rom D+I was monitored in vitro. Under physiological conditions of pH, it is possible to observe that approximately 80% of DIZE was controlled released during the first four hours. Interestingly, no further release was detected after this period. 7 / 18 Ocular Inserts of DIZE to Treat Glaucoma in Rats Fig 2. Differential scanning calorimetry curves of DIZE, placebo inserts and DIZE inserts. Peaks at 64.3C and 314.9C on P+I curve are attributed to evaporation of residual water and degradation of the main polymeric chain, respectively. On DIZE curves, peaks at 98.3C and 137.0C are attributed to loss of water from the salt and peaks at 175.1C and 214.0C indicates the decomposition of DIZE. Peaks of decomposition of DIZE were not detected in D+I curves; n = 3 per group. doi:10.1371/journal.pone.0133149.g002 Effects of the controlled release of DIZE on IOP of PF-562271 price glaucomatous rats Functionally, the biological efficiency of our inserts to release DIZE was tested PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19747074 in an experimental model of glaucoma induced by intraocular injection of HA. Fig 5A shows the IOP of all experimental groups during the period of 6 weeks. The IOP of control glaucomatous 
animals and of glaucomatous animals treated with P+I were significantly higher than control animals treated or not with P+I. After one week of the treatment with D+I, the IOP of glaucomatous animals lowered at the levels of control animals. Importantly, this effect lasted for the following four weeks of evaluation. No significant changes in IOP were observed in non-glaucomatous animals treated with inserts containing DIZE. Of note, the anti-glaucomatous effects of the inserts containing DIZE were not accompanied by alterations in MAP. Effects of the controlled release of DIZE on RGC of glaucomatous rats The IOP lowering effects of DIZE were followed by preservation of RGC. As seen in Fig 6, we found that control glaucomatous animals and glaucomatous rats treated with P+I presented a large reduction in the number of RGC. Micrograph of the lateral side of the insert. Micrograph of the surface of the insert. D+I are uniform without granular particles in the middle or in the surface and measuring approximately 40 m of thickness. doi:10.1371/journal.pone.0133149.g003 glaucomatous rats + placebo inserts–GLAU+P+I: 476.4 11.0 cells). This effect was completely restored by the inserts containing DIZE. Again, no significant changes were induced by D+I in non-glaucomatous animals. Additionally, the reduction in the number of RGC caused by elevated IOP was accompanied by a severe loss of neural fibers with consequent increase in the optic nerve head cupping. These effects were abolished by the treatment with inserts containing DIZE. Altogether, these data indicate that controlled release of DIZE induced a neuroprotection by decreasing the loss of RGC and neural fibers. Biodistribution studies Radiolabeling of DIZE with 99mTc showed a radiochemical purity of 81.39% 0.51%. It was observed that the amount of 99mTc-DIZE that began to clear from the corneal region reaching 9 / 18 Ocular Inserts of DIZE to Treat Glaucoma in Rats Fig 4. In vitro release of DIZE. In vitro release of DIZE from DIZE inserts showing that approximately 80% of the drug was controlled released in four hours. doi:10.1371/journal.pone.0133149.g004 the gastrointestinal tract via PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19748686 the nasolacrimal drainage system was significantly higher in animals treated with 99mTc-DIZE eye drops than in those rats treated with 99mTc-D+I. Quantitatively, the animals
