BET bromodomain inhibitors regulate keratinocyte plasticity.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.

The Discovery of N-(1-Methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6- ((methylamino)methyl)pyrimidin-4-yl)oxy)-1H-indole-1-carboxamide (Acrizanib), a VEGFR-2 Inhibitor Specifically Designed for Topical Ocular Delivery, as a Therapy for Neovascular Age-Related Macular Degeneration.

A noninvasive topical ocular therapy for the treatment of neovascular or "wet" age-related macular degeneration would provide a patient administered alternative to the current standard of care, which requires physician administered intravitreal injections. This manuscript describes a novel strategy for the use of in vivo models of choroidal neovascularization (CNV) as the primary means of developing SAR related to efficacy from topical administration. Ultimately, this effort led to the discovery of acrizanib (LHA510), a small-molecule VEGFR-2 inhibitor with potency and efficacy in rodent CNV models, limited systemic exposure after topical ocular administration, multiple formulation options, and an acceptable rabbit ocular PK profile.

Discovery of Oral VEGFR-2 Inhibitors with Prolonged Ocular Retention That Are Efficacious in Models of Wet Age-Related Macular Degeneration.

The benefit of intravitreal anti-VEGF therapy in treating wet age-related macular degeneration (AMD) is well established. Identification of VEGFR-2 inhibitors with optimal ADME properties for an ocular indication provides opportunities for dosing routes beyond intravitreal injection. We employed a high-throughput in vivo screening strategy with rodent models of choroidal neovascularization and iterative compound design to identify VEGFR-2 inhibitors with potential to benefit wet AMD patients. These compounds demonstrate preferential ocular tissue distribution and efficacy after oral administration while minimizing systemic exposure.

The influence of aqueous content in small scale salt screening--improving hit rate for weakly basic, low solubility drugs.

Salt screening and selection is a well established approach for improving the properties of drug candidates, including dissolution rate and bioavailability. Typically during early development only small amounts of compound are available for solid state profiling, including salt screening. In order to probe large areas of experimental space, high-throughput screening is utilized and is often designed in a way to search for suitable crystallization parameters within hundreds or even thousands of conditions. However, the hit rate in these types of screens can be very low. In order to allow for selection of a salt form early within the drug development process whilst using smaller amounts of compounds, a screening procedure taking into account the compounds properties and the driving forces for salt formation is described. Experiments were carried out on the model compounds clotrimazole, cinnarizine itraconazole and atropine. We found an increase in crystalline hit rate for water-insoluble drugs crystallized from solutions that included at least 10% aqueous content. Conversely it was observed that compounds with greater water solubility did not benefit from aqueous content in salt screening, instead organic solvents lead to more crystalline screening hits. Results from four model compounds show that the inclusion of an aqueous component to the salt reaction can enhance the chance of salt formation and significantly improve the crystalline hit rate for low water soluble drugs.

Interlaced optical force-fluorescence measurements for single molecule biophysics.

Combining optical tweezers with single molecule fluorescence offers a powerful technique to study the biophysical properties of single proteins and molecules. However, such integration into a combined, coincident arrangement has been severely limited by the dramatic reduction in fluorescence longevity of common dyes under simultaneous exposure to trapping and fluorescence excitation beams. We present a novel approach to overcome this problem by alternately modulating the optical trap and excitation beams to prevent simultaneous exposure of the fluorescent dye. We demonstrate the dramatic reduction of trap-induced photobleaching effects on the common single molecule fluorescence dye Cy3, which is highly susceptible to this destructive pathway. The extension in characteristic fluorophore longevity, a 20-fold improvement when compared to simultaneous exposure to both beams, prolongs the fluorescence emission to several tens of seconds in a combined, coincident arrangement. Furthermore, we show that this scheme, interlaced optical force-fluorescence, does not compromise the trap stiffness or single molecule fluorescence sensitivity at sufficiently high modulation frequencies. Such improvement permits the simultaneous measurement of the mechanical state of a system with optical tweezers and the localization of molecular changes with single molecule fluorescence, as demonstrated by mechanically unzipping a 15-basepair DNA segment labeled with Cy3.

Cavity ringdown strain gauge.

Biconical tapered single-mode fiber, which is common in many telecommunications components, offers an alternative sensor to typical optical fiber strain gauges that are susceptible to temperature and pressure effects and require expensive and sophisticated signal acquisition systems. Cavity ringdown spectroscopy, a technique commonly applied to high-sensitivity chemical analysis, offers detection sensitivity advantages that can be used to improve strain measurement with biconical tapers. Combining these two technologies in a spatially extended resonator, we demonstrate a minimum detectable change in ringdown time of 0.08%, corresponding to a minimum detectable displacement of 4.8 nm, and a sensitivity to strain as small as 79 n epsilon/square root(Hz) over a 5-mm taper length.

Trace moisture detection using continuous-wave cavity ring-down spectroscopy.

We have developed an instrument to measure trace concentrations of small hydride species in gases using continuous-wave cavity ring-down spectroscopy with near-infrared diode laser excitation. An rms baseline equivalent absorbance of 9.2 x 10(-11) cm(-1)/square root(n) is found, where n is the number of ring-down transients. When the 1396.376-nm absorption line of water is used, this corresponds to a noise equivalent moisture concentration in nitrogen gas of 68 pptv/square root(n). Water vapor concentration is detected over a range extending from 3 to 1000 ppbv and found to depend linearly on the concentration as determined by a calibrated commercial moisture sensor.