Mutants were selected by plating appropriate dilutions of overnight cultures of S. aureus ISP794 on brain heart infusion agar without any antibiotic or with garenoxacin or ciprofloxacin at concentrations one-, two-, four-, and eightfold the MIC of each drug. For selection with garenoxacin, large (150- by 15-mm) petri dishes were used to plate 1011 to 1012 CFU. Each plating was done in duplicate and repeated at least twice. Selection plates were incubated at 37°C. The frequency of selection of resistant mutants was calculated as the ratio of the number of resistant colonies at 48 h to the number of cells inoculated. Selected colonies were subcultured once on brain heart infusion agar plates containing the selecting concentration of garenoxacin and, if necessary, once more on brain heart infusion agar without any antibiotic and then stored at −70°C in 10% glycerol in brain heart infusion broth.[2]
S. aureus ISP794 was serially passaged on brain heart infusion agar containing twofold-increasing concentrations of garenoxacin to define the highest level of resistance achievable. Selection began at the MIC of garenoxacin for ISP794. At each step, several mutant colonies were subcultured on brain heart infusion agar plates containing the selecting concentration of garenoxacin before being stored at −70°C and passaged at a twofold higher antibiotic concentration.[2]
The new quinolone garenoxacin (BMS-284756), which lacks a C-6 fluorine, was examined for its ability to block the growth of Staphylococcus aureus. Measurement of the MIC and the mutant prevention concentration (MPC) revealed that garenoxacin was 20-fold more potent than ciprofloxacin for a variety of ciprofloxacin-susceptible isolates, some of which were resistant to methicillin. The MPC for 90% of the isolates (MPC90) was below published serum drug concentrations achieved with recommended doses of garenoxacin. These in vitro observations suggest that garenoxacin has a low propensity for selective enrichment of fluoroquinolone-resistant mutants among ciprofloxacin-susceptible isolates of S. aureus. For ciprofloxacin-resistant isolates, the MIC at which 90% of the isolates tested were inhibited was below serum drug concentrations while the MPC90 was not. Thus, for these strains, garenoxacin concentrations are expected to fall inside the mutant selection window (between the MIC and the MPC) for much of the treatment time. As a result, garenoxacin is expected to selectively enrich mutants with even lower susceptibility.[3]

The resultant colonies were screened for susceptibility to tetracycline (MIC, ≤3 μg/ml) and change in susceptibility to garenoxacin (MIC, >0.064 μg/ml) or ciprofloxacin (MIC, >0.25 μg/ml). The MICs of ciprofloxacin and garenoxacin were determined for colonies that were tetracycline susceptible and had a changed susceptibility to garenoxacin or ciprofloxacin. Direct DNA sequencing of the PCR product of the appropriate region amplified from chromosomal DNA was used to confirm the presence of the expected mutations.[2]
Ciprofloxacin was dissolved in sterile water to give a final concentration of 10 mg/ml. Levofloxacin and gatifloxacin stock solutions were prepared similarly, except that about a 1/10 volume of 1 M NaOH was added to help dissolve both compounds. Garenoxacin was dissolved in 0.001 M acetic acid as a 5-mg/ml stock solution. Stock solutions were divided into 1-ml aliquots and stored at −80°C. Dilution series were prepared with autoclaved water. Solutions were occasionally stored at −20°C for several weeks.[3]
To assess the potency of garenoxacin against ciprofloxacin-resistant isolates, we measured the MICs and the MPCs for a diverse panel of isolates comprising 18 MRSA and 4 MSSA isolates. The MIC ranged between 0.04 and 4.8 μg/ml, the modal MIC was between 0.8 and 1.2 μg/ml, and the MIC90 was 3.2 μg/ml, similar to data in one report (15) and slightly higher than that in another (21). The range of MPCs was 3.2 to >29 μg/ml, the MPC90 was >19.6 μg/ml, and the modal MPC ranged between 9.6 and 12.8 μg/ml. These values, which are listed in Table 2, are 30- to 100-fold higher than those observed for ciprofloxacin-susceptible isolates.[3]

[1]. In vitro susceptibilities to and bactericidal activities of garenoxacin (BMS-284756) and other antimicrobial agents against human mycoplasmas and ureaplasmas. Antimicrob Agents Chemother. 2003 Jan;47(1):161-5.

[2]. Dual targeting of DNA gyrase and topoisomerase IV: target interactions of garenoxacin (BMS-284756, T-3811ME), a new desfluoroquinolone. Antimicrob Agents Chemother. 2002 Nov;46(11):3370-80.

[3]. Mutant prevention concentration of garenoxacin (BMS-284756) for ciprofloxacin-susceptible or -resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2003 Mar;47(3):1023-7.

[4]. Activities of garenoxacin against quinolone-resistant Streptococcus pneumoniae strains in vitro and in a mouse pneumonia model. Antimicrob Agents Chemother. 2004 Mar;48(3):765-73.

See also: Garenoxacin (annotation moved to).

C23H20N2O4F2

426.4127

522.127

223652-82-2

Garenoxacin;194804-75-6

124094

Typically exists as solid at room temperature

1.421g/cm3

581.5ºC at 760mmHg

305.5ºC

5.38

3

11

5

36

863

1

C[C@@H]1C2=C(C=C(C=C2)C3=C(C4=C(C=C3)C(=O)C(=CN4C5CC5)C(=O)O)OC(F)F)CN1.CS(=O)(=O)O

UPHLDCUEQOTSAD-RFVHGSKJSA-N

InChI=1S/C23H20F2N2O4.CH4O3S/c1-11-15-5-2-12(8-13(15)9-26-11)16-6-7-17-19(21(16)31-23(24)25)27(14-3-4-14)10-18(20(17)28)22(29)30;1-5(2,3)4/h2,5-8,10-11,14,23,26H,3-4,9H2,1H3,(H,29,30);1H3,(H,2,3,4)/t11-;/m1./s1

3-Quinolinecarboxylic acid, 1-cyclopropyl-8-(difluoromethoxy)-7-((1R)-2,3-dihydro-1-methyl-1H-isoindol-5-yl)-1,4-dihydro-4-oxo-, monomethanesulfonate

BMS-284756 mesylate; BM-284756; BMS284756; Garenoxacin; Garenoxacin mesilate.

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)