Safety, tolerability, and bioavailability of topical SAR 1118, a novel antagonist of lymphocyte function-associated antigen-1: a phase 1b study.

PURPOSE: A growing body of evidence points to a role for inflammation mediated by lymphocyte function-associated antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 in the pathogenesis of diabetic macular oedema. This phase 1b clinical trial assessed the safety, tolerability, and pharmacokinetics of topically administered SAR 1118, a novel LFA-1 antagonist, in human subjects. METHODS: In this prospective, randomized, double-masked trial, 13 subjects scheduled for vitrectomy received one of three concentrations of topical SAR 1118 (0.1, 1.0, or 5.0%) twice daily for 1 week before surgery. Undiluted aqueous and vitreous samples were collected at surgery and analysed for the concentration of the medication. RESULTS: All subjects completed the entire course of medication. The only adverse events reported were instillation site irritation (4/13, 31%) and dysgeusia (3/13, 23%). These were mild and transient, occurring at the highest dose. Mean concentrations (ng/ml) of SAR 1118 in the aqueous humour were 0.25, 37.2, and 101.1 for the 0.1%, 1.0%, and 5.0% dose groups, respectively. SAR 1118 was below the level of detection (0.5 ng/ml) for all vitreous samples except in a single subject who had a history of prior vitrectomy and a dislocated intraocular lens. CONCLUSIONS: Topical SAR 1118 was safe and well tolerated, and dose-dependent levels of drug were detected in aqueous. However, vitreous levels were below the threshold of detection with the concentrations tested. Further investigation of this medication for posterior segment applications would require intravitreal delivery or chemical modification of the drug.

Light and inherited retinal degeneration.

Light deprivation has long been considered a potential treatment for patients with inherited retinal degenerative diseases, but no therapeutic benefit has been demonstrated to date. In the few clinical studies that have addressed this issue, the underlying mutations were unknown. Our rapidly expanding knowledge of the genes and mechanisms involved in retinal degeneration have made it possible to reconsider the potential value of light restriction in specific genetic contexts. This review summarises the clinical evidence for a modifying role of light exposure in retinal degeneration and experimental evidence from animal models, focusing on retinitis pigmentosa with regional degeneration, Oguchi disease, and Stargardt macular dystrophy. These cases illustrate distinct pathophysiological roles for light, and suggest that light restriction may benefit carefully defined subsets of patients.

Evidence for common sites of contact between the antisigma factor SpoIIAB and its partners SpoIIAA and the developmental transcription factor sigmaF in Bacillus subtilis.

The activity of the developmental transcription factor sigmaF in Bacillus subtilis is governed by a switch involving the dual function protein SpoIIAB. SpoIIAB is an antisigma factor that forms complexes with sigmaF and with an alternative partner protein SpoIIAA. SpoIIAB is also a protein kinase that can inactivate SpoIIAA by phosphorylating it on a serine residue. We sought to identify amino acids in SpoIIAB that are involved in the formation of the SpoIIAB-SpoIIAA complex by screening for mutants that were defective in the activation of sigmaF. This genetic screen, in combination with biochemical analysis and the construction of loss-of-side-chain (alanine substitution) mutants, led to the identification of amino acid side-chains in the N-terminal region of SpoIIAB that could contact SpoIIAA. Unexpectedly, the same amino acid side-chains (R20 and N50) that appear to touch SpoIIAA are required for binding to, and may represent sites of contact with, sigmaF. We propose that the N-terminal region of SpoIIAB forms a binding surface that is responsible for the formation of both the SpoIIAB-SpoIIAA and the SpoIIAB-sigmaF complexes, and that in some cases the same amino acid side-chains contact both partner proteins. N50 is also the defining residue of a region of amino acid sequence homology known as the N-box that is shared by SpoIIAB and related serine protein kinases, as well as by members of a mechanistically dissimilar family of protein kinases that undergo autophosphorylation at a histidine residue. We discuss the implications of this finding for the mechanism of histidine autophosphorylation.

The kinase activity of the antisigma factor SpoIIAB is required for activation as well as inhibition of transcription factor sigmaF during sporulation in Bacillus subtilis.

The activity of the developmental transcription factor sigmaF in the spore-forming bacterium Bacillus subtilis is controlled by SpoIIAB, which sequesters sigmaF in an inactive complex. sigmaF is released from the SpoIIAB-sigmaF complex by the action of SpoIIAA, which triggers the dissociation of the complex. SpoIIAB is also a protein kinase that phosphorylates SpoIIAA on serine residue 58 (S58). This phosphorylation inactivates SpoIIAA and thus indirectly prevents the activation of sigmaF. Here, we report the identification of a patch of amino acid residues located in the vicinity of the adenosine nucleotide binding pocket of SpoIIAB that is required for the phosphorylation of SpoIIAA. A lysine substitution (E104K) at one of these residues (Glu104) markedly impaired the capacity of SpoIIAB to phosphorylate SpoIIAA in vitro as well as during sporulation. Kinetic analysis and evidence from the construction of alanine substitution mutants indicates that the side-chains of these amino acids could be contact sites for the SpoIIAA substrate during the phosphorylation reaction. Importantly, E104K and other kinase mutants blocked the activation of sigmaF during sporulation. This is paradoxical, because a mutant of SpoIIAA (S58A) that cannot be phosphorylated is known to cause higher than normal levels of sigmaF activity during sporulation. In resolution of this paradox, we present biochemical evidence indicating that SpoIIAA directly attacks the SpoIIAB-sigmaF complex and that SpoIIAA is phosphorylated as a result of this reaction. Consistent with this idea, mutations impairing kinase function of SpoIIAB were found to be epistatic to a mutation causing the S58A substitution in SpoIIAA; that is, cells producing mutant forms of both proteins were blocked in the activation of sigmaF. We conclude that phosphorylation of SpoIIAA plays a dual role in the sigmaF pathway, and that the kinase function of SpoIIAB is required for the activation as well as the inhibition of sigmaF during sporulation.