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Drug delivery by topical application has higher patient acceptance and lower morbidity than intraocular injection, but many ophthalmic treatments are unable to enter the eye or reach the posterior segment after topical application. The first stage towards posterior segment delivery after topical application is ocular surface penetration and existing models are in vivo or use large quantities of tissue. We therefore developed a novel ex vivo model using discs of porcine and human cornea and sclera (5 mm diameter) to assess penetration of a candidate neuroprotective siRNA. siRNA against caspase 2 or control solutions of known penetrance were applied to the corneal epithelial surface and trans-corneal penetration and corneal adsorbance measured at fixed time points. To demonstrate that leakage did not occur, we applied dextran blue, which should not penetrate the intact cornea and did not do so in our model. Fluorescein penetration (0.09%) was less than rhodamine B (6.98%) at 60 min. siCASP2 penetration was 0.01% by 60 min. When the applied siCASP2 was washed off after 2 min, (representing lacrimal drainage) 0.071% penetrated porcine cornea by 60 min and 0.0002% penetrated human cornea and 0.001% penetrated human sclera. Our ex vivo model rapidly and cost-effectively assesses transcorneal penetration of candidate topical therapies, allowing rates of trans-corneal penetration for potential therapies such as siRNA to be evaluated with small quantities of human or animal tissue. Ocular penetration of topically-applied drugs remains a pharmaceutical challenge. Small interfering ribonucleic acid (siRNA) molecules have the potential to treat a wide range of ophthalmic pathologies 1 , generating an interest in agents to enhance their penetration after topical application 2. In particular, siRNA against caspase-2 (siCASP2) prevents retinal ganglion cell death and is in Phase 3 clinical trials for non-arteritic anterior ischaemic optic neuropathy (NCT02341560; ClinicalTrials.gov) 3, 4. Current application of siRNA requires intraocular injection, which is invasive, carries a risk of infection and is less acceptable to patients than other methods of drug delivery. Diffusion of compounds through the eye after topical application is hindered by the presence of anatomical barriers including the tear film, cornea, conjunctiva, sclera, choroid, aqueous, lens and vitreous 5. Having passed through these barriers, compounds are additionally cleared by vascular or aqueous drainage 5, 6. The bloodaqueous and the blood-retinal barriers provide challenges for systemic drug delivery in addition to systemic toxicity 7-9. As a result, after initial topical application approximately 1/100,000 of compounds such as steroids including prednisolone acetate will reach the back of the eye 5, 10. The therapeutic efficacy of such dilutions may not be sufficient for biologically relevant treatment effects. However, topical delivery offers the potential for noninvasive treatment of intra-ocular diseases with greater patient acceptance and reduced compl...

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Contrary to a previous report, the alkylation of ferrocene with t-butyl chloride under Friedel-Crafts conditions proceeds smoothly, and mono-, 1, 1'-di-1,3-di-, 1,1',3-tri-, and 1,1',3,3'-tetra-t-butylferrocenes can be isolated crystalline. 1, 1'-Di-t-alkylferrocenes are also prepared from the corresponding t-alkylcyclopentadienes, which are obtained by treating cyclopentadienylmetal halides with t-alkyl halides. Alkylferrocenes containing t-alkyl groups separated from the nucleus by one and two carbon atoms are described. From the reaction between pivaloyl chloride and ferrocene in the presence of aluminium chloride various products, including l,l'-dipivaloyl-3,3'-di-t-butylferrocene, are obtained. A synthesis of neopentylcyclopentadiene via ethoxymethylcyclopentadiene is recorded.ALKYL derivatives of cyclopentadiene have been used for the preparation of the corresponding 1,l'-dia1kylferrocenes.l By treating cyclopentadienyl-sodium or -potassium in either liquid ammonia or organic solvents with n-and s-alkyl halides the alkylcyclopentadienes are obtained in high yield 2 but attempts to prepare t-butylcyclopentadiene from cyclopentadienyl-sodium, -lithium, or -potassium and t-butyl chloride were unsuccessful. Riemschneider and Grabitz could not make cyclopentadienylpotassium react with t-butyl bromide, and similar failures to cause reaction between t-butyl chloride and other organosodium compounds have been reported. Homeyer et aL4 could not alkylate malonic ester, and Hamrick et aZ.5 noted the absence of reaction between sodium diphenylmethide and t-butyl chloride. In contrast to these findings, a recent German patent claims 50--80% yields of t-alkylcyclopentadienes from cyclopentadienylsodium and t-alkyl bromides. It has been found by the author, and independently by Riemschneider and Nehring,' that if the alkali-metal derivative of cyclopentadiene is replaced either by a cyclopentadienylmagnesium halide or by cyclopentadienylzinc chloride, then alkylation with t-alkyl chlorides is readily effected. The t-alkylcyclopentadienes listed in Table 1 were all prepared from B.P. 733,129.