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

Against tested strains, Garenoxacin (BMS284756) (0-8 days) inhibits mycoplasmas and ureaplasmas with MIC90s ≤0.25 μg/mL [1].
S. aureus wild type and mutants are inhibited by genoxacin (48 h) with MICs ranging from 0.0128 to 4.0 μg/mL[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[2].
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]

Against the wild-type strain and mutants carrying a single mutation in a mouse pneumonia model with S. pneumonia infection, geldanoxin (12.5–50 mg/kg; s.c.; once) exhibits remarkable efficacy[4].
When BALB/c female mice are exposed to experimental secondary pneumococcal pneumonia caused by S. pneumoniae D-979, the viable cell counts in the lungs are reduced and their survival is significantly prolonged when garenoxacin (10 and 30 mg/kg; p.o.; once) is administered[5].

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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: Ureaplasma spp., M. pneumonia, M. fermentans, and M. hominis.
Incubation Time: 48 hours for M. hominis, 24 hours for Ureaplasma spp., and 4–8 days for M. pneumonia
Result: demonstrated inhibition against strains of M. pneumonia, M. fermentans, M. hominis, and Ureaplasma spp. with MIC90s of 0.031 μg/mL, ≤0.008 μg/mL, ≤0.008 μg/mL, and 0.25 μg/mL, 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 unbound serum concentration-time curve over 24 h divided by the MIC (fAUC0–24/MIC) is one of the most important predictors for the clinical efficacy of fluoroquinolones (Craig, 1998). In this secondary pneumococcal pneumonia model following IAV infection, oral garenoxacin (10 and 30 mg/kg) had fAUC0–24/MIC ratios of 71.7 and 288 in the serum and fAUC0–24/MIC ratios of 106 and 381 in the lungs, resulting in effective bacterial eradication and excellent efficacy (Table 1). Although it is not clear yet whether the 3 quinolones show similar efficacy in the same fAUC0-24/MIC in the secondary pneumococcal pneumonia model or not, the fAUC/MIC90 ratio of garenoxacin at a clinical dose in human for S. pneumoniae is ≥352, which is also greater than those of levofloxacin (15.5) and moxifloxacin (107) (Chein et al., 1997, Takagi et al., 2008, Watanabe et al., 2012, Zeitlinger et al., 2003). It was considered that the potent antibacterial activity and favorable pharmacokinetic profile of garenoxacin reflected its excellent therapeutic effect on experimental secondary pneumococcal pneumonia following IAV infection. Although moxifloxacin (30 mg/kg) decreased the viable cells in the lung to a similar extent as garenoxacin despite of lower fAUC0-24/MIC than that of garenoxacin, its mortality of 40% was larger than that of garenoxacin (Table 1). Further study would be required for clarifying the difference in the target value of fAUC/MIC among the quinolones. [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.

Garenoxacin is a quinolinemonocarboxylic acid that is 1,4-dihydroquinoline-3-carboxylic acid that is substituted by a cyclopropyl group at position 1, an oxo group at position 4, a (1R)-1-methyl-2,3-dihydro-1H-isoindol-5-yl group at position 7, and a difluoromethoxy group at position 8. It has a role as an antibacterial drug and a non-steroidal anti-inflammatory drug. It is a quinolone antibiotic, a quinolinemonocarboxylic acid, an organofluorine compound, a member of cyclopropanes, an aromatic ether and a member of isoindoles.
Garenoxacin, a quinolone antibiotic, is being investigated for the treatment of gram-positive and gram-negative bacterial infections.

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Drug Indication
Investigated for use/treatment in bacterial infection.

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In addition to determining the MBCs for a subgroup of organisms, we sought to evaluate the dynamics of killing by garenoxacin of a representative isolate each of M. pneumoniae and M. hominis, since the MBCs had shown that garenoxacin had bactericidal effects against these organisms. Due to the slower growth rate of mycoplasmas, especially M. pneumoniae, with a generation time of 6 h, the usual 24-h duration of time-kill studies had to be lengthened in order to demonstrate an effect. We were able to demonstrate that garenoxacin has concentration-dependent bactericidal activity against M. pneumoniae after 24 to 96 h of incubation. Garenoxacin also demonstrated bactericidal activity against M. hominis after 24 h of incubation at four to eight times the MIC and after 48 h of incubation at two times the MIC. The regrowth of ≤2 log10 CFU observed in the presence of some of the lower concentrations of garenoxacin could have been due to a very small population of viable organisms that survived and that were allowed to propagate over time, perhaps aided by the degradation and inactivation of garenoxacin after prolonged incubation for several days in the case of M. pneumoniae. This is the first demonstration of the bactericidal effects of an antimicrobial agent against M. pneumoniae by time-kill studies modified from those commonly used to evaluate the effects of antimicrobials against other bacteria. The present study has shown that garenoxacin is a promising drug for the treatment of infections caused by Mycoplasma and Ureaplasma species. Further clinical evaluation should be pursued. [1]

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In summary, garenoxacin interacts similarly with both DNA gyrase and topoisomerase IV and has generated novel mutations expanding the range of the QRDR to both the amino-terminal domain of GrlB and the carboxy-terminal domain of GyrA. This novel desfluoroquinolone, with its high potency and low frequency of selection of resistant mutants, may thus prove advantageous in clinical settings, decreasing the possibility of selection of new resistant mutants. Strains with previously selected multiple quinolone resistance mutations, as is now common with methicillin-resistant clinical isolates of S. aureus, however, exhibit cross-resistance to garenoxacin that may limit the utility of this antibiotic for treatment of strains with established resistance to earlier quinolones. [2]

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For the isolates that were already resistant to ciprofloxacin, the MIC90 of garenoxacin was 3.2 μg/ml, roughly eight times higher than the MPC90 for the susceptible isolates. This observation suggests that multiple mutations were present in some of the resistant strains, as has been documented by other studies. Since the MIC for resistant isolates is below achievable serum drug concentrations (Fig. 2C), it is conceivable that treatment of ciprofloxacin-resistant S. aureus with garenoxacin might sometimes cure infection. However, the MPC90 of the isolates already resistant to ciprofloxacin was >19.6 μg/ml, which is well above the serum drug levels achieved with garenoxacin, even if daily doses are raised to 600 mg (Fig. 2C). Indeed, the MPC-based pharmacodynamics of garenoxacin and the mutants (Fig. 2C) are similar to those of ciprofloxacin and fully susceptible isolates (Fig. 2A). Since ciprofloxacin selected resistant mutants rapidly, we predict that additional mutations will be fixed in S. aureus if garenoxacin is used against ciprofloxacin-resistant strains. Those mutations would then preclude the use of garenoxacin in combination therapy. [3]

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In vivo, garenoxacin is as potent as TVA in terms of survival rates among mice infected with wild-type strains and resistant strains with single mutations and is slightly more effective than TVA against the mutants with double parC and gyrA mutations: 50 mg of garenoxacin per kg prolonged survival, whereas 200 mg of TVA per kg was ineffective. A comparison of the activity of garenoxacin with that of CIP, a well-characterized and widely distributed quinolone, showed that garenoxacin was far more effective. This was as expected, given the poor in vitro activity of CIP. The in vivo activity of garenoxacin is due to its better in vitro activity against wild-type and fluoroquinolone-resistant S. pneumoniae strains relative to that of CIP and its better activity against mutants with double and triple mutations compared to that of TVA. However, other factors, and particularly PK-PD parameters, are involved in the efficacies of quinolones in vivo. Forrest et al. and Hyatt et al. reported that the AUC/MIC ratio was the main parameter associated with bacterial eradication and clinical cure among patients with nosocomial pneumonia, with a minimal clinically effective ratio of 125. The favorable PK-PD parameters of garenoxacin thus contribute to its efficacy. Compared to CIP, garenoxacin has a longer half-life, larger AUCs, and superior in vitro activity, especially against S. pneumoniae; and garenoxacin yielded the highest AUC/MIC ratios in mouse serum and lung tissue samples. These PK and PD parameters are also very favorable for TVA, explaining why this quinolone is as effective as garenoxacin. Our pharmacokinetic data for garenoxacin closely match the mouse survival data, suggesting that serum protein binding has little influence on the therapeutic outcome, even though the level of serum protein binding reaches about 80% in mice (D. R. Andes and W. A. Craig, Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-309, p. 10, 2003). This might be explained by the weak binding of garenoxacin to serum proteins. Moreover, inflammatory cells in lungs may serve as a reservoir, releasing garenoxacin in serum. TVA also shows high-level serum protein binding, while its efficacy is related to its good PK behavior. TVA was an interesting comparator in this mouse model of pneumococcal pneumonia, but it is clinically less relevant than garenoxacin because it has been withdrawn from the market. In conclusion, garenoxacin is highly effective in a mouse model of pneumonia induced by both quinolone-susceptible and quinolone-resistant strains of S. pneumoniae. Garenoxacin could thus be a useful option for the empirical treatment of community-acquired respiratory tract infections. [4]

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Previous studies have shown that β-lactam induced no improvement in survival from secondary bacterial pneumonia, despite effective bacterial eradication (McCullers, 2004), and that improved survival with macrolide-treatment was mediated by decreased inflammation (Karlström et al., 2009). Hara et al. (2011) has reported that garenoxacin has anti-inflammatory activity through its capacity to alter the secretion of interleukin 8 from both a human lung epithelial cell line and a human monocyte cell line, although in the case of lipopolysaccharide-stimulated cell. The improved outcome with garenoxacin might be related to not only bacterial eradication due to its greater fAUC0-24/MIC but also to the suppression of the inflammatory response. Thus, these data suggest a potential role for garenoxacin for the treatment of secondary pneumococcal pneumonia after influenza. Further studies would be required to better understand the influence of garenoxacin on inflammatory response and clinical efficacy in patients with secondary pneumococcal pneumonia. [5]

C23H20F2N2O4

426.412713050842

426.139

C, 64.78; H, 4.73; F, 8.91; N, 6.57; O, 15.01

194804-75-6

Garenoxacin Mesylate hydrate;223652-90-2; Garenoxacin mesylate; 223652-82-2; 194804-75-6

124093

Solid powder

1.4±0.1 g/cm3

581.5±50.0 °C at 760 mmHg

226-227°; mp 234-235°

305.5±30.1 °C

0.0±1.7 mmHg at 25°C

1.631

2.28

2

8

5

31

771

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

NJDRXTDGYFKORP-LLVKDONJSA-N

InChI=1S/C23H20F2N2O4/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/h2,5-8,10-11,14,23,26H,3-4,9H2,1H3,(H,29,30)/t11-/m1/s1

1-Cyclopropyl-8-(difluoromethoxy)-7-[(1R)-1-methyl-2,3-dihydro-1H-isoindol-5-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid

BMS-284756; BMS 284756; BMS284756; tradename: Geninax.

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)

DMSO : ~2 mg/mL ( ~4.69 mM )

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.)