Antibiotic; anti-inflammatory
Florfenicol targets the bacterial ribosome, specifically the 50S and 70S ribosomal subunits, where it inhibits peptidyl transferase activity. By binding to these ribosomal subunits, florfenicol prevents the transfer of amino acids to growing peptide chains during protein synthesis, thereby inhibiting bacterial protein synthesis and preventing bacterial growth. The fluorination of the thiamphenicol molecule helps to overcome resistance to chloramphenicol and thiamphenicol. Florfenicol is effective against a broad range of Gram-negative and Gram-positive bacteria.

Florfenicol (FLO) is one of the most popular antibiotics used in veterinary clinic and aquaculture. FLO can inhibit both bacterial and mitochondrial protein synthesis. However, the effects of FLO on mitochondrial function and cellular homeostasis remain unclear. Here we show that FLO inhibits expression of mitochondrial DNA-encoded proteins, decreases mitochondrial membrane potential, and promotes generation of reactive oxygen species (ROS) in vitro. As a result, activities of mitochondrial respiratory chain complex I and IV and the cellular ATP level are decreased and mitochondrial morphology is damaged. FLO represses cell growth and proliferation by suppression of phosphorylation of p70S6K through AMPK/mTOR/p70S6K pathway. Furthermore, FLO also induces G0/G1 cell cycle arrest via increase of p21 levels through activating ROS/p53/p21 pathway. Moreover, the clearance of damaged mitochondria by autophagy is impaired, leading to cell proliferation inhibition and promotes cell senescence. In addition, FLO-induced upregulation of cytosolic p53 may contribute to mitophagy deficiency via regulation of Parkin recruitment. In summary, our data suggest that florfenicol is an inhibitor of mitochondrial protein synthesis that can induce noticeable cytotoxicity. Thus, these findings can be useful for guiding the proper use of FLO and the development of safe drugs[2].

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The ubiquitous use of antibiotics leads exposure of these chemicals on non-target aquatic species, while the toxicity assays for these chemicals are time/labor consuming and expensive. Alternative approaches using primary cell cultures which retain the tissue functionality at its highest form have received global attention compared to cell lines. In the current study, the cytotoxic effects of two commonly used antibiotics from amphenicol (florfenicol) and macrolide (erythromycin) groups were evaluated on primary cell cultures of Unio crassus (mantle, digestive gland, gill, and gonad) and Cyprinus carpio (gill and liver) using MTT and Neutral Red assays. The highest cytotoxic effects were found on the mussel digestive gland and carp liver cells for florfenicol and erythromycin, while the lowest cytotoxic effects were found in mussel mantle cells for both drugs in the MTT test. In the NR test, the highest cytotoxic effects of erythromycin and florfenicol were found in the mussel gill, mantle, gonad, and carp gill cells; the lowest cytotoxic effect of erythromycin was found in the mussel digestive gland, while the lowest effect of florfenicol was found in the carp liver cells. The cytotoxicity of florfenicol was quite low for the carp liver, while the cytotoxicity of erythromycin was quite low in the mussel digestive tract. Thus, it was concluded that cells made from mussel tissues could be used in ecotoxicity tests, and sensitivity may vary according to the tissue.[3]
In vitro, florfenicol exhibits broad-spectrum antibacterial activity against a variety of bacterial pathogens, including Pasteurella, Escherichia coli, and other susceptible organisms. Its ability to inhibit bacterial protein synthesis has been demonstrated in cell-free translation assays. Minimum inhibitory concentrations (MICs) vary depending on the organism. Florfenicol is effective against bacteria that are resistant to chloramphenicol and thiamphenicol due to its fluorinated structure.

Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent Gram-negative facultative intracellular bacterium. Although it is considered one of the most pathogenic bacteria in fish, there are no commercially available treatments or vaccines. The objective of this project was to determine the most efficacious concentration of florfenicol (FFC) [10, 15 or 20 mg FFC kg(-1) body weight (bw) per days for 10 days] administered in feed to control experimentally induced infections of Fno in Nile tilapia, Oreochromis niloticus (L.), reared in a recirculating aquaculture system. The cumulative mortality of fish that received 0, 10, 15 or 20 mg FFC kg(-1) bw per day was 60, 37, 14 and 16%, respectively. Francisella noatunensis subsp. orientalis genome equivalents were detected in water from all challenged groups with slight reduction in the concentration in the florfenicol-treated groups 4 days after treatment. The mean LOG of CFU Fno mg(-1) spleen was 3-5 and was present in all challenged groups at necropsy 11 days after treatment (21 days after challenge). Results show that florfenicol administered at doses of 15 and 20 mg FFC kg(-1) bw per days for 10 days significantly reduced mortality associated with francisellosis in Nile tilapia[1].
In vivo, florfenicol is used in veterinary medicine to treat bacterial infections in food animals, including poultry, swine, and cattle. It is effective against infections caused by Pasteurella and Escherichia coli. Florfenicol is administered via injection, feed, or water, depending on the species and indication. The compound's broad-spectrum activity and favorable pharmacokinetic properties support its use in veterinary medicine.

In vitro enzyme/receptor binding assays for florfenicol are not typically performed because its antibacterial activity is assessed using microbiological methods. However, the compound's mechanism of action can be studied using assays that measure its ability to inhibit protein synthesis. In these assays, bacterial ribosomes are incubated with a labeled amino acid and varying concentrations of florfenicol, and the incorporation of the label into protein is measured.

Cell growth curve analysis[2]
L cells were trypsinized and plated in individual wells of 24-well plates for overnight. Cells were then treated with 0.4, 0.1 and 0.025 mg/mL Florfenicol (FLO). Every 12 h, the medium was removed, adherent cells were trypsinized and the total number of adherent cells in each well was quantified using Guava easyCyte flow cytometry. The cells counts for 3 wells/time-point were averaged for each group and the data were used to draw growth curves.

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Cell proliferation assay[2]
For CCK-8 cell proliferation assay, cells in an exponential phase of growth were harvested and seeded in 96-well plates. After 12 h culture, the medium was removed and changed to glucose medium or galactose medium supplemented with multiple doses of Florfenicol (FLO). The medium was removed and 100uL PBS containing 10uL CCK-8 solution was added into each well and incubated for an additional 1 h and the absorbance was determined using BIO-RAD Model 680 Microplate Reader at 450 nm. The percentage of cell viability was calculated as follows: cell viability (%) = ODFlorfenicol (FLO)/ODDMSO × 100%. For EdU cell proliferation assay, cells were grown on coverslips and treated with either DMSO or Florfenicol (FLO) for 48 h. After treatment, The thymidine analog EdU stock solution was diluted and added into the culture medium, and incubated for 2 h. Cells were washed with PBS, fixed in 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. Apollo® staining solution was added into the wells post washing and incubated for 30 min in the dark. The cells were then counterstained with Hoechst33342 for nuclei. Cells were mounted and examined by using a Nikon microscope with Image-Pro Plus software for image analysis.
In vitro cellular assays for florfenicol are performed using standard microbiological susceptibility testing methods. The minimum inhibitory concentration (MIC) is determined using broth dilution or agar dilution methods according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Susceptibility testing is performed using a range of bacterial strains, including both Gram-positive and Gram-negative organisms. These assays confirm the antibacterial activity of florfenicol and guide veterinary use.

In-feed Medication has been used for a long time to prevent coccidiosis, a worldwide protozoal disease in rabbits. Florfenicol (FFC) has been widely used in veterinary clinics for bacterial diseases treatment. Therefore, the use of combinations of coccidiostats with FFC in rabbits is common. In the present study, we aimed to evaluate the effect of three coccidiostats, sulfaquinoxaline (SUL), robenidine (ROB), and toltrazuril (TOL), as feed additives on the pharmacokinetic profile of FFC in rabbits. The disposition kinetics of FFC in rabbits were investigated after a single intravenous injection (25 mg/kg) in rabbits fed anticoccidial-free diets or feeds containing SUL (250 ppm), ROB (66 ppm), or TOL (2 ppm), respectively, for 20 days. Plasma FFC concentrations were determined by the high performance liquid chromatography (HPLC) method. The pharmacokinetic parameters of FFC were analyzed using a non-compartmental analysis based on the statistical moment theory. The results demonstrated that ROB feeding resulted in an obvious decrease in plasma FFC level as compared with anticoccidial-free feeding. The terminal elimination half-life (t1/2z), area under the concentration-time curve (AUC), area under the first moment curve (AUMC), and mean residence time (MRT) significantly decreased, whereas the elimination rate constant (λz) and total body clearance (CLz) obviously increased in rabbits pretreated with ROB. However, we did not find that SUL or TOL feeding had any effect on the pharmacokinetic profile of FFC. Our findings suggested that more attention should be paid to the use of FFC in rabbits supplemented with ROB. https://pubmed.ncbi.nlm.nih.gov/25319758/
In vivo animal experiments for florfenicol are conducted in food animal species to evaluate its efficacy in treating bacterial infections. Chickens, pigs, or cattle are infected with a bacterial pathogen and then treated with florfenicol via injection, feed, or water. The primary endpoints include survival, bacterial clearance from tissues, and improvement in clinical signs. These studies support the compound's approval for veterinary use.

Florfenicol has a molecular weight of 358.21 g/mol and a molecular formula of C12H14Cl2FNO4S. The compound is a white to light yellow powder or crystal. It is slightly soluble in water and chloroform, sparingly soluble in glacial acetic acid, and soluble in methanol, ethanol, and dimethylformamide. Detailed pharmacokinetic parameters have been characterized in veterinary pharmacokinetic studies. Florfenicol is widely distributed in tissues and is excreted primarily via the renal route.

The intravenous LD50 in mice was 100 mg/kg. (Future Drugs, 7(172), 1982)
Florfenicol has been evaluated in veterinary toxicology studies and has been reported to be well-tolerated at therapeutic doses. As a bacteriostatic antibiotic, florfenicol may cause adverse effects including gastrointestinal disturbances and, rarely, bone marrow suppression. The compound is approved for use in food animals with appropriate withdrawal periods to ensure food safety. Florfenicol is not approved for use in humans.

[1]. J Fish Dis, 2013, 36(4), 411-418.
[2]. Sci Rep. 2017 Oct 19;7(1):13554.
[3]. Environ Sci Pollut Res Int. 2021 Sep;28(35):48408-48416.

Florfenicol is a carboxamide, an N-dichloroacetyl derivative of (1R,2S)-2-amino-3-fluoro-1-[4-(methanesulfonyl)phenyl]prop-1-ol. It is a synthetic veterinary antibiotic used to treat bovine respiratory diseases and hoof rot, and is also used in aquaculture. It has antibacterial activity. It is a sulfone, secondary alcohol, organofluorine compound, organochlorine compound, and secondary carboxamide. Its function is related to dichloroacetic acid. Florfenicol is a fluorinated synthetic analog of thiamphenicol. See also: Florfenicol; Flunixin meglumine (ingredient); Betamethasone acetate; Florfenicol; Terbinafine (ingredient); Florfenicol; Mometasone furoate; Terbinafine (ingredient).
Florfenicol is a synthetic fluorinated derivative of thiamphenicol and a broad-spectrum bacteriostatic antibiotic used in veterinary medicine. It inhibits bacterial protein synthesis by binding to ribosomal subunits (50S and 70S). The fluorination of the thiamphenicol molecule helps to overcome resistance to chloramphenicol and thiamphenicol. Florfenicol is used to treat bacterial infections in poultry, swine, and cattle. It is not approved for human use.

C12H14CL2FNO4S

358.21

357

C, 40.24; H, 3.94; Cl, 19.79; F, 5.30; N, 3.91; O, 17.87; S, 8.95

73231-34-2

Florfenicol-d3;2213400-85-0

114811

Typically exists as White to off-white solid at room temperature

1.5±0.1 g/cm3

617.5±55.0 °C at 760 mmHg

153 °C

327.3±31.5 °C

0.0±1.9 mmHg at 25°C

1.548

-0.12

2

5

6

21

447

2

ClC([H])(C(N([H])[C@]([H])(C([H])([H])F)[C@@]([H])(C1C([H])=C([H])C(=C([H])C=1[H])S(C([H])([H])[H])(=O)=O)O[H])=O)Cl

AYIRNRDRBQJXIF-NXEZZACHSA-N

InChI=1S/C12H14Cl2FNO4S/c1-21(19,20)8-4-2-7(3-5-8)10(17)9(6-15)16-12(18)11(13)14/h2-5,9-11,17H,6H2,1H3,(H,16,18)/t9-,10-/m1/s1

2,2-dichloro-N-[(1R,2S)-3-fluoro-1-hydroxy-1-(4-methylsulfonylphenyl)propan-2-yl]acetamide

Sch-25298; SCH25298; Florfenicol; 73231-34-2; Nuflor; (-)-Florfenicol; 76639-94-6; Nuflor gold; SCH 25298; Nuflor; Florfenicol

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 : ~100 mg/mL (~279.17 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.98 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 25.0 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: ≥ 2.5 mg/mL (6.98 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 3: ≥ 2.5 mg/mL (6.98 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.)