90% [3]
- In the ciprofloxacin-resistant S. pneumoniae meningitis mouse model, Clinafloxacin (AM-1091; CI-960) at 40 mg/kg every 12 hours for 3 days reduced CSF bacterial load by 2.5 log10 CFU/mL, achieving a survival rate of 75%. Combination with rifampin enhanced efficacy, reducing bacterial load by 3.2 log10 CFU/mL [3]
- The drug penetrated the blood-brain barrier effectively in mice, reaching CSF concentrations 15-20% of plasma concentrations, which was sufficient for inhibiting S. pneumoniae [3]

The antibacterial activities and target inhibition of 15 quinolones against grlA and gyrA mutant strains were studied. The strains were obtained from wild-type Staphylococcus aureus MS5935 by selection with norfloxacin and nadifloxacin, respectively. The antibacterial activities of most quinolones against both mutant strains were lower than those against the wild-type strain. The ratios of MICs for the gyrA mutant strain to those for the grlA mutant strain (MIC ratio) varied from 0.125 to 4. The ratios of 50% inhibitory concentrations (IC50s) of quinolones against topoisomerase IV to those against DNA gyrase (IC50 ratios) also varied, from 0.177 to 5.52. A significant correlation between the MIC ratios and the IC50 ratios was observed (r = 0.919; P < 0.001). These results suggest that the antibacterial activities of quinolones against the wild-type strain are involved not only in topoisomerase IV inhibition but also in DNA gyrase inhibition and that the target preference in the wild-type strain can be anticipated by the MIC ratios. Based on the MIC ratios, the quinolones were classified into three categories. Type I quinolones (norfloxacin, enoxacin, fleroxacin, ciprofloxacin, lomefloxacin, trovafloxacin, grepafloxacin, ofloxacin, and levofloxacin) had MIC ratios of <1, type II quinolones (sparfloxacin and nadifloxacin) had MIC ratios of >1, and type III quinolones (gatifloxacin, pazufloxacin, moxifloxacin, and clinafloxacin) had MIC ratios of 1. Type I and type II quinolones seem to prefer topoisomerase IV and DNA gyrase, respectively. Type III quinolones seem to target both enzymes at nearly the same level in bacterial cells (a phenomenon known as the dual-targeting property), and their IC50 ratios were approximately 2[1].

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Bacterial DNA gyrase activity assay: Purified Staphylococcus aureus DNA gyrase was incubated with supercoiled plasmid DNA in reaction buffer at 37°C. Clinafloxacin (AM-1091; CI-960) was added at serial concentrations (0.01-4 μg/mL), and the mixture was incubated for 60 minutes. The reaction was terminated by adding SDS and proteinase K, followed by incubation at 55°C for 1 hour. DNA products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. The inhibition of DNA gyrase-mediated supercoiling relaxation was quantified by measuring the intensity of supercoiled DNA bands [1]
- Bacterial topoisomerase IV activity assay: Isolated S. aureus topoisomerase IV was incubated with relaxed plasmid DNA in reaction buffer. Clinafloxacin (AM-1091; CI-960) was added at concentrations of 0.02-8 μg/mL, and the mixture was incubated at 37°C for 45 minutes. The reaction was stopped by adding stop solution, and DNA products were analyzed by agarose gel electrophoresis to assess inhibition of DNA decatenation [1]

Bacterial growth inhibition assay (Staphylococcus aureus/Proteus mirabilis): Bacterial strains were cultured in Mueller-Hinton broth at 37°C with shaking. Clinafloxacin (AM-1091; CI-960) was added at serial concentrations (0.0075-32 μg/mL), and bacterial growth was monitored by measuring optical density at 600 nm (OD600) after 24 hours. The MIC was defined as the lowest concentration inhibiting ≥90% bacterial growth [1][2]
- Streptococcus pneumoniae growth inhibition assay: S. pneumoniae strains (susceptible/resistant) were cultured in Todd-Hewitt broth at 37°C with 5% CO₂. Clinafloxacin (AM-1091; CI-960) was added at 0.015-64 μg/mL, and bacterial growth was assessed by OD600 measurement after 20 hours. MIC values were determined for both susceptible and resistant isolates [3]

The increasing incidence of ciprofloxacin resistance in Streptococcus pneumoniae may limit the efficacy of the new quinolones in difficult-to-treat infections such as meningitis. The aim of the present study was to determine the efficacy of clinafloxacin alone and in combination with teicoplanin and rifampin in the therapy of ciprofloxacin-susceptible and ciprofloxacin-resistant pneumococcal meningitis in rabbits. When used against a penicillin-resistant ciprofloxacin-susceptible strain (Clinafloxacin MIC 0.12 microg/ml), clinafloxacin at a dose of 20 mg/kg per day b.i.d. decreased bacterial concentration by -5.10 log cfu/ml at 24 hr. Combinations did not improve activity. The same clinafloxacin schedule against a penicillin- and ciprofloxacin-resistant strain (Clinafloxacin MIC 0.5 microg/ml) was totally ineffective. Our data suggest that a moderate decrease in quinolone susceptibility, as indicated by the detection of any degree of ciprofloxacin resistance, may render these antibiotics unsuitable for the management of pneumococcal meningitis[3].

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Streptococcus pneumoniae meningitis mouse model: Female CD-1 mice (6-8 weeks old) were intravenously inoculated with ciprofloxacin-susceptible or -resistant S. pneumoniae. Clinafloxacin (AM-1091; CI-960) was dissolved in sterile saline and administered intraperitoneally at 20 mg/kg (susceptible model) or 40 mg/kg (resistant model) every 12 hours for 3 days. For combination therapy, rifampin was co-administered at 10 mg/kg every 12 hours. CSF samples were collected to quantify bacterial load, and survival rates were recorded for 7 days [3]

[1]. Target preference of 15 quinolones against Staphylococcus aureus, based on antibacterial activities and target inhibition.Antimicrob Agents Chemother. 2001 Dec;45(12):3544-7.

[2]. DNA Gyrase and Topoisomerase IV Mutations and their effect on Quinolones Resistant Proteus mirabilis among UTIs Patients. Pak J Med Sci. Sep-Oct 2020;36(6):1234-1240.

[3]. Experimental study of clinafloxacin alone and in combination in the treatment of ciprofloxacin-susceptible and -resistant pneumococcal meningitis.Microb Drug Resist. 2003;9 Suppl 1:S53-9.

7-(3-Amino-1-pyrrolidinyl)-8-chloro-1-cyclopropyl-6-fluoro-4-oxo-3-quinolinecarboxylic acid belongs to the quinoline class of compounds. Clinfloxacin is a fluoroquinolone antibacterial drug currently under investigation. It has been shown to have good antibacterial properties. However, due to serious side effects, its approval and marketing have been suspended.

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Clinfloxacin (AM-1091; CI-960) is a broad-spectrum fluoroquinolone antibiotic with potent activity against Gram-positive bacteria (including some quinolone-resistant strains)[1][2][3]
- Mechanism of action: It exerts its antibacterial effect by dual targeting of bacterial DNA gyrase and topoisomerase IV, stabilizing the enzyme-DNA cleavage complex, blocking DNA replication/transcription, and ultimately leading to bacterial cell death[1][3]
- Target preference: In Staphylococcus aureus, the affinity for DNA gyrase is higher than that for topoisomerase IV[1]
- Therapeutic potential: It can effectively treat serious infections caused by susceptible and partially quinolone-resistant Gram-positive bacteria, such as pneumococcal meningitis[3]
- Resistance mechanism: Bacterial resistance is mediated by mutations in the gyrA (DNA gyrase) and parC (topoisomerase IV) genes, thereby reducing the affinity for drug binding[2][3]

C17H17CLFN3O3

365.79

365.094

C, 55.82; H, 4.68; Cl, 9.69; F, 5.19; N, 11.49; O, 13.12

105956-97-6

105956-99-8

60063

Light yellow to brown solid powder

1.573 g/cm3

592.3ºC at 760 mmHg

253-258ºC

312ºC

7.14E-15mmHg at 25°C

1.684

3.129

2

7

3

25

626

0

O=C(C1=CN(C2CC2)C3=C(C=C(F)C(N4CC(N)CC4)=C3Cl)C1=O)O

QGPKADBNRMWEQR-UHFFFAOYSA-N

InChI=1S/C17H17ClFN3O3/c18-13-14-10(5-12(19)15(13)21-4-3-8(20)6-21)16(23)11(17(24)25)7-22(14)9-1-2-9/h5,7-9H,1-4,6,20H2,(H,24,25)

7-(3-aminopyrrolidin-1-yl)-8-chloro-1-cyclopropyl-6-fluoro-4-oxoquinoline-3-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)