STUDY ON PHARMACOKINETICS/PHARMACODYNAMICS MODEL OF DOXYCYCLINE AGAINST AEROMONAS HYDROPHILA IN SERUM OF CHANNEL CATFISH EX VIVO

In order to determine reasonable dosage regimens of doxycycline in treating the haemorrhagic septicemia of channel catfish (Ictalurus punctatus), we integrated of pharmacokinetics in vivo and pharmacodynamics ex vivo to study the antibacterial activity of doxycycline against Aeromonas hydrophila in channel catfish serum. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of doxycycline against A. hydrophila in extract broth and serum were measured by using the micro-dilution method, in which the final concentration of A. hydrophila was 106 CFU/mL and the incubating temperature was 28℃. The pharmacokinetics study of doxycycline was investigated after single oral gavage administration in channel catfish at water temperature of (281)℃. Blood samples were collected at every different intervals before and after administration, and then made them to be plasma and serum. The concentrations of doxycycline in channel catfish plasma were determined by means of Ultra Performance Liquid Chromatography (UPLC) with UV detection method. The plasma samples were extracted with perchloric acid, then the extracts were centrifuged and the supernatant was sent to UPLC-UV after purified by 0.22 m filter. 5 L extract was separated on a reversed phase C18 reversion chromatographic column (1.7 m, 2.1 mm50 mm) at 45℃, which was then eluted with 0.01 mol/L sodium dihydrogen phosphateacetonitrile (4∶1) at a flow of 0.300 mL/min. The UV detection was at 350nm. Pharmacokinetic parameters were analyzed with the pharmacokinetic computer program 3P97. We learnt the pharmacodynamics effect of doxycycline by detecting the number of A. hydrophila in channel catfish serum sampled at different times. Pharmacodynamics parameters were analyzed with the pharmacodynamic computer program kinetica 4.4. The Pharmacokinetics/pharmacodynamics model parameters were calculated with the MICserum and pharmacokinetic in vivo parameters. The accurate MIC of doxycycline against A. hydrophila in broth and serum were both 2.0 g/mL, which revealed that doxycycline had high bactericidal activity to A. hydrophila in different mediums. Doxycycline was absorbed fast and eliminated slowly after oral administration (20 mg/kg), the Cmax was 1.72 g/mL at 2.57h, elimination half-life T(1/2) was 38.63h. A. hydrophila were killed obviously and inhibited for only 3h by doxycycline in 1-8h serum, and A. hydrophila regrew after about 6h. However, the doxycycline in 4h serum could kill A. hydrophila to 103 level and continuously inhibited for 12h. The doxycycline in serum collected after 12h could hardly kill A. hydrophila, and A. hydrophila quickly regrew to 108-1010 level. Half-effective concentration parameter (EC50) was 16.95h, which meant doxycycline produced 50% of the maximal antibacterial effect when the concentration of doxycycline was 1.41 g/mL in serum. The PK-PD model parameter Cmax/MICserum was 0.86, AUC024h /MICserum was 20.57h. We estimated the optimal dose ranged from 10.68 to 41.42 mg/kg per kilo of body weight through the Inhibitory Effect Sigmoid Emax. It concluded that when doxycycline was employed to treat the haemorrhagic septicemia of channel catfish, dosage regime should be fed with mixed feed which contained doxycycline 41.42 mg per kilo of body weight, once a day. When doxycycline was employed to prevent the disease, dosage regime should be fed mixed feed which contained doxycycline 10.68 mg per kilo of body weight, once a day.