Gyrase ( IC50 = 1.25 μg/mL ); TOPO IV ( IC50 = 1.5-2.5 μg/mL ); Quinolone

Garenoxacin (BMS284756) (0-8 days) inhibits the growth of tested strains of mycoplasma and ureaplasma with MIC90s ≤0.25 μg/mL[1].
Garenoxacin (48 h) has MICs of 0.0128-4.0 μg/mL, which inhibits both wild type and mutant S. aureus[2].
Garenoxacin has an IC50 of 1.25 to 2.5 μg/mL for topoisomerase IV and 1.25 μg/mL for gyrase from S. aureus, respectively.
Garenoxacin has a low tendency to selectively enrich fluoroquinolone-resistant mutants from S. aureus isolates that are susceptible to ciprofloxacin[3].

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The in vitro susceptibilities to garenoxacin (BMS-284756), an investigational des-fluoroquinolone, and eight other agents were determined for 63 Mycoplasma pneumoniae, 45 Mycoplasma hominis, 15 Mycoplasma fermentans, and 68 Ureaplasma sp. isolates. Garenoxacin was the most active quinolone, inhibiting all isolates at Researchers determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 x 10(-12) to 4.0 x 10(-11)) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 micro g/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin, its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5' terminus of grlB and the 3' terminus of gyrA. [2]

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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 (MPC(90)) 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 MPC(90) 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]

Garenoxacin (12.5-50 mg/kg; s.c.; once) is very effective against both the wild-type strain and mutants carrying a single mutation in a mouse pneumonia model with S. pneumonia infection[4].
Garenoxacin (10 and 30 mg/kg; p.o.; once) significantly increases the time that BALB/c female mice survive experimental secondary pneumococcal pneumonia caused by S. pneumoniae D-979[5] while also reducing the number of viable cells in the lungs.

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The pulmonary pharmacokinetic parameters in mice infected with strain P-4241 and treated with garenoxacin or TVA (25 mg/kg of body weight) were as follows: maximum concentration of drug in serum (C(max); 17.3 and 21.2 micro g/ml, respectively), C(max)/MIC ratio (288 and 170, respectively), area under the concentration-time curve (AUC; 48.5 and 250 microg. h/ml, respectively), and AUC/MIC ratio (808 and 2000, respectively). Garenoxacin at 25 and 50 mg/kg was highly effective (survival rates, 85 to 100%) against the wild-type strain and mutants harboring a single mutation. TVA was as effective as garenoxacin against these strains. TVA at 200 mg/kg and garenoxacin at 50 mg/kg were ineffective against the mutant with the parC and gyrA double mutations and the mutant with the gyrA, parC, and parE triple mutations. The efficacy of garenoxacin was reduced only when strains bore several mutations for quinolone resistance. [4]

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In a pneumococcal pneumonia murine model following influenza virus infection, garenoxacin was more effective than other fluoroquinolones and demonstrated high levels of bacterial eradication in the lung, low mortality, and potent histopathological improvements. Garenoxacin could potentially be used for the treatment of secondary pneumococcal pneumonia following influenza. [5]

Topoisomerase IV assay. [2]
The reaction mixture (20 μl) for decatenation assays contained 50 mM Tris-HCl (pH 7.7), 5 mM MgCl2, 5 mM dithiothreitol, 50 μg of bovine serum albumin per ml 250 mM potassium glutamate, 1 mM ATP, 100 ng of kinetoplast DNA, and various amounts of GrlA and GrlB. Following incubation of the reaction mixtures at 37°C for 1 h, the reactions were terminated by addition of EDTA to a final concentration of 50 mM, and the products were analyzed by electrophoresis in 1% agarose. Gels were stained with ethidium bromide following electrophoresis.

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DNA gyrase assay. [2]
DNA supercoiling activity was assayed in buffer containing 75 mM Tris-HCl (pH 7.5), 7.5 mM MgCl2, 7.5 mM dithiothreitol, 2mM ATP, 75 μg of bovine serum albumin per ml, 30 mM KCl, 250 mM potassium glutamate, and 2 μg of tRNA with 0.5 μg of relaxed pBR322 as the substrate in a total volume of 20 μl. The reaction was carried out at 30°C for 1 h and stopped by addition of EDTA to a final concentration of 50 mM, and the products were analyzed by electrophoresis in 1% agarose as for the topoisomerase IV assays.

Cell Line: M. pneumonia, M. fermentans, M. hominis and Ureaplasma spp.
Incubation Time: 24 h for Ureaplasma spp., 48 h for M. hominis, 4 to 8 days for M. pneumonia
Result: Showed inhibition with MIC90s of 0.031 μg/mL, ≤0.008 μg/mL, ≤0.008 μg/mL and 0.25 μg/mL against M. pneumonia, M. fermentans, M. hominis and Ureaplasma spp. strains, respectively.

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Drug susceptibility determinations.[2]
MICs were determined in duplicate at least twice on Trypticase soy agar containing serial twofold dilutions of antibiotics, and growth was scored after 24 and 48 h of incubation at 37°C. MICs of nalidixic acid were used to screen for gyrA mutations, MICs of novobiocin were used to screen for grlB mutations, and MICs of ethidium bromide were used to screen for NorA overexpression. In genetic tests when twofold differences were encountered, they were confirmed by repetitive testing.

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Frequency of selection of mutants. [2]
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.

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Stepwise selection of resistant mutants. [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]

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Antibiotic treatment [4]
Therapy was initiated 18 h after challenge with the wild-type virulent penicillin-susceptible strain (P-4241) and with the quinolone-resistant mutants (mutants with single parC, gyrA, and efflux mutations and the mutant with double parC and gyrA mutations). Treatment was initiated 3 h after challenge with the parE and the parC gyrA parE clinical strains. Garenoxacin and TVA were administered as six subcutaneous (s.c.) injections at doses of 12.5, 25, and 50 mg/kg. TVA was given at doses of 50, 100, and 200 mg/kg to mice challenged with the mutant with the double mutations. Infected, untreated control mice received the same volume of isotonic saline. Each treatment group comprised 15 animals. The observation period was 10 days. Death rates were recorded daily, and the cumulative survival rates were compared.

Animal Model: Swiss mice with S. pneumonia infection[4].
Dosage: 12.5, 25 and 50 mg/kg
Administration: Subcutaneous injection, once
Result: Significantly improved the survival rate.

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Bactericidal activity in vivo [4]
The protocol used to study bactericidal activity in vivo was the same as that used for the mouse survival studies. The total CFU counts recovered from whole-lung homogenates were determined 6 h after the first treatment, which was initiated 18 h after bacterial challenge, and 12 h after the second, fourth, and sixth treatments at doses of 12.5 and 25 mg of garenoxacin per kg. Three mice were used for each dose and time point. Mice were killed by intraperitoneal injection of sodium pentobarbital and were exsanguinated by cardiac puncture; blood was used for culture. The lungs were removed and homogenized in 1 ml of normal saline. Serial 10-fold dilutions of the homogenates were plated on Columbia agar. Blood was cultured in brain heart infusion broth. After overnight culture, colonies were counted on agar plates seeded with lung samples, and blood cultures were examined for turbidity. Results are expressed as the mean ± standard deviation log10 CFU per lung and as the number of positive or negative blood cultures for groups of three mice each.

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Determination of garenoxacin concentrations in serum and lungs and PK analysis [4]
Antibiotics were administered as a single s.c. dose of 25 mg of garenoxacin or TVA per kg to both infected and uninfected mice. Infected mice were treated at 18 h postinfection. Serum and lung samples were collected from groups of six mice at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after drug administration. All samples were stored at −20°C and protected from light to avoid garenoxacin degradation during analysis. Lung samples were crushed in liquid nitrogen with a magnetic crusher. Serum samples (100 μl) and lung tissue samples (20 to 50 mg of lung powder, as measured precisely) were prepared by mixing an internal standard with methanolic acid (100 and 500 μl, respectively). After precipitation or diffusion, vortexing or ultrasonic mixing, and centrifugation, 50 μl of the upper phase was injected into a high-performance liquid chromatographic system. The total drug concentration was determined by use of an octadecyl silyl column (Novapak C18; 4.6 by 150 mm) coupled to a spectrofluorometric detector operating at excitation and emission wavelengths of 280 and 415 nm, respectively. The mobile phase was a mixture of acetonitrile, sodium citrate buffer solution (pH 3.5), and water (22/15/63; vol/vol) with 0.2% triethylamine, adjusted to pH 4. The flow rate was 1.0 ml/min. The limits of quantification were 0.02 μg/ml and 0.05 μg/g for serum and lung tissue samples, respectively, and measurements were linear over the ranges of 0.2 to 10.0 μg/ml and 0.5 to 50.0 μg/g for serum and lung tissue samples, respectively. The coefficients of variation for quality control were below 10% for both serum and lung tissue samples. The pharmacokinetic (PK) parameters for TVA were evaluated as described elsewhere

The area under the free serum concentration-time curve divided by the minimum inhibitory concentration (fAUC0–24/MIC) over 24 hours is one of the most important predictors of the clinical efficacy of fluoroquinolones (Craig, 1998). In the influenza virus-induced secondary pneumococcal pneumonia model established in this study, the fAUC0–24/MIC ratios in serum after oral administration of rhinoxacin (10 and 30 mg/kg) were 71.7 and 288, respectively, and in the lungs, the fAUC0–24/MIC ratios were 106 and 381, respectively, indicating that it can effectively clear bacteria and has excellent efficacy (Table 1). Although it remains unclear whether these three quinolones have similar fAUC0-24/MIC efficacy in secondary pneumococcal pneumonia models, garenofloxacin, at clinical doses, has an fAUC/MIC90 ratio ≥352 against Streptococcus pneumoniae, higher than levofloxacin (15.5) and moxifloxacin (107) (Chein et al., 1997; Takagi et al., 2008; Watanabe et al., 2012; Zeitlinger et al., 2003). The potent antibacterial activity and favorable pharmacokinetic characteristics of garenofloxacin are considered to reflect its excellent therapeutic effect on secondary pneumococcal pneumonia following influenza virus infection. Although moxifloxacin (30 mg/kg) has a similar effect to garenofloxacin in reducing lung viable cells, and its fAUC0-24/MIC value is lower than that of garenofloxacin, its mortality rate (40%) is higher (Table 1). Further research is needed to clarify the differences in the target fAUC/MIC values for quinolone drugs. [5]

[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.

[5]. Therapeutic effects of garenoxacin in murine experimental secondary pneumonia by Streptococcus pneumoniae after influenza virus infection. Diagn Microbiol Infect Dis. 2014 Feb;78(2):168-71.

Garenofloxacin is a quinoline monocarboxylic acid, namely 1,4-dihydroquinoline-3-carboxylic acid, with a cyclopropyl group substituted at position 1, an oxo group substituted at position 4, a (1R)-1-methyl-2,3-dihydro-1H-isoindole-5-yl substituted at position 7, and a difluoromethoxy group substituted at position 8. It is an antibacterial drug and a nonsteroidal anti-inflammatory drug. It is a quinolone antibiotic, a quinoline monocarboxylic acid, an organofluorine compound, a cyclopropane compound, an aromatic ether compound, and an isoindole compound. Garenofloxacin is a quinolone antibiotic, and its efficacy in treating Gram-positive and Gram-negative bacterial infections is currently under investigation. [Drug Indications] It has been studied for the treatment of bacterial infections. In addition to determining the minimum bactericidal concentration (MBC) for some microorganisms, we also attempted to evaluate the bactericidal kinetics of garenofloxacin against representative isolates of Mycoplasma pneumoniae and Mycoplasma hominis, as MBC has already demonstrated the bactericidal activity of garenofloxacin against these microorganisms. Because mycoplasmas (especially Mycoplasma pneumoniae) have a slow growth rate, with a generation time of 6 hours, the 24-hour time-kill curve study typically needs to be extended to confirm its efficacy. We demonstrated that garenofloxacin exhibits concentration-dependent bactericidal activity against Mycoplasma pneumoniae after incubation ranging from 24 to 96 hours. Garenofloxacin also showed bactericidal activity against Mycoplasma hominis after incubation at concentrations 4 to 8 times the MIC for 24 hours, and after incubation at concentrations 2 times the MIC for 48 hours. Regeneration of ≤2 log10 CFU was observed in the presence of certain low concentrations of garenofloxacin, possibly due to the survival and proliferation of a very small number of surviving microorganisms over time. For Mycoplasma pneumoniae, degradation and inactivation of garenofloxacin after prolonged incubation for several days may have promoted the proliferation of these microorganisms. This is the first time that the bactericidal effect of an antimicrobial drug on Mycoplasma pneumoniae has been confirmed by a time-effect bactericidal experiment modified from the commonly used method for evaluating the effects of antimicrobial drugs on other bacteria. This study suggests that garenofloxacin is a promising drug for the treatment of mycoplasma and ureaplasma infections. Further clinical evaluation should be conducted. [1] In summary, garenofloxacin interacts similarly with DNA gyrase and topoisomerase IV and produces novel mutations that extend the quinolone resistance determination region (QRDR) to the N-terminal domain of GrlB and the C-terminal domain of GyrA. This novel defluoroquinolone drug has high potency and low selection frequency of resistance mutants, which may be advantageous in clinical applications and reduce the likelihood of new resistance mutants emerging. However, garenofloxacin exhibits cross-resistance with strains that have previously been selected for multiple quinolone resistance mutants (e.g., the currently common methicillin-resistant Staphylococcus aureus clinical isolates), which may limit the application of this antibiotic in treating strains that have developed resistance to earlier quinolone drugs. [2]

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For isolates resistant to ciprofloxacin, the MIC90 of garenofloxacin is 3.2 μg/ml, which is about 8 times that of the MPC90 of sensitive isolates. This observation suggests that some resistant strains have multiple mutations, consistent with reports from other studies. Since the MIC of resistant isolates is lower than the achievable serum drug concentration (Figure 2C), it can be inferred that treatment with garenofloxacin for ciprofloxacin-resistant Staphylococcus aureus may sometimes cure the infection. However, the MPC90 of ciprofloxacin-resistant isolates is >19.6 μg/ml, which, even with a daily dose increased to 600 mg, is far higher than the serum drug concentrations achievable with garanodexacin (Figure 2C). In fact, the pharmacodynamics of MPC-based garanodexacin and its mutant strains (Figure 2C) are similar to those of ciprofloxacin and its fully susceptible strains (Figure 2A). Because ciprofloxacin can rapidly screen for resistant mutants, we predict that if garanodexacin is used to treat ciprofloxacin-resistant strains, more mutations will be fixed in Staphylococcus aureus. These mutations will render garanodexacin unsuitable for combination therapy. [3]

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In vivo experiments showed that in mice infected with wild-type strains and single-mutant resistant strains, the survival rate of garenofloxacin was comparable to that of TVA, and its efficacy against mutant strains carrying the parC and gyrA double mutations was slightly better than that of TVA: 50 mg/kg body weight of garenofloxacin prolonged the survival time of mice, while 200 mg/kg body weight of TVA had no effect. Comparison of the activity of garenofloxacin with that of ciprofloxacin (a well-defined and widely used quinolone drug) showed that garenofloxacin was far more effective than ciprofloxacin. This result was expected given the poor in vitro activity of ciprofloxacin. The in vivo activity of garenofloxacin stems from its superior in vitro activity against wild-type and fluoroquinolone-resistant Streptococcus pneumoniae strains compared to ciprofloxacin, and its superior activity against double-mutant and triple-mutant strains compared to retinoic acid (TVA). However, the in vivo efficacy of quinolone drugs is also affected by other factors, particularly pharmacokinetic/pharmacodynamic (PK-PD) parameters. Forrest et al. and Hyatt et al. reported that the AUC/MIC ratio is a key parameter associated with bacterial clearance and clinical cure in patients with hospital-acquired pneumonia, with a minimum clinically effective ratio of 125. Therefore, the favorable PK-PD parameters of garenoxacin contribute to its efficacy. Compared to ciprofloxacin (CIP), garenoxacin has a longer half-life, a larger AUC value, and superior in vitro activity, especially against Streptococcus pneumoniae; it exhibits the highest AUC/MIC ratio in mouse serum and lung tissue samples. These pharmacokinetic (PK) and pharmacodynamic (PD) parameters are also highly favorable for retinoic acid (TVA), explaining why this quinolone is as effective as garenoxacin. Our obtained pharmacokinetic data for garafloxacin were in excellent agreement with mouse survival data, indicating that serum protein binding had little impact on treatment outcomes, even when serum protein binding was as high as approximately 80% in mice (DR Andes and WA Craig, Abstract of the 43rd Interdisciplinary Conference on Antimicrobial Agents and Chemotherapy, Abstract A-309, p. 10, 2003). This is likely due to the weak binding of garafloxacin to serum proteins. Furthermore, inflammatory lung cells may act as a drug reservoir, releasing garafloxacin into the serum. TVA also exhibited high serum protein binding, and its efficacy was associated with its favorable pharmacokinetic behavior. TVA was an interesting control in this mouse model of pneumococcal pneumonia, but its clinical relevance is less than that of garafloxacin due to its withdrawal from the market. In conclusion, garafloxacin demonstrated high efficacy in mouse models of pneumonia caused by both quinolone-sensitive and drug-resistant Streptococcus pneumoniae strains. Therefore, garafloxacin may be an effective option for empirical treatment of community-acquired respiratory infections. [4] Previous studies have shown that although β-lactams can effectively clear bacteria, they do not improve the survival rate of secondary bacterial pneumonia (McCullers, 2004), while macrolides improve survival by reducing inflammation (Karlström et al., 2009). A study by Hara et al. (2011) reported that garenofloxacin has anti-inflammatory activity, and its mechanism of action is through altering the secretion of interleukin-8 by human lung epithelial cell lines and human mononuclear cell lines (albeit in lipopolysaccharide-stimulated cells). The improvement in the efficacy of garenofloxacin may be related not only to its higher fAUC0-24/MIC value leading to bacterial clearance, but also to its inhibition of the inflammatory response. Therefore, these data suggest that garenofloxacin may have a potential role in the treatment of secondary pneumococcal pneumonia after influenza. Further research is needed to better understand the effects of garenofloxacin on the inflammatory response and clinical efficacy in patients with secondary pneumococcal pneumonia. [5]

C24H26F2N2O8S

540.53

540.137

C, 53.33; H, 4.85; F, 7.03; N, 5.18; O, 23.68; S, 5.93

223652-90-2

194804-75-6; 223652-90-2 (mesylate hydrate); 223652-82-2 (mesylate)

157690

Off-white to gray solid powder

581.5ºC at 760 mmHg

2.29E-14mmHg at 25°C

5.316

4

12

5

37

863

1

O=C(C1=CN(C2CC2)C3=C(C=CC(C4=CC5=C([C@@H](C)NC5)C=C4)=C3OC(F)F)C1=O)O.CS(=O)(O)=O.O

IGTHEWGRXUAFKF-NVJADKKVSA-N

InChI=1S/C23H20F2N2O4.CH4O3S.H2O/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);1H2/t11-;;/m1../s1

1-cyclopropyl-8-(difluoromethoxy)-7-[(1R)-1-methyl-2,3-dihydro-1H-isoindol-5-yl]-4-oxoquinoline-3-carboxylic acid;methanesulfonic acid;hydrate

BMS-284756-01; BMS284756-01; T-3811ME; 223652-90-2; Garenoxacin mesylate; Garenoxacin mesilate; Geninax; T3811ME; Garenoxacin mesylate; Garenoxacin mesylate hydrate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: 2~100 mg/mL (4.7~185.0 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.63 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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