Bacterial DNA gyrase (inhibition of DNA gyrase is the principal mechanism of action). [1]
The primary molecular target of Danofloxacin Mesylate is bacterial DNA gyrase (topoisomerase II), an essential enzyme for bacterial DNA replication. DNA gyrase introduces negative supercoils into DNA, which is necessary for the unwinding of the DNA double helix during replication and transcription. By inhibiting DNA gyrase, Danofloxacin prevents bacterial DNA replication and transcription, leading to bacterial cell death. As a fluoroquinolone, Danofloxacin also inhibits topoisomerase IV, which is involved in the decatenation of replicated DNA molecules, further contributing to its antibacterial activity. Danofloxacin is a third-generation quinolone that has been optimized for veterinary use, with enhanced activity against respiratory pathogens in livestock. The compound's fluoroquinolone structure allows for broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria, including the pathogens responsible for respiratory disease in chickens (e.g., Pasteurella multocida, Mycoplasma gallisepticum), cattle (e.g., Mannheimia haemolytica, Pasteurella multocida), and pigs (e.g., Actinobacillus pleuropneumoniae, Pasteurella multocida).

Danofloxacin is a synthetic antibacterial agent of the fluoroquinolone class, acts principally by the inhibition of bacterial DNA-gyrase, which is necessary for supercoiling of DNA to provide a suitable spatial arrangement of DNA within the bacterial cell. The minimum inhibitory concentration of danofloxacin against 90% (MIC90) of contemporary European and North American field isolates of Pasteurella haemolytica, Pasteurella multocida and Haemophilus somnus, the most important bacterial respiratory pathogens of cattle, is 0.125 μg/ml.

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The minimum inhibitory concentration (MIC) of Danofloxacin Mesylate against 90% (MIC90) of contemporary European and North American field isolates of Pasteurella haemolytica was 0.125 μg/mL (Europe) and 0.06 μg/mL (North America); MIC90 against Pasteurella multocida was 0.06 μg/mL for both origins; MIC90 against Haemophilus somnus was 0.06 μg/mL for both origins. MIC range for all isolates was ≤0.008 μg/mL to 2 μg/mL. [1]
In comparison, MIC90 values of other antibacterials (oxytetracycline, trimethoprim/sulphamethoxazole, amoxycillin, erythromycin) against these isolates were generally much higher (e.g., oxytetracycline MIC90 >64 μg/mL for many Pasteurella isolates). [1]
In vitro antibacterial activity of Danofloxacin Mesylate has been extensively characterized against veterinary pathogens. Against Pasteurella multocida, MIC₉₀ values are typically 0.03-0.12 μg/mL. Against Mannheimia haemolytica, MIC₉₀ values are 0.06-0.25 μg/mL. Against Actinobacillus pleuropneumoniae, MIC₉₀ values are 0.03-0.12 μg/mL. Against Mycoplasma species, MIC₉₀ values are 0.1-0.5 μg/mL. Danofloxacin shows good activity against both susceptible and resistant strains of these pathogens, though cross-resistance with other fluoroquinolones can occur. The compound's activity is bactericidal, with a minimum bactericidal concentration (MBC) typically within 2-4 times the MIC. Danofloxacin shows concentration-dependent killing, with higher concentrations achieving more rapid bacterial killing. The compound's activity is enhanced at alkaline pH and is reduced in the presence of divalent cations (Mg²⁺, Ca²⁺), which can chelate the compound and reduce its cellular uptake. Danofloxacin's in vitro activity has been validated using CLSI guidelines for veterinary susceptibility testing.

Danofloxacin shows protective dose (PD50) of 0.38, 0.8, 2.42 mg/kg for P. multocida, E. coli and S. choleraesuis in in vivo mouse protection assay.
In vivo activity of Danofloxacin Mesylate has been demonstrated in animal models of respiratory disease. In chickens experimentally infected with P. multocida or M. gallisepticum, oral or injectable administration of Danofloxacin at doses of 2.5-10 mg/kg once daily for 3-5 days significantly reduces mortality, clinical signs, and bacterial isolation from respiratory tissues. In cattle with experimentally induced pneumonia caused by M. haemolytica or P. multocida, Danofloxacin administered subcutaneously at doses of 6-10 mg/kg once daily for 3 days reduces fever, improves clinical scores, and reduces lung lesion scores. In pigs with A. pleuropneumoniae or P. multocida infections, Danofloxacin at 2.5-5 mg/kg once daily for 3 days is effective in controlling the disease. In clinical use, Danofloxacin mesylate is widely used in veterinary medicine for the treatment of respiratory disease in poultry, cattle, and pigs. The compound's pharmacokinetic properties, including good tissue penetration and long half-life in target species, contribute to its clinical efficacy.

For in vitro antibacterial susceptibility testing with Danofloxacin, the following protocol is used following CLSI guidelines: bacterial isolates (e.g., P. multocida, M. haemolytica, A. pleuropneumoniae) are cultured on appropriate agar media. A bacterial suspension is prepared in sterile saline to a turbidity equivalent to a 0.5 McFarland standard (approximately 1-2 × 10⁸ CFU/mL). The suspension is diluted 1:100 in cation-adjusted Mueller-Hinton broth (CAMHB) for non-fastidious organisms, or in CAMHB with 2.5-5% lysed horse blood for fastidious organisms like M. haemolytica. Danofloxacin is serially diluted two-fold in 96-well microtiter plates in CAMHB to achieve final concentrations ranging from 0.002 to 64 μg/mL. The bacterial suspension is added to each well, and the plates are incubated at 35-37°C for 16-20 hours (or 24 hours for fastidious organisms). The MIC is determined as the lowest concentration of the compound that completely inhibits visible bacterial growth. Quality control strains (e.g., E. coli ATCC 25922, S. aureus ATCC 29213, P. aeruginosa ATCC 27853) are included in each run. For Mycoplasma susceptibility testing, a microbroth dilution method with SP-4 medium is used.

For in vitro cell-based assays with Danofloxacin, the following typical protocol is used: for studies on antibacterial mechanism, bacterial cells are cultured to mid-log phase and treated with Danofloxacin at concentrations 2-4 × MIC. DNA gyrase activity is measured by assessing DNA supercoiling using a DNA gyrase assay kit. Bacterial DNA fragmentation is assessed by agarose gel electrophoresis. For cytotoxicity studies, mammalian cell lines (e.g., HEK293 or HepG2) are cultured in DMEM with 10% FBS at 37°C in 5% CO₂. Cells are seeded in 96-well plates at 10,000 cells per well and treated with Danofloxacin at concentrations ranging from 0.1 to 1000 μg/mL for 24-72 hours. Cell viability is assessed using the MTT assay to determine the CC50. For assessment of cellular uptake, bacteria or mammalian cells are treated with Danofloxacin, lysed, and the intracellular concentration is measured by HPLC or LC-MS/MS. For assessment of efflux, cells are treated with the compound in the presence or absence of efflux pump inhibitors (e.g., reserpine, verapamil) to assess the contribution of efflux to resistance.

Dissolved in 2.5% or 5% water solution; 0-10 mg/kg; p.o or s.c. Mice model

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Kinetics study I: Twelve cross-bred calves (90-133 kg) were used in a cross-over design. On day 0, a single intravenous (IV) injection of Danofloxacin Mesylate at 1.25 mg/kg was given to group A; subcutaneous (SC) injection at 1.25 mg/kg to group B; intramuscular (IM) injection at 1.25 mg/kg to group C. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h post-dose. Groups B and C then received four more daily doses (SC or IM) at 24-h intervals, with blood sampling repeated on days 2 and 4. The treatment and sampling were repeated on days 12 and 24 with routes rotated. Calves were re-weighed on days 12 and 24 to recalculate dose. [1]
Kinetics study II: Thirty-six cross-bred calves (223-291 kg) received a single IM injection of Danofloxacin Mesylate at 1.25 mg/kg. Groups of calves were slaughtered at 1, 2, 4, 8, 12, and 24 h post-dose. Before slaughter, heparinized blood samples were collected from the jugular vein. After slaughter, lungs were removed, and pooled lung tissue samples (approx. 500 g per calf) were homogenized and aliquots stored for analysis. [1]
For in vivo animal studies with Danofloxacin, the following general protocol is used: for efficacy studies in chickens, 3-week-old specific pathogen-free chickens are infected intranasally or intratracheally with P. multocida or M. gallisepticum. After 24-48 hours, chickens are treated orally or by injection with Danofloxacin at doses of 2.5, 5, and 10 mg/kg once daily for 3-5 days. Clinical signs, mortality, and bacterial isolation from respiratory tissues are assessed. For cattle studies, calves are infected intratracheally with M. haemolytica or P. multocida. After 24-48 hours, calves are treated subcutaneously with Danofloxacin at doses of 6, 8, and 10 mg/kg once daily for 3 days. Clinical signs, rectal temperature, and lung lesion scores are assessed at necropsy. For pig studies, pigs are infected intranasally with A. pleuropneumoniae or P. multocida, and treated with Danofloxacin at 2.5-5 mg/kg once daily for 3 days. Blood samples are collected for pharmacokinetic analysis, and tissue samples (lung, liver, kidney, muscle) are collected at various time points for residue analysis to determine withdrawal times.

After IV administration at 1.25 mg/kg, plasma concentrations declined biexponentially with a terminal elimination half-life (t1/2) of 4.01 h (harmonic mean), systemic plasma clearance (CLp) of 0.468 L/h·kg, volume of distribution at steady state (Vdss) of 2.48 L/kg. [1]
After IM administration at 1.25 mg/kg, mean Cmax in plasma was 0.47 μg/mL (first dose), Tmax ~1.0 h, AUC(0-∞) 2.8 μg·h/mL, t1/2 ~3.86 h. After SC administration, mean Cmax was 0.37 μg/mL, AUC(0-∞) 2.6 μg·h/mL. Bioavailability was 101% (IM) and 94% (SC) relative to IV. No significant differences in AUC between routes; IM gave higher Cmax (statistically significant for first and third doses). [1]
After a single IM dose of 1.25 mg/kg, mean peak lung concentration was 1.44 μg/g, which was 4.1 times greater than peak plasma concentration (0.35 μg/mL). Tmax for both lung and plasma was 1.0 h. AUC(0-∞) for lung was 7.4 μg·h/g, 3.7 times greater than that for plasma (2.0 μg·h/mL). Elimination half-life from lung (peak to 12 h) was 3.5 h, similar to plasma (3.3 h). A slower later elimination phase in lung with t1/2 ~4.3 h was suggested. [1]
Plasma protein binding of Danofloxacin Mesylate was concentration-dependent: 50±3% at 50 ng/mL, 42±3% at 100 ng/mL, 34±1% at 250 ng/mL, 29±1% at 500 ng/mL, and 26±1% at 1000 ng/mL. [1]
The pharmacokinetic properties of Danofloxacin have been characterized in target species. After oral administration in chickens, Danofloxacin is rapidly absorbed with peak plasma concentrations reached within 1-2 hours. Bioavailability is approximately 60-80%. In cattle, after subcutaneous injection, peak plasma concentrations are reached within 1-2 hours, and bioavailability is approximately 80-100%. In pigs, after intramuscular injection, peak concentrations are reached within 1-2 hours, with bioavailability of approximately 70-90%. Danofloxacin has a large volume of distribution (3-6 L/kg), indicating extensive tissue distribution. It penetrates well into respiratory tissues, achieving concentrations in lung tissue that are 2-10 times higher than plasma concentrations. Plasma protein binding is moderate (approximately 40-60%). The elimination half-life is approximately 4-8 hours in chickens, 6-10 hours in cattle, and 5-8 hours in pigs, allowing for once-daily dosing. Danofloxacin is primarily excreted unchanged in the urine (approximately 50-60%) and feces (approximately 20-30%). Metabolism occurs through glucuronidation and N-oxidation. The compound's pharmacokinetics are linear over the therapeutic dose range. Withdrawal times for meat and eggs have been established based on residue depletion studies.

Safety evaluation in various age groups of cattle in laboratory and field studies demonstrated that Danofloxacin Mesylate injectable has a wide margin of safety when the recommended dose is administered by IM or SC routes. No adverse effects were observed in field usage, and no adverse effects upon reproductive performance were observed following treatment of breeding cattle. [1]
The toxicity profile of Danofloxacin is favorable for veterinary use. In target species, the compound is generally well-tolerated at therapeutic doses. Common adverse effects are mild and include gastrointestinal disturbances (diarrhea, vomiting) and injection site reactions (swelling, pain). As with other fluoroquinolones, Danofloxacin can cause articular cartilage damage in juvenile animals (particularly in dogs and horses), but this is less of a concern in poultry, cattle, and pigs at therapeutic doses. The compound has a wide therapeutic index, with the NOAEL (no observed adverse effect level) in rats being approximately 50-100 mg/kg/day. The compound is not genotoxic or carcinogenic in standard toxicological studies. Danofloxacin is not recommended for use in animals with known hypersensitivity to fluoroquinolones. It should be used with caution in animals with renal or hepatic impairment. Proper withdrawal times must be observed to prevent residues in meat and eggs for human consumption.

J Vet Pharmacol Ther.1991 Dec;14(4):400-10;J Med Chem.1992 Feb 21;35(4):611-20.

Danofloxacin mesylate belongs to the quinoline class of compounds.

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Danofloxacin Mesylate is a fluoroquinolone antibacterial developed for veterinary use. Its mechanism of action is inhibition of bacterial DNA gyrase. It is indicated for the treatment of bacterial respiratory disease in cattle, particularly against Pasteurella haemolytica, Pasteurella multocida, and Haemophilus somnus. The MIC90 values against these pathogens (0.06-0.125 μg/mL) are substantially lower than the plasma and lung concentrations achieved after parenteral administration of 1.25 mg/kg, supporting clinical efficacy. [1]
Danofloxacin Mesylate (CP 76136-27) (CAS# 119478-55-6) is a fluoroquinolone antibiotic for respiratory disease in chickens, cattle, and pigs. It has a molecular formula of C20H24FN3O6S and a molecular weight of 453.48 g/mol. It primarily inhibits bacterial DNA gyrase and is freely soluble in water. Future research could focus on developing new formulations for improved delivery, investigating its activity against emerging respiratory pathogens, and optimizing dosing regimens to reduce the emergence of bacterial resistance.

C19H20FN3O3.CH4O3S

453.48

453.136

C, 52.97; H, 5.33; F, 4.19; N, 9.27; O, 21.17; S, 7.07

119478-55-6

Danofloxacin;112398-08-0;Danofloxacin-d3 mesylate;1217860-94-0

71334

Solid powder

569.3ºC at 760 mmHg

337-339ºC

298.1ºC

8.41E-14mmHg at 25°C

2.654

2

10

3

31

772

2

S(C([H])([H])[H])(=O)(=O)O[H].FC1C([H])=C2C(C(C(=O)O[H])=C([H])N(C2=C([H])C=1N1C([H])([H])[C@]2([H])C([H])([H])[C@@]1([H])C([H])([H])N2C([H])([H])[H])C1([H])C([H])([H])C1([H])[H])=O

APFDJSVKQNSTKF-FXMYHANSSA-N

InChI=1S/C19H20FN3O3.CH4O3S/c1-21-7-12-4-11(21)8-22(12)17-6-16-13(5-15(17)20)18(24)14(19(25)26)9-23(16)10-2-3-10;1-5(2,3)4/h5-6,9-12H,2-4,7-8H2,1H3,(H,25,26);1H3,(H,2,3,4)/t11-,12-;/m0./s1

1-Cyclopropyl-6-fluoro-1,4-dihydro-7-((1S,4S)-5-methyl-2,5-diazabicyclo(2.2.1)hept-2-yl)-4-oxo-3-quinolinecarboxylic acid, monomethanesulfonate

CP 76,136-27; CP-76,136-27; Danofloxacin monomethanesulfonate.

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)

Water : 91~100 mg/mL(~220.52 mM)
DMSO : ~20 mg/mL (~44.10 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (4.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2 mg/mL (4.41 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 20.0 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 3: ≥ 2 mg/mL (4.41 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 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Saline: 30 mg/mL

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Solubility in Formulation 5: 50 mg/mL (110.26 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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