| Calcitonin (CT) is a peptide hormone which is composed of 32 amino-acids. The activity of salmon calcitonin is the highest in the different source of calcitonins. It can reduce blood calcium reduce, inhibit the loss of bone calcium and significantly relieve pain by inhibit the activity of osteoclast. Due to its large molecular weight and bad liposolubility, it is difficult to get through the biomembrane and susceptible to gastrointestinal enzymatic hydrolysis, easy to degradate in the low PH and has the first pass effect of liver. So the bioavailability of salmon calcitonin in the oral administration is low (generally less than 10%).Therefore, in order to improve the bioavailability and the oral absorption of salmon calcitonin, this study was taken to establish a submicron drug carrier system and a site-specific colon system by using natural polymers and bioadhesive agent materials.The yAGA2-r-sCT microspheres were manufactured by the method of spray drying ,with naturalβ-cyclodextrin and arabic gum as wall materials, with HPMC and carbopol as bioadhesive agent. The commercially enteric-coated capsules were used as positioning carrier to improve the local concentration. The contact time of the drug with the colon were extended, the bioavailability was improved and the side effects were reduced.In the experiment, the method of in vitro analysis of capsules of colon site-specific sustained release microspheres of recombinant saccharomyces cerevisiae expressing salmon calcitonin was established firstly. The formulation factors as types and amounts ofβ-cyclodextrin and arabic gum,the ratios of bioadhesive agent and core materials were studied. The optimum process and formulation of yAGA2-r-sCT sustained-release microspheres with spray-drying technology was determined, which meets the design demand.The serum calcium concentration of the hypercalcemia rat model induced by Tsuruoka's process was measured by LX20 Beckman Full Automatic Biochemistry Analyzer. yAGA2-r-sCT microspheres as sustained-release salmon calcitonin(sCT) delivery system was orally administrated to rats at a single dosage of 0.5 g·kg-1. Miacalcic (Mia) was intramuscularly administrated to rats at a single dosage of 100 IU·kg-1. The plasma concentration of sCT was measured by ELISA and the pharmacokinetics parameters were calculated and analyzed by DAS 2.0 program.Result : po yAGA2-r-sCT microspheres 0.5 1 g·kg-1,iv pamidronate(Pam) 5 mg·kg-1 and im Mia 100 /IU·kg-1 reduced concentration of calcium in serum obviously and there was no difference of mean changes of calcium at the time point of 1 hour among the three kinds of preparations ( P > 0.05) . The four-parameter regression equation was described as following: OD=-7E-07C3+0.0003C2+0.0073C-0.0761(r=0.9985). The detectable concentration was ranging from 5 to 200μg·L-1.Minimum detectable concentration was 5μg(?)L-1. the intra-day and inter-day RSD were less than 8.6%. The major pharmacokinetics parameters of yAGA2-r-sCT microspheres and Mia were showen as following: Tmax were (3.01±0.57),(0.99±0.30 ) h respectively;Cmax were (107.25±20.03) and (131.83±26.96)μg·L-1 respectively;T1/2αwere (2.30±0.38),(0.07±0.22) h respectively;T1/2βwere (3.85±0.68),(2.50±0.51 ) h respectively ; Ka were (0.42±0.23) , (14.26±0.21) h-1 respectively;AUC0-t were (800.72±131.36),(601.29±112.02)μg(?)h·L-1 respectively;AUC0-∞were (861.31±158.65),( 617.06±130.27)μg(?)h·L-1 respectively;MRT0-t were (5.46±1.071),(4.29±0.75) h;MRT0-∞were (6.66±1.23),(4.91±1.20) h respectively.The results showed that the yAGA2-r-sCT microspheres had the best effect on the decreased serum calcium level and the process of determination with ELISA was stable and reliable results can be obtained.yAGA2-r-sCT microspheres showed characteristic of sustained-release and higher effect compared with Mia. |
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