| In this paper, for the rational usage of doxycycline(DOTC) and control the residues, the safety of doxycycline in channel catfish(Ictalurus punctatus), comparative pharmacokinetics, new dosage form, pharmacokinetics-pharmacodynamics model and the rapid determination of doxycycline were investigated.1. The Serum Biochemical Parameters and the damage of DOTC on channel catfish red blood cells were studied at different concentrations of DOTC(87.5,175,350 mg/kg). The results showed that the content of TG, T-CHO. ALP, AST, CRE, and BUN did not change obviously in the whole experimental stage, and compared with the control group, these parameters was not significant different(P>0.05). There was significant difference on ALT between the three administrational group and the control group in the first 4d, the micronucleus rate and nuclear anomalies rate of red blood cells became increasing, however, they were not significant different. The results indicated that the channel catfish liver cells could be damaged on a certain concentration of DOTC, and it has no cytotoxic effect on channel catfish at the concentration level of our experiment.2. Study on pharmacokinetics and residues of doxycycline in channel catfish.(1)The pharmacokinetics of DOTC in channel catfish was researched after a single oral administration at a dose of 20 mg·kg-1 in the different temperature (18±1℃and 28±1℃), and the parameters were compared with DOTC in johnny carp(Carassius auratus) at the same condition of experiment. In consequence, the concentration-time course of DOTC in channel catfish and johnny carp can all be described by a two-compartment open model after a single oral administration at the different temperature. The elimination rate was markedly different in different water temperature at the same tissues or different water temperature at the same tissues(P<0.05). At 18℃, the distribution and eliminate of DOTC was slower than at 28℃in plasma and tissues. The concentration of DOTC in tissues at 28℃were all higher than the lower temperature groups, and the Cmax(2) was exceeded than the corresponding Cmax(i). In blood, compare DOTC in johnny carp with channel catfish, the rate for drug absorption and elimination were slower, T1/2 Ka and Tp were much more faster and T1/2βwas slower, and AUC was only 1/3 of it in channel fish.(2)The residues of doxycycline in channel catfish was researched after a singleoral administration at a dose of 20 mg·kg-1 in the different temperature (18±1℃and 28±1℃). The results indicated that the depletion speed of DOTC in channel catfish closely relates with the water temperature. To compare with other tissues, the elimination of DOTC residue in muscle and skin was slowest, liver was the main reservoir of DOTC residue in channel catfish. And if take an edible tissue of muscle and skin as target tissue in this experiment, the withdrawl period should not be less than 22 d和19 d at 18℃and 28℃respectively.3. The preparation and evaluation of DOTC chitosan nanoparticles freeze-dried powder.(1)DOTC-CS-NPs freeze-dried powder was prepared by using freeze-drying and ionic gelation method. The preparation was optimized by orthogonal experimental design with encapsulation efficiency as the index. The particle morphology, size and potential were determined respectively by transmission electron microscopy, particle and potentiometric analyzer. The result showed that The DOTC-CS-NPs freeze-dried powder has a feasible process of preparation, characteristic of good stabilities, and significant slow-release characteristic. The optimal preparation process of DOTC-CS-NPs was 2.5mg/mL chitosan solution,1.2mg/mL sodium tripolyphosphate solution,8mg DOTC and pH 5.5. The DOTC-CS-NPs freeze-dried powder had a fluffy surface and good solubility. The DOTC-CS-NPs were discrete and uniform spheres under transmission electron microscopy with a mean particle size of (126.3±17.8) nm. The encapsulation efficiency and drug loading of DOTC-CS-NPs were (56.52±2.1)% and (16.83±0.27)%, respectively. The ultraviolet, thermal and humid stabilities were measured. Compared to DOTC raw material drug, the ultraviolet degradation of DOTC-CS-NPs freeze-dried powder in 10 d under bright lights of (4500±500) Lx was decreased by 20.37%, and the thermal degradations in 10 d were decreased by 16.75% at 40℃and 29.53% at 60℃, while the humid degradations in 10 d were decreased by 19.43% at 75% and 25.88% at 92.5%.The release speed from fast to slow was artificial gastric juice, artificial intestinal juice and pH7.4 buffer phosphate. When the pH of artificial gastric juice was from 2 to 4, the release speed of freeze-dried powder decreased as the pH of artificial gastric juice increased. (2)The pharmacokinetics and tissue distribution of DOTC-CS-NPs freeze-dried powder and DOTC raw material drug in channel catfish were researched. After a single oral administration at a dose of 20 mg·kg-1 at (25±1)℃. In consequence, The double-peak phenomenons of plasma, liver, kidney, muscle were turned into single-peak. Compared to DOTC raw material drug, the peak concentrations of plasma and tissues decreased and the peak times prolonged. In plasma, the peak time and terminal elimination half-life of DOTC-CS-NPs freeze-dried powder prolonged, and the peak concentration decreased. The area under concentration-time curve was larger, and the relative bioavailability of DOTC-CS-NPs freeze-dried powder was 116% of the DOTC raw material drug.4. Study on pharmacokinetics-pharmacodynamics model(PK-PD model) of DOTC against Aeromonas hydrophila(A.h).(1)an in vitro PK-PD model was developed, and the PK-PD relationship of DOTC against A.h was described by the model. In consequence, in the in vitro model with elimination half-life of 38.61h, A.h was inhibited for only 3h by 2 MIC DOTC, and A.h regrew after the model worked about for 3h.4,8 and 16 MIC DOTC showed continously inhibitive action on A.h. It concluded that the inhibiting effect of DOTC against A.h was closely related to its concentration, and continous inhabitation could be achieved at the condition of Cmax/MIC>3.96.(2)An ex vitro PK-PD model of DOTC against A.h was established. For the rational usage of DOTC to treat the haemorrhagic septicemia of channel catfish, the integration of pharmacokinetics in vivo and pharmacodynamics ex vivo was used to study the antibacterial activity of DOTC against A.h in channel catfish serum ex vivo. The data were analyzed with the pharmacokinetic computer program kinetica 4.4 and 3p97. The results indicated that DOTC was absorbed fast and eliminated slowly after oral administration(20mg·kg-1), the Cmax was 1.72μg/mL at 2.57h, Elimination half-life (T(1/2)β)was 38.63h. EC50 was 16.95h. The PK-PD model parameter Cmax/MICserum was 0.86, AUCo→24h/MICserum was 20.57h. We estimated the optimal dose ranged from 10.68 to 41.42 mg per kilo of body weight through the Inhibitory Effect Sigmoid Emax. The conclusion is that when DOTC was employed to treat the haemorrhagic septicemia of channel catfish, dosage regime should be fed mixed feed which contained DOTC 41.42 mg per kilo of body weight,once a day. When DOTC was employed to provent the disease, dosage regime should be fed mixed feed which contained DOTC 10.68 mg per kilo of body weight,once a day.5. The improved two-step carbodiimide method of connecting DOTC hapten with carrier protein BSA to make artificial antigen of DOTC. at the same time, use the same method to connect DOTC with the carrier protein OVA to make artificial coating antigen. On the one hand, UV scanning analysis and animal immune tests confirm the success in preparation of artificial antigen. On the other hand, the titer of the antiserum acquired from animal tests is 2.048×106, and the combination ratio of DOTC and BSA is 3:1 by calculating. We established a ci-ELISA for detecting the residues of DOTC through the artificial antigen above. Comparing with HPLC method for determination of residues of DOTC, the repeatability, sensitivity, and accuracy parameters showed this ci-ELISA method could meet the needs of the actual detection of DOTC.
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