Preparation Of Florfenicol Nanocrystals And Studies On The Properties Of Florfenicol Nanocrystals Both In Vitro And In Vivo

FF is a new kind of amido alcohol antibiotic.It has the advantages of wide antibacterial spectrum and no regenerative anemia disorder,which is widely used in veterinary animal husbandry.However,in practical applications,FF has some disadvantage,such as poor water solubility and low bioavailability,which causes poor curative effects.In this paper,FF-NC were prepared by top-down technology in order to improve its water solubility and bioavailability,so as to increase its efficiency and reduce its toxicity.In addition,the absorption and IVIVC of FF-NC were investigated to provide theoretical basis for the large-scale production of the preparation.The main research contents and results are as follows:?1?Preformulation study of FFA determination method of FF in nanocrystals was established and validated by methodology.The properties of FF were investigated,including specific rotation,oil-water partition coefficient,solubility and dissolution rate.Besides,the effects of strong light,high humidity,and high temperature on the stability of the FF drug were also researched.The results show that the determination method has good reproducibility and stability.In addition,the excipients have no interference to the determination.The measured specific rotation of the original drug was-18°,which was in accordance with the provisions of"florfenicol"of the Chinese veterinary pharmacopoeia?2015 edition?.The oil-water partition coefficient of the original drug was measured to be 1.70,indicating that FF belongs to the BCS class II.The solubility of the original drug in water was only 1.159 mg·mL-1,and the cumulative dissolution percentage in one hour was less than 50%when water was used as the dissolution medium.Stress testing results indicated that high temperature,high humidity as well as strong illumination were little effect on the stability of the active pharmaceutical ingredients.?2?Study on the preparation technology of FF-NCThe miniaturized media grinding method was used to conduct single-factor investigations on the formulation factors?such as stabilizer type,drug amount and stabilizer dosage?as well as process factors?such as grinding media dosage and temperature?under a lab-scale.The formulation was optimized by box-behnken design-response surface method,then the optimized formulation will be prepared by enlargement and spray drying.The particle size,zeta potential,saturation solubility,and dissolution of FF-NC were also investigated after redissolution.Besides,the powder pro,accelerated stability and long-term stability of solidified products were investigated.Morphology and crystal form of FF-NC were characterized by FTIR,DSC,XRD,SEM technology.The final preparation formula were as follows:appropriate amount of HPMC _E55 and SDS were swelling in water so as to create a polymer solution,then was added into the mixer at a dispersion speed of 800 rpm.At the same time,FF were added for premixing 30 min.Then,the FF-NC suspension was obtained after grinding in chamber.FF-NC suspension was solidified by spray drying to obtain solid powder,which was blue opalescent after reconstitution.The average particle size and PDI of three batches of cured products was 265.8±18.5 nm and 0.163±0.023separately.After redissolution,the particle size and PDI of FF-NC were276.4±19.4 nm and 0.166±0.011,resepectively.Angle of repose and bulk density measurement results show that the fluidity of the cured product conforms the requirements in the production process.Accelerated and long-term stability test results show that under the above conditions,the dissolution rate,drug content,and particle size of the cured product did not changed significantly.SEM results show that FF-NC was spherical and evenly distributed.DSC and XRD results showed that the crystal form of nanoparticles in FF-NC was amorphous.In the medium with different pH,the solubility and dissolution rate of nanocrystals were significantly improved compared with the original drug,moreover,the dissolution rate was less affected by p H.?3?Study on the pharmacodynamics of FF and FF-NC in vitroIn vitro bacteriostatic experiments were performed to investigate the effects of FF and FF-NC on the MIC and MBC of E.coli and staphylococcus aureus.The size of bacteriostatic ring was also determined by Oxford cup method.MTT assay was used to investigate the cytotoxicity of FF and FF-NC on RAW 264.7 cells.Effects of FF and FF-NC on inflammatory factors TNF-?,IL-6 and NO content were investigated using Elisa and Gress reagent method.The results showed that the MIC of FF-NC against E.coli and S.aureus was 77.5%and 80.0%of the original drug,respectively.The MBC of FF-NC was comparable to that of FF and the diameter of inhibition zone was about 4 times of FF and 1.3 times of FF commercially available premix.Results of MTT assay indicated that the FF and FF-NC did not show significant cytotoxicity on RAW264.7 cells within the concentration range of 20-100?g·mL^-1.During the same low dose concentration(5?g·mL^-1),the inhibition rate of TNF-?by nanocrystals was 15.55%higher than that of FF,which was similar to the inhibition rate of 25?g·mL-1 of FF?29.58%?.For the LPS group,the inhibition rates of IL-6 secretion in FF group reached 15.82%,24.92%and 49.93%separately,while the inhibition rates in FF-NC group at the same dose concentration reached 21.27%,35.81%,and 70.46%,respectively.Both groups showed dose-dependent inhibition of IL-6,and the inhibition of FF-NC was stronger than FF at the same dose concentration.The measurement results of NO content showed that the inhibitory effect of 5?g·mL-1 FF-NC on NO production was equivalent to that of 100?g·mL-1 FF,and returned NO content to normal cell levels.?4?Investigation of FF and FF-NC absorption in small intestine in ratsSingle-pass intestinal perfusion model was used to investigated the absorption charateristics of FF and FF-NC in different segments of intestine,and the oral toxicity of the preparation was investigated through histological examination to initially evaluate safety of the preparation.The results showed that the main absorption segment of FF-NC and FF were duodenum,followed by jejunum and ileum.Compared to FF group,the K_a value of FF-NC were improved by 2.28,1.25 and 10.1-fold in the duodenum,jujenum and ileum separately,while P_effff value was improved by 1.59,1.17 and 7.0-fold in the above segment of intestine.The absorption of FF-NC in all parts of the small intestine was higher than that of FF,And nanocrystals mainly improves the absorption of FF in ileum.Histological evaluation of the small intestine showed no significant effects on the intestinal morphology,which mean that the FFand FF-NC possess low oral toxicity.?5?Study on the pharmacokinetics of FF-NC and IVIVCWith FF as the control,the pharmacokinetics study on FF-NC was caried out using rats by oral gavage.In combination with the results of dissolution characteristics in vitro,Wagner-Nelson formula was used to establish the IVIVC.According to the pharmacokinetic parameter of oral aiministration,the in vivo pharmacokinetic behavior of FF and FF-NC fitted the one-compartment model.In addition to T_max,the major pharmacokinetic parameters of FF-NC group(C_max,T_1/2,AUC_0-t,MRT,CL,V)were significantly different compared with the FF group.The CL of the FF-NC groups and FF groups were 0.53 L·h-1·kg-1 and 2.48 L·h-1·kg-1,respectively.AUC_0-t,C_maxax and MRT of the FF-NC groups were 38.51mg·L-1·h-1,9.51 g·mL-1 and 3.23 h separately,which increased by 4.62,2.06 and 1.68 times compared with the FF group.The results of IVIVC investigation showed that the in vitro dissolution of FF-NC in the simulated intestinal fluid medium had a good correlation with the drug absorption fraction in vivo.Therefore,the law of drug absorption in vivo can be predicted based on the in vitro dissolution.To sum up,FF-NC drug delivery system was constructed to improve the water solubility and bioavailability of FF,so as to enhance antibacterial and anti-inflammatory effects.At the same time,in vivo intestinal absorption and in vivo-in vitro correlation models were established.This will provides certain experimental reference for the development and application of poorly soluble drugs.