| Objective Design and production the "half-moon" sustained-release film of Pilocarpine nitrate to meet the anatomy of conjunctival sac; Observe application of it in rabbit eyes pharmacokinetic characteristics and local toxicity; Evaluate the relative bioavailability and the local security; Research and development the new dosage form of nitrate Pilocarpine, to provide a safe, reliable and effective sustained release drugs on the treatment of glaucoma.Methods 1.54 healthy New Zealand rabbits with either sex, weight 2-3kg and no eye disease by Slit-lamp microscope examination, were randomly divided into 9 groups of 6 rabbits (6 eyes), all the right eye were experimental group, and the left eye as the control. Experimental group conjunctival sac implanted in 1% pilocarpine sustained-release film, the left eye trickle-down 1% pilocarpine nitrate eye drops 50μl. After administration, respectively 0.5,1,2,3,5,7,10,15 and 24h quantitative analysis of aqueous extract. Dichloromethane extract aqueous humor contained in the drug, using reversed-phase high performance liquid chromatography (RP-HPLC) determination of the drug concentration in aqueous, the pharmacokinetic parameters calculated with 3P87 pharmacokinetic software to calculate obtain. Comparing the two formulations of pilocarpine pharmacokinetics features.2.12 healthy New Zealand rabbits with either sex, weight 2-3kg and no eye disease by Slit-lamp microscope examination, were randomly divided into A, B two groups of 6 rabbits (6 eyes), Each rabbit's right eye to give 1% sustained-release film of Pilocarpine nitrate or eye drops, and the left eye as the blank control group. Single-dose stimulation test:after Single-dose was given to all the rabbits' right eyes, then at 1,2,4,24,48 and 72 hours respectively check rabbits' eyes by slit-lamp microscopy. Repeated stimulation test administration:sustained-release film was given every 24 hours once(based on experimental results of pharmacokinetics), eye drops was given four times a day (based on clinical treatment characteristics:8,12,16, and 20 o'clock medication), For 5 days, each time before and last time administered after at the 1,2,4,24,48 and 72 hours, respectively check rabbits' conjunctiva, cornea and iris by slit-lamp microscopy, In accordance with "Draize" standard irritation score.3.9 healthy New Zealand rabbits with either sex, weight 2-3kg and no eye disease by Slit-lamp microscope examination, were randomly divided into A, B two groups of 6 rabbits (6 eyes), Were randomly divided into normal group 3 (6); the remaining 6 rabbits'(12 eyes) right eye as the experimental group, the left eye as the control, Experimental group conjunctival sac implanted in 1% pilocarpine sustained-release film, the left eye trickle-down 1% pilocarpine nitrate eye drops 50μl. sustained-release film was given every 24 hours once, eye drops was given four times a day,30 days after, killed the rabbit by ear vein air embolism, to take tissue samples of Conjunctiva and cornea then observe the local toxicity by light microscopy and scanning electron microscopy examination.Result 1.RP-HPLC can be separated with Pilocarpine nitrate and other impurities.The minimum detectable concentration was 0.05μg/ml. The average recovery rate of pilocarpine nitrate from aqueous humor was 99.1%. The peak concentrations and half-life of pilocarpine in aqueous humor were 6.55±0.32μg/ml at 3h and 11.13h, respectively, in the experimental group. Whereas, the peak concentrations and half-life of pilocarpine in aqueous humor were 4.33±0.29μg/ml at 0.4h and 1.93h, respectively, in the control group. The peak concentration of pilocarpine in aqueous humor in the experimental group was 1.51(P<0.05) time higher than the control group.The area under the curve of drug concentration-time (AUCo-iso) in the experimental group was 1.92 time higher than the control group.2. Single-dose stimulation test:There is no any irritation symptoms in all rabbit eyes by slit lamp examination. Repeated stimulation test: One rabbit conjunctival edema in experimental group; Control group, one rabbit conjunctival edema, tears, and with a little white discharge occurs in rabbit eyes. Accordance to the "Draize" criteria, the experimental group and the control group were 0.17 and 0.33, all less than 3. Therefore considered that neither sustained-release film nor eye drops is irritation to rabbit eyes and no significant difference between the two groups (P> 0.05).3. Local toxicity test:Conjunctiva inflammatory cell counts by light microscope, In the normal group, experimental group and control group the number of inflammatory cells were 35.83±5.98,58.17±6.55 and 113.17±6.85, After SNK pairwise comparison tests, according to a= 0.05 level, the control group average maximum, followed by the experimental group and control group is the lowest, difference between the pairwise mean are statistically significant. The scanning electron microscope found that the edge of corneal epithelial cells are irregular, cell shrinkage, cell spacing widened, loss of microvilli, cell empty and dark cells increased, but the control group corneal epithelial damage badly than the experimental group (P<0.05). Conclusion compared with 1% pilocarpine eye drops, the peak time of 1% pilocarpine nitrate half-moon-release film is delayed, peak concentration of it was increased, the elimination half-life of it is extended, suggesting its bioavailability increased, the action time prolonged, then played slow-release role. Either single or repeated stimulation test are no irritant reaction. Continuous administration a month later, conjunctivitis cell proliferation,corneal epithelial micro-structure changes, but lighter than the control group, indicating it was a local safe and reliable new formulations. |
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