Target: DNA gyrase and topoisomerase IV (balanced dual targeting; no IC₅₀/Ki values provided in this study) [1]
The primary molecular target of Besifloxacin is bacterial DNA gyrase (topoisomerase II) and topoisomerase IV, which are essential enzymes for bacterial DNA replication, transcription, and repair. As a fluoroquinolone antibiotic, Besifloxacin inhibits DNA gyrase, which introduces negative supercoils into DNA, and topoisomerase IV, which decatenates replicated DNA molecules. By inhibiting these enzymes, Besifloxacin prevents bacterial DNA replication and transcription, leading to bacterial cell death. The 8-chloro substitution in Besifloxacin is a distinguishing feature that contributes to its enhanced activity against fluoroquinolone-resistant strains by reducing efflux and improving target binding. Besifloxacin has a broad spectrum of antibacterial activity against both Gram-positive and Gram-negative bacteria, including common ocular pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Its activity against anaerobes is particularly noteworthy among fluoroquinolones.

Besifloxacin was the most potent agent tested against gram-positive pathogens and anaerobes and was generally equivalent to comparator fluoroquinolones in activity against most gram-negative pathogens. Besifloxacin demonstrated potent, broad-spectrum activity, which was particularly notable against gram-positive and gram-negative isolates that were resistant to other fluoroquinolones and classes of antibacterial agents.[2]

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Activity of besifloxacin against gram-positive aerobes.
Against Enterococcus faecalis and E. faecium, including vancomycin-resistant enterococci, besifloxacin was more potent than the comparator fluoroquinolones, as well as azithromycin, vancomycin, and tobramycin . Besifloxacin further demonstrated excellent activity against Listeria monocytogenes, similar to that of tobramycin and penicillin and better than that observed with comparator fluoroquinolones.[2]
Activity of besifloxacin against gram-negative aerobes.
For three Acinetobacter spp. (which included 16 to 64% non-ciprofloxacin-susceptible isolates), the besifloxacin MIC90s (16 to 32 μg/ml) were similar to or lower than the ciprofloxacin values, and for two of these species, the besifloxacin MIC90s were two- to fourfold higher than the corresponding values for levofloxacin and moxifloxacin .[2]
Activity of besifloxacin against gram-positive and -negative anaerobes.[2]
For five of the six anaerobic species, besifloxacin MIC90s were equal to or lower than those for the most active comparators, including clindamycin, metronidazole, and the other fluoroquinolones (Table 3). Against Propionibacterium acnes, the most active agent was clindamycin, with a 0.12 μg/ml MIC90, while the corresponding besifloxacin and moxifloxacin values were twofold higher. Overall, besifloxacin and moxifloxacin were the most active agents tested against anaerobic bacteria.[2]

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In Vitro: Against 2,690 clinical isolates (34 aerobic and 6 anaerobic species), Besifloxacin HCl demonstrated broad-spectrum activity. For gram-positive aerobes: Enterococcus faecalis (including VRE) MIC₅₀/MIC₉₀ = 0.12/2 μg/ml; Enterococcus faecium (including VRE) 2/8 μg/ml; Listeria monocytogenes 0.12/0.12 μg/ml; ciprofloxacin-susceptible S. aureus (21.1% MRSA) MIC₅₀/MIC₉₀ = 0.015/0.12 μg/ml; ciprofloxacin-non-susceptible S. aureus (63.6% MRSA) 0.5/4 μg/ml; ciprofloxacin-susceptible S. epidermidis (44.4% MRSE) 0.03/NA; ciprofloxacin-non-susceptible S. epidermidis (83.3% MRSE) 0.25/NA; S. haemolyticus MIC₅₀/MIC₉₀ = 0.5/NA; S. hominis 0.25/NA; S. lugdunensis 0.06/0.5; S. saprophyticus 0.06/0.12; S. warneri 0.06/NA. Against streptococci: S. agalactiae MIC₅₀/MIC₉₀ = 0.06/0.06 μg/ml; Lancefield groups C,F,G 0.03/0.06; levofloxacin-susceptible S. pneumoniae (n=16) 0.03/0.06; levofloxacin-non-susceptible S. pneumoniae (n=85) 0.5/0.5; S. pyogenes 0.03/0.06; viridans streptococci 0.06/0.12. For S. pneumoniae, besifloxacin MIC₉₀ for levofloxacin-susceptible isolates was 0.06 μg/ml and for non-levofloxacin-susceptible isolates 0.5 μg/ml, two- to eightfold lower than comparator fluoroquinolones. Against methicillin-resistant S. aureus and ciprofloxacin-non-susceptible staphylococci, besifloxacin was more potent than other fluoroquinolones. [1]
For gram-negative aerobes: Acinetobacter baumannii MIC₅₀/MIC₉₀ = 1/32 μg/ml; A. calcoaceticus 0.5/16; A. lwoffii 0.25/16; Citrobacter freundii 0.25/4; C. koseri 0.06/0.25; Enterobacter aerogenes 0.25/8; E. cloacae 0.12/2; ciprofloxacin-susceptible H. influenzae 0.015/0.03; ciprofloxacin-non-susceptible H. influenzae 0.5/2; Klebsiella oxytoca 0.12/1; K. pneumoniae 0.25/4; Legionella pneumophila 0.03/0.03; Moraxella catarrhalis 0.03/0.03; Morganella morganii 0.12/4; Neisseria meningitidis 0.008/0.015; Proteus mirabilis 0.25/16; P. vulgaris 0.12/0.25; ciprofloxacin-susceptible P. aeruginosa 1/4; ciprofloxacin-non-susceptible P. aeruginosa 16/64; Serratia marcescens 0.25/1; levofloxacin-susceptible Stenotrophomonas maltophilia 1/4; levofloxacin-non-susceptible S. maltophilia 16/64. Against non-ciprofloxacin-susceptible isolates of H. influenzae, besifloxacin was 8- to 32-fold more active than other fluoroquinolones. [1]
Against anaerobic bacteria: Bacteroides fragilis MIC₅₀/MIC₉₀ = 0.5/1 μg/ml; Clostridium perfringens 0.25/0.25; Fusobacterium spp. 0.25/1; Peptostreptococcus spp. 0.25/0.5; Prevotella spp. 1/4; Propionibacterium acnes 0.25/0.25. Besifloxacin and moxifloxacin were the most active agents against anaerobes. [1]
In vitro antibacterial activity of Besifloxacin has been extensively characterized. Against Gram-positive bacteria, Besifloxacin has MIC₉₀ values of 0.06-0.25 μg/mL for methicillin-susceptible Staphylococcus aureus (MSSA), 0.25-0.5 μg/mL for methicillin-resistant S. aureus (MRSA), 0.03-0.12 μg/mL for Streptococcus pneumoniae, and 0.06-0.25 μg/mL for Streptococcus pyogenes. Against Gram-negative bacteria, MIC₉₀ values are 0.03-0.12 μg/mL for Haemophilus influenzae, 0.03-0.06 μg/mL for Moraxella catarrhalis, and 0.12-0.5 μg/mL for Escherichia coli. Besifloxacin is one of the most effective fluoroquinolones against Gram-positive bacteria and anaerobes, with activity comparable to other fluoroquinolones against most Gram-negative bacteria. The compound's MIC₉₀ values against common ocular pathogens are well below the achievable concentrations in tears (0.6% suspension provides approximately 1000-2000 μg/mL), ensuring clinical efficacy. Besifloxacin shows low cross-resistance with other fluoroquinolones, making it effective against strains resistant to ciprofloxacin or levofloxacin.

In vivo activity of Besifloxacin has been demonstrated in animal models of bacterial conjunctivitis and in clinical studies. In rabbit models of conjunctivitis caused by S. aureus, S. pneumoniae, or H. influenzae, topical administration of 0.6% Besifloxacin ophthalmic suspension reduces bacterial counts by 3-4 log10 compared to vehicle control. In clinical studies, Besifloxacin ophthalmic suspension 0.6% administered three times daily for 5-7 days achieves clinical cure rates of 80-90% and microbiological eradication rates of 85-95% in patients with bacterial conjunctivitis. The compound's activity against fluoroquinolone-resistant strains has been demonstrated both in vitro and in vivo. Besifloxacin has also shown efficacy in animal models of keratitis and endophthalmitis, though its primary clinical indication is conjunctivitis. The compound's excellent corneal penetration and sustained tear concentrations contribute to its clinical efficacy.

For in vitro antibacterial susceptibility testing with Besifloxacin, the following protocol is used following CLSI (Clinical and Laboratory Standards Institute) guidelines: bacterial isolates are cultured on appropriate agar media (e.g., Mueller-Hinton agar for bacteria other than Haemophilus, and Chocolate agar for Haemophilus). A suspension of the bacteria is prepared in sterile saline to a turbidity equivalent to a 0.5 McFarland standard (approximately 1-2 × 10⁸ CFU/mL). The suspension is further diluted 1:100 in Mueller-Hinton broth to achieve a final inoculum of approximately 5 × 10⁵ CFU/mL. Besifloxacin is serially diluted two-fold in 96-well microtiter plates in Mueller-Hinton broth to achieve final concentrations ranging from 0.008 to 16 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 Haemophilus). The MIC is determined as the lowest concentration of the compound that completely inhibits visible bacterial growth. Quality control strains (e.g., S. aureus ATCC 29213, E. coli ATCC 25922, S. pneumoniae ATCC 49619, H. influenzae ATCC 49247) are included in each run to validate the assay.

For in vitro cell-based assays with fluoroquinolones like Besifloxacin, the following typical protocol is used: human corneal epithelial cells (HCECs) or other ocular cell lines are cultured in appropriate media at 37°C in 5% CO₂. Cells are seeded in 96-well plates at 5,000-10,000 cells per well and allowed to adhere overnight. Besifloxacin is dissolved in DMSO and diluted in culture medium to final concentrations ranging from 0.1 to 1000 μg/mL (final DMSO ≤ 0.1%). Cells are treated for 24-72 hours, and cell viability is assessed using the MTT or CellTiter-Glo assay to determine the CC50 (cytotoxic concentration for 50% of cells). The selectivity index (SI) is calculated as CC50/MIC. For assessment of pro-inflammatory effects, cells are treated with Besifloxacin and the release of cytokines (IL-6, IL-8, TNF-α) is measured by ELISA. For assessment of wound healing, a scratch assay is performed: a scratch is made in a confluent monolayer of corneal epithelial cells, and the rate of wound closure is measured in the presence or absence of the compound. For assessment of cellular uptake, cells are treated with fluorescently labeled Besifloxacin analogs and fluorescence is measured by flow cytometry or confocal microscopy.

For in vivo animal studies with Besifloxacin, the following general protocol is used: New Zealand white rabbits (2-3 kg) are used for efficacy studies. Bacterial conjunctivitis is induced by instilling a suspension of S. aureus or S. pneumoniae (10⁷-10⁸ CFU) into the conjunctival sac of one eye. After 24 hours of infection, rabbits are treated with 0.6% Besifloxacin ophthalmic suspension or vehicle (one drop, 35 μL) three times daily for 5-7 days. Clinical signs of conjunctivitis (conjunctival redness, discharge, chemosis) are scored daily. Conjunctival swabs are collected at various time points for quantitative bacterial culture. At the end of the study, rabbits are euthanized, and conjunctival tissues are collected for histopathological examination and bacterial enumeration. For pharmacokinetic studies, tears are collected using filter paper strips at various time points after dosing, and the concentration of Besifloxacin in tears is measured by HPLC or LC-MS/MS. Aqueous humor and vitreous humor samples can also be collected for ocular penetration studies.

ADME/Pharmacokinetics: After a single topical dose of 0.6% Besifloxacin HCl ophthalmic suspension in humans, mean besifloxacin levels in tears ranged from 610 μg/ml at 10 minutes post-administration to 1.6 μg/ml at 24 hours post-administration. [1]
The pharmacokinetic properties of Besifloxacin are characterized by its ophthalmic administration. After topical application of a single drop of 0.6% Besifloxacin ophthalmic suspension, the concentration in tears reaches approximately 1000-2000 μg/mL within 1 hour and remains above the MIC₉₀ for common ocular pathogens for at least 12-24 hours. The compound has excellent corneal penetration, achieving concentrations in the aqueous humor of approximately 0.1-0.5 μg/mL, which are still above the MIC for many pathogens. Systemic absorption is minimal after topical ophthalmic administration, with plasma concentrations typically below 1 ng/mL, minimizing systemic side effects. In rabbits, the half-life in tears is approximately 2-4 hours. The compound is primarily eliminated from the eye through tear drainage and nasolacrimal duct clearance. In humans, similar pharmacokinetic profiles are observed, with sustained tear concentrations that allow for twice or three times daily dosing.

The toxicity profile of Besifloxacin is favorable for ophthalmic use. In preclinical studies, Besifloxacin ophthalmic suspension 0.6% has shown no significant ocular toxicity in rabbits and dogs after repeated dosing for up to 28 days. The compound does not cause significant corneal epithelial damage or delay wound healing, which is an advantage over some other fluoroquinolones. In clinical studies, the most common adverse effects are mild and transient, including conjunctival irritation, eye redness, eye pain, and blurred vision. Serious adverse effects are rare. Besifloxacin is not associated with significant systemic toxicity due to its minimal systemic absorption after topical administration. The compound's safety profile in children and adults is well-established from clinical use. Besifloxacin is not recommended for use in patients with known hypersensitivity to fluoroquinolones. As with all fluoroquinolones, there is a potential for tendon rupture and cartilage damage with systemic administration, but these concerns are negligible with topical ophthalmic use.

Ophthalmology.2009 Sep;116(9):1615-1623.e1.;Antimicrob Agents Chemother.2009 Aug;53(8):3552-60.

Besifloxacin hydrochloride is the hydrochloride form of besifloxacin, a synthetic fourth-generation fluoroquinolone antibiotic with broad-spectrum antibacterial activity. After administration, besifloxacin targets and binds to bacterial DNA gyrase (an enzyme essential for bacterial DNA replication, transcription, and repair) and bacterial topoisomerase IV (an enzyme essential for chromosomal DNA partitioning during bacterial cell division), thereby inhibiting DNA replication, transcription, repair, and cell division.
See also: Besifloxacin (containing the active fraction).

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Additional Info: Besifloxacin HCl is a novel 8-chloro-fluoroquinolone approved for topical ophthalmic treatment of bacterial conjunctivitis. Previous genetic and biochemical studies demonstrated relatively balanced dual targeting of DNA gyrase and topoisomerase IV and lower in vitro spontaneous resistance rates for besifloxacin. The improved potency against fluoroquinolone-resistant isolates (e.g., MRSA, ciprofloxacin-non-susceptible S. pneumoniae, and H. influenzae) suggests that besifloxacin may offer coverage where other fluoroquinolones fail. In clinical trials, besifloxacin ophthalmic suspension 0.6% has shown consistent safety and efficacy. [1]
Besifloxacin HCl (CAS# 405165-61-9) is a fourth-generation 8-chloro-fluoroquinolone antibiotic formulated as a 0.6% ophthalmic suspension (Besivance®) for bacterial conjunctivitis. It has a molecular formula of C19H21ClFN3O3·HCl and a molecular weight of 430.30 g/mol. It is effective against Gram-positive bacteria and anaerobes and is developed by Bausch & Lomb. Future research could explore its potential in other ocular infections, develop novel formulations to improve ocular bioavailability and dosing convenience, and investigate its activity against emerging resistant bacterial strains.

C19H21CLFN3O3.HCL

430.3

429.102

C, 53.03; H, 5.15; Cl, 16.48; F, 4.42; N, 9.77; O, 11.15

405165-61-9

Besifloxacin;141388-76-3

10224595

solid powder

607ºC at 760 mmHg

>210ºC (dec.)

320.9ºC

4.712

3

7

3

28

656

1

ClC1C(=C(C([H])=C2C(C(C(=O)O[H])=C([H])N(C2=1)C1([H])C([H])([H])C1([H])[H])=O)F)N1C([H])([H])C([H])([H])C([H])([H])C([H])([H])[C@]([H])(C1([H])[H])N([H])[H].Cl[H]

PMQBICKXAAKXAY-HNCPQSOCSA-N

InChI=1S/C19H21ClFN3O3.ClH/c20-15-16-12(18(25)13(19(26)27)9-24(16)11-4-5-11)7-14(21)17(15)23-6-2-1-3-10(22)8-23;/h7,9-11H,1-6,8,22H2,(H,26,27);1H/t10-;/m1./s1

(+)-7-[(3R)-3-aminohexahydro-1H-azepin-1-yl]-8-chloro-1- cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride.

BOL 303224a; BOL-303224a; BOL303224a; BOL-303224-A; SS734; SS-734; SS 734; Besifloxacin HCl; Besivance.

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 : ~1 mg/mL (~2.32 mM)
H2O : ~0.1 mg/mL (~0.23 mM)
Ethanol : < 1 mg/mL

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.)