Preparation Of Doxycycline Enteric-coated Pellets And Studies On Bioequivalence Of Doxycycline In Piglets

Doxycycline is a member of the semisynthetic tetracycline antibiotics group, which shows 2-4 times greater antibacterial activity than tetracycline and minimum toxicity currently found in tetracycline group. Doxycycline is characterized by its high effectiveness and broad spectrum, thus widely used in clinical therapy. In respect of veterinary applications, there are only three main medicinal formulations:tablet, soluble powder and injection. The application of other formulations was less reported. Doxycycline is unstable when dissolved in water. It causes a few gastrointestinal side-effects after oral administration. The enteral pellets have prominent advantages, with its current preparation methods, materials and equipments quite sophisticated. This paper describes the preparation of doxycycline enteric-coated pellets and the bioequivalence study.According to the physical and chemical properties and spectral characteristics of Doxycycline, this paper established UV-spectrophotometric (UV) method for rapid investigation of doxycycline enteric-coated pellets drug release and high performance liquid chromatography (HPLC) for precise determination of Doxycycline concentrations in materials, preparations and biological samples. Reliability of these methods were confirmed by related methodology. The UV method establish double standard curves in both 0.1 mol/L hydrochloric acid (pH=1) and pH=6.8 buffer solution (750mL of 0.1 mol·L-1 hydrochloric acid solution added with 250mL of 0.2 mol·L-1 phosphatic sodium solution) to research doxycycline enteric-coated pellets release behavior. At 268nm, the correlation between absorbance(A) and Doxycycline concentration(C) are described by the following equations when the Doxycycline concentration ranged from 1μg·ml-1 to 40μg·ml-1:A1=0.0360 C + 0.0279, while at 272nm the equations:A2=0.0293 C + 0.0319. The released amount of drug in acidic solutions and buffer solutions met the requirement of enteral preparations described in the 2005 version of Pharmacopoeia of the People’s Republic of China volumeⅡ(C.P.2005Ⅱ),whereas in buffer solutions the rapid release met the physiological characteristics that doxycycline was mainly absorbed in forepart of small intestine. Basing on the chromatographic conditions described in C.P.2005II the content of doxycycline in materials, preparations were determined. Experimental results show the content of doxycycline in materials and test preparations were 97.32±0.75% and 99.68±0.71% respectively, with the RSD of recovery, precision, stability lower than 5%. For assaying the concentration of the drug concentration, the pig plasma samples were applied to chromatographic column Dikma C18 (250 mm×4.6 mm,5μm) and washed with mobile phase of acetonitrile-methanol-0.01 mol·L-1 oxalic acid solution (68:21:11) (pH value of 6.7 adjusted with phosphoric acid), the drug content were detected at the wavelength of 350 nm. The chromatographic conditions resulted in an excellent chromatogram, with no impure peaks affecting the measurement, indicating a great accuracy and coefficient of recovery under these conditions.This paper describes the preparation of doxycycline enteric-coated pellets by multifunctional extrusion-spheronization machine. The cores of pellets were made using starch and dextrin as diluent, HPMC water solution as adhesive. Based on the result from preliminary tests, an orthogonal-testing method considering five factors and four levels was designed with A:diluents (dextrin starch), B:adhesives (HPMC solutions), C:extrusion speed, D:roll speed and E:roll time as investigation factors and with yields(24～30 mesh), bulk density,sphericity, fragility as estimate indicators. An eigenvalue method was applied endowing different weighing to estimate target and the feature vector W calculated by Rad method were 0.750,0.036,0.107 and 0.107 which corresponded to four estimate targets following the colligation score method to calculate "score" indices. All indices were applied to Analysis of Variance based on SPSS, which concluded that factors A and D have significant influences on score (P< 0.05). The influencing degree of each factor on the score was as follows:A>D>C>E>B. The factor B has significant influences on fragility (P<0.05). Finally, the formula and preparation process of pellet cores were determined:7% dextrin starch as diluent,3% HPMC solution as adhesives, a small amount of micronization silica powder as lubricant,40rpm of extrusion speed,850rpm of spheronization speed,60 seconds of spheronization time. The reproducibility test showed an excellent reproducibility of pellet cores in a comprehensive level resulting from the optimization scheme. HPMCP 55s was used for coating the pellet cores. The coating parameters confirmed by the investigation of different factors in the coating process were as follows:500g of feeding,6% HPMCP organic coating liquid,200rpm of spheronization speed,20Hz of Blasting frequency,40℃of coating temperature、20 kg·cm-2 of spraying pressure, 9ml·min-1 of fluid supply speed,20% of weight increase and 2 hours of drying in 40℃after coating. In this coating condition, the release of pellets met the requirement and the coating of pellet cores showed good reproducibility. The resulted pellets present uniform and smooth surfaces. The stability tests indicated that doxycycline enteric-coated pellets would remain effective when stored in room temperature with hermetic and dark condition. The tentative expiration period was 2 years.A randomized, two-way, crossover bioequivalence study in 6 healthy young pigs was conducted to compare doxycycline enteric-coated pellets with doxyprex(?) premix. The drug was given in a single dose of 10 mg·kg-1.bw, and then 1.5ml of blood samples were collected at 0 min,5min、0.25,0.5,0.75,1,1.5,2,4,6,8,12,24,48,72h from vein of ear verge. Doxycycline content in plasma was determined by high-performance liquid chromatography assay. The relative bioavailability and bioequivalence of the two forms of doxycycline were calculated by DAS and compared by SPSS. The pharmacokinetic parameters of doxycycline enteric-coated pellets were as follows: AUC0→∞=15.93±5.17μg·ml-1·h, AUC0→72=14.70±4.83μg·ml-1·h,Cmax=1.71±0.79μg·ml-1, Tmax=5+1.095h。The pharmacokinetic parameters of doxyprexTM premix were as follows:AUC0→∞=15.59±5.47μg·ml-1·h, AUC0→72=14.79±5.38μg·ml-1·h,Cmax=1.82±0.51μg·ml-1,Tmax=4.33±1.366h. The relative bioavailability of doxycycline enteric-coated pellets was 107.08±47.40%. Variance analysis and two one-sided t-test were performed to Napierian logarithm of parameters Cmax, AUC0→72 and AUC0→∞Nonparametric test was performed to Tmax. The results showed that the parameters had no significant statistical differences. The two preparations of doxycycline are bioequivalent.