Nonclinical Development of ENV905 (Difluprednate) Ophthalmic Implant for the Treatment of Inflammation and Pain Associated with Ocular Surgery.

PURPOSE: Topical corticosteroids are widely used in the treatment of inflammation and pain after ocular surgery, but they possess several shortcomings, including frequent dosing and low patient adherence. We evaluated the efficacy and pharmacokinetics of ENV905 (difluprednate or DFBA) Ophthalmic Implant, a single-dose drug delivery system, compared with 0.05% Durezol. METHODS: PRINT® technology was used to fabricate ENV905 implants for either intracameral (IC) or subconjunctival (SCJ) delivery of extended-release DFBA. A postoperative inflammation model and ocular pharmacokinetics studies of ENV905 or Durezol were conducted in albino rabbits for a maximum of 12 weeks. RESULTS: Suppression of ocular inflammation was marked for both IC and SJC ENV905 compared with placebo, and it was superior or equivalent to that observed with QID Durezol. Concentrations of desacetyl difluprednate (DFB, active metabolite) peaked on day 1 and tapered over time for ENV905, with IC ENV905 delivering DFB to the target tissue at the time of greatest inflammation, whereas SJC produced a longer duration of exposure. Durezol eyes demonstrated consistent exposure over time with maximal exposure in the cornea. Although the pharmacokinetic profile differed for the two routes, efficacy was similar. CONCLUSION: ENV905 was well tolerated and demonstrated a robust reduction in ocular inflammation with targeted drug delivery. The results from these studies show that ENV905 provides a sustained therapeutic effect after a single dose. By resolving low patient compliance and eliminating the peaks and troughs in drug concentration, sustained drug delivery via ENV905 may further improve the overall control of postoperative inflammation and pain.

High-content RNA interference assay: analysis of tau hyperphosphorylation as a generic paradigm.

High-content analysis methods provide the opportunity to interrogate specific cellular end points in living cells. When coupled with high-throughput RNA interference (ht-RNAi) loss of function screens, high-content analyses are a powerful discovery tool for the identification of new genes and pathways involved in specific disease-relevant cellular functions. The most common readout is a fluorescence measurement, usually based on a green fluorescent protein reporter (or some derivative thereof ) or a fluorescently labeled antibody. Here, we describe a specific approach to the development of a high-content assay for the hyperphosphorylation of tau protein that is compatible with RNAi screens. The goal of this chapter is to provide a generic paradigm, using hyperphosphorylation of tau protein as an example, to serve as a blueprint for the investigation of additional cellular end points or protein functions for those interested in performing high-content screens.

High-content siRNA screening of the kinome identifies kinases involved in Alzheimer's disease-related tau hyperphosphorylation.

BACKGROUND: Neurofibrillary tangles (NFT), a cardinal neuropathological feature of Alzheimer's disease (AD) that is highly correlated with synaptic loss and dementia severity, appear to be partly attributable to increased phosphorylation of the microtubule stabilizing protein tau at certain AD-related residues. Identifying the kinases involved in the pathologic phosphorylation of tau may provide targets at which to aim new AD-modifying treatments. RESULTS: We report results from a screen of 572 kinases in the human genome for effects on tau hyperphosphorylation using a loss of function, high-throughput RNAi approach. We confirm effects of three kinases from this screen, the eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), and the A-kinase anchor protein 13 (AKAP13) on tau phosphorylation at the 12E8 epitope (serine 262/serine 356). We provide evidence that EIF2AK2 effects may result from effects on tau protein expression, whereas DYRK1A and AKAP13 are likely more specifically involved in tau phosphorylation pathways. CONCLUSIONS: These findings identify novel kinases that phosphorylate tau protein and provide a valuable reference data set describing the kinases involved in phosphorylating tau at an AD-relevant epitope.

Genomic and Proteomic Biomarker Discovery in Neurological Disease.

Technology for high-throughout scanning of the human genome and its encoded proteins have rapidly developed to allow systematic analyses of human disease. Application of these technologies is becoming an increasingly effective approach for identifying the biological basis of genetically complex neurological diseases. This review will highlight significant findings resulting from the use of a multitude of genomic and proteomic technologies toward biomarker discovery in neurological disorders. Though substantial discoveries have been made, there is clearly significant promise and potential remaining to be fully realized through increasing use of and further development of -omic technologies.