The development of an innervated epithelial barrier model using a human corneal cell line and ND7/23 sensory neurons.

The corneal epithelium is a highly innervated tissue and hence in vitro models that mimic the effects of chemicals or radiation (e.g. ultra violet) on this important barrier should include consideration of the potential role of innervation. A sensory neural cell line, ND7/23, was incorporated into a 2D and 3D model of a corneal epithelium, using a human corneal cell line, and effects on barrier integrity were neither adverse nor stimulatory. In the 3D model the nerve cell bodies were separated from the corneal epithelium, via a porous polycarbonate insert membrane. The ND7/23 cells were induced to form neurites and cease division when cultured in the keratinocyte medium employed for the corneal cells. In the absence of calcium, the epithelial barrier function was lost, shown by enhanced fluorescein leakage and relocation of ZO-1 and E-cadherin from the cell membrane. At 60 microM calcium, and above, the corneal cells formed tight junctions, with peripheral membrane location of ZO-1 and E-cadherin. The presence of the ND7/23 cells did not compromise or enhance the time taken to form these junctions, when monitored at 24-h intervals over 72 h. Both male- and female-derived human corneal cell lines showed a similar tight junction functional response to different medium calcium concentrations in the presence or absence of the ND7/23 cells. Once differentiated in keratinocyte medium, patch-clamped ND7/23 cells were capable of producing a whole-cell current when exposed to low pH (5.4), indicative of the presence of active pH-gated ion channels.

The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer .

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.