| Florfenicol, the chloramphenicol antibiotic, belongs to broad-spectrum antibiotic. F atoms and methyl-sulfoxide (CH3SO2-) of florfenicol replace hydroxyl group (-OH) and the chloramphenicol nitro (-NO2) of the chloramphenicol in the structure. Florfenicol shows better antibacterial effect than chloramphenicol, and does not cause irreversible regeneration obstacles anemia at the same time. Unfortunately, florfenieol relatively poor aqueous solublility (1.335±0.030 mg/ml)hampers bioavailability. Then, the study of the water-soluble florfenicol has been intense in veterinary medicine field. At present, the main physical solubilization technology are solid dispersion, micronized, molecular inclusion and micro-emulsion. But the organic solvent used has irritation and toxicity, and can’t achieve the desired solubilizing effect. Accordingly, the water-soluble florfenicol prodrug which was preparated by chemical has good application prospect.The molecule of florfenicol has a secondary hydroxyl group that can be reacted with various substances to be water-soluble prodrug. In order to improve florfenicol solubility in water, we used phosphorus oxychloride for reagents to synthesis of florfenicol phosphate prodrug in this study. And selected the best technology of florfenicol phosphate synthesis process by uniform design test. The best process was:The phosphorus oxychloride (10 ml) and a certain amount of florfenicol reacted for 1 h at room temperature, then hydrolyzed by slow 20 ml water under the conditions of -20 to -15℃,and then stand in a refrigerator overnight. This test used reverse crystallization to recrystallized product to get florfenicol phosphate crystals.In this study, the melting point of florfenicol phosphate was 147.8～149.5℃, was white needle-like crystals with high purity. We analysis the new material by UV, IR, MS and HPLC. The UV absorption spectra showed that there may be phenyl, hydrocy (-OH), chorine group atom (-X) and carbonyl (-C=O) on the florfenicol prodrug. The retention time of florfenicol was 10.12 min and the florfenicol phosphate was 2.22 min. That was shown by HPLC analysis. The infrared absorption spectrum measurement showed that the prodrug and florfenicol had a similar structure, but the prodrug possessed the phosphorus ester groups. The mass spectrum shows clearly that there were florfenicol phosphate and the molecular weight was 438. Florfenicol phosphate can be hydrolyzed to florfenicol under acid or alkaline solution.In summary, florfenicol phosphate could be confirmed successful by the synthesis.The results of the solubility for florfenicol and florfenicol phosphate in pure water at 25℃ show that the solubility of florfenicol is 1.335±0.030 mg/ml, but the florfenicol phosphate esters is 695.997±0.210 mg/ml. Compared with florfenicol, the water-soluble of florfenicol phosphate increased obviously and improved more than 520 times. The melting point of forfenicol and forfenicol-phosphate were close, forfenicol was 153.2～153.7℃,and forfenicol-phosphate was 147.8～149.5℃. However, florfenicol phosphate appeared carbonation phenomenon at 127.9～0.4℃. At room temperature, saturated florfenicol was pH 5.2.But 10 mg/ml prodrug aqueous solution pH were 1.6, shown strongly acidic, so it could not make intravenous fluids. The stability test of florfenicol phosphat shown that it can’t tolerate on the acid-base, and affected by high temperature and humidity greatly. It can aqueous solution of fully hydrolyzed for 2 h in 37 ℃.Compare the pharmacokinetic for florfenicol phosphate sterile aqueous solution and florfenicol ordinary injection and both were administrated on health rabbits by intramuscular injection. Plasma drug concentration were determined by HPLC. Pharmacokinetic parameters were analyzed by DAS2.0 and SPSS19.0. The result shown that both group data complied with one-absorbtion and two-compartmet model (weight=1/CC). The main parameters of pharmacokinetic were as follows:The group of florfenicol sterile aqueous solution:Ka was 0.018±0.002 mg/min,T1/2a was 56.764±4.35 min, T1/2p was 253.875±65.445 min, AUC(0-∞) was 1744.209±41.755 mg/L*min, Cmax was 8.409±0.07 mg/L, Tmax was 60±0 min,the fitting equation was:C=46.315*e-0.012t +1.618*e-0.003t-47.884*e-0.018t; The group of florfenicol phosphate sterile aqueous solution: Ka was 0.02±0 mg/min,T1/2a was 48.045±4 min,T1/2β was 240.402±64.215 min, AUC(0-∞) was 2107.482±93.96 mg/L*min, Tmax was 70±17.321min, Cmax was 9.586±0.199 mg/L, the fitting equation was:C=57.617e-0.014t+3.662*e-0.003t-61.468*e-0.002t. The difference of Cmax and AUC(o-∞) was significant. The result indicated that florfenicol phosphate bioavailability has improved significantly.The study was based on prodrug design principles, and synthesised of florfenicol phosphate successfully. The method was simple, the yield was higher, conditions was unrigorous and suitable for industrial production. Compared with florfenicol, florfenicol phosphate increased solubility significantly, improved bioavailability in rabbits effectively. The experimental results has laid a solid foundation for florfenicol phosphate clinical application, and better play antibacterial properties for florfenicol. And provided a new way of thinking for further develop the florfenicol drugs. |
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