| Objective:To investigate and evaluate the pharmacokinetic behavior of two sustained-release formulations-sodium alginate microspheres(SAM) and nano-suspension (NS) of micromolecular compound J2 in vitro and in vivo; To examine the effect of nano-suspension J2 on preventing rat allograft rejection via subconjunctival injection and its influence on rat T cell subsets as well as to investigate its pharmacodynamics and mechanism.Method:①Release behavior in vitro:the shape and size of J2-SAM was observed with optical microscope; Laser Particle Size Analyzer was used to scan the particle size of J2-NS; the drug loading of J2-SAM and J2-NS and its cumulative release in vitro were measured by HPLC.②Pharmacokinetic study in vivo:J2-SAM and J2-NS were injected in rabbits'conjunctiva and drug concentration of the cornea, aqueous humor, iris, and peripheral blood was detected by HPLC on Id,3d, 1w,2w,3w,4w respectively after injection. The main pharmacokinetic parameters were calculated by pharmacokinetic software-WinNonlin.③Pharmacodynamic study:allogeneic penetrating keratoplasty model was established using the adult female Wistar rats as donors and SD rats as recipients. Rats were divided into 4 groups randomly(n=16). Group A:SD rats were injected 0.05ml placebo subconjunctivally after autograft. Group B:allograft rats were injected 0.05ml placebo subconjunctivally after surgery. Group C:allograft rats were injected 0.05ml placebo subconjunctivally and administered 1% CsA 5 times a day after surgery. Group D:allograft rats were injected 0.1% J2-NS 0.05ml subconjunctivally after surgery. The graft rejection index (RI) and the survival time of animals in each group were recorded. The cornea and ipsilateral submandibular lymph nodes were examined pathologically on 7d,14d respectively after surgery.④Mechanism study:allogeneic penetrating keratoplasty model was established using the adult female Wistar rats as donors and SD rats as recipients. Group A:normal SD rats were injected 0.05ml placebo subconjunctivally, n=6. Group B:autograft rats were injected 0.05ml placebo subconjunctivally after surgery, n=6. Group C:allograft rats were injected 0.05ml placebo subconjunctivally after surgery, n=6. Group D:allograft rats were injected J2-NS 0.05ml subconjunctivally after surgery, n=6. Peripheral blood T lymphocyte subsets of each group were tested and compared by Flow cytometry Technology on 3d, 1w,2w,3w after operation.Results:①Release behavior in vitro:the mean diameters of J2-SAM and J2-NS were 165.61±37.943μm and 170nm respectively; drug loading was 0.12% and 0.1%.Two kinds of sustained release formulation showed significant release effect, steady release and no burst in vitro. Sustained release time all maintained more than 30d and cumulative release was 39.19% and 32.91% respectively in the 30th day.②Pharmacokinetic study in vivo: J2-SAM and J2-NS were injected in rabbit subconjunctiva and the drugs were released in ocular organizations slowly. The drug concentration was higher in cornea and lower in aqueous humor and iris, it also could be detected in blood. The sustained release formulation had long-term sustained-release effect in the eye.③Pharmacodynamic study: there was no rejection happened in group A in the observation period; the survival time of group B, C, D was 10.75±1.60d,16.00±3.79d and 14.33±2.93d respectively; Group C and D showed no significant difference, but all showed significant difference with group A and B. There were mainly infiltrated by polymorphonuclear leukocytes in corneal stroma of allograft groups at 7d after operation, lymphocytes at 14d. The cellular components and angiogenesis in all layers of cornea of group D were less compared with group B at the same period. The ipsilateral lymph nodes were hyperplasia after the allograft. However, the treatment groups were weaker than untreated group B. By immunohistochemistry dye, there was no CD4+, CD8+T cell infiltration in group A and there showed brown material depositing in nuclear membrane of T cells in stroma after transplantation. There were more positive cells in paracortex and interfollicular areas of ipsilateral submandibular lymph nodes, especially in group B.④Mechanism study:There were no significant difference of the total CD3+T cells, CD4+T cells, CD8+T cells and CD4+/CD8+T cells ratio of peripheral blood lymphocytes in group B during the observed time. There were no significant difference of the total CD3+T cells, CD4+T cells, CD8+T cells in group C at 3d and 1w after operation and significant difference at 2w and 3w whose cell numbers all increased. There were no significant difference of the total CD3+T cells, CD4+T cells, CD8+T cells in group B at 1w and 2w after operation in group D. The horizontal comparison of the same time:The total CD3+T lymphocytes of group D was significantly less than group C at 3d, 1w,2w after operation whereas there was no significant difference at 3w. There were smaller numbers of CD4+T lymphocytes in group D than group C at 3d and 1w but with no significant difference. The ratio of CD4+/CD8+ had no significant difference in group D compared with group C at 3d, 1w,3w.Conclusion:①J2-SAM and J2-NS exhibit uniform size that can be good drug carriers and have wonderful sustained release potency in vitro.②J2-SAM and J2-NS can be released slowly in eyes and sustain long-term stable drug concentration by single subconjunctival injection. There is no significant difference of pharmacokinetic parameters between the two slow-release formulations. However, J2-NS is more convenient to inject and it is easier to control the injection dosage of J2-NS than J2-SAM.③Subconjunctival injection of J2-NS can inhibit rat corneal allograft rejection, which has similar pharmacodynamic action with CsA. It also can reduce cell proliferation and inhibit the immune response in cornea and ipsilateral submandibular lymph nodes.④Rat corneal autograft does not irritate proliferation of peripheral blood T lymphocytes whereas it increases significantly in corneal allograft rejection. J2 inhibits T lymphocyte proliferation and then inhibits the T cell-mediated corneal allograft rejection. |
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