Implementation of a registry and open access genetic testing program for inherited retinal diseases within a non-profit foundation.

The Foundation Fighting Blindness is a 50-year old 501c(3) non-profit organization dedicated to supporting the development of treatments and cures for people affected by the inherited retinal diseases (IRD), a group of clinical diagnoses that include orphan diseases such as retinitis pigmentosa, Usher syndrome, and Stargardt disease, among others. Over $760 M has been raised and invested in preclinical and clinical research and resources. Key resources include a multi-national clinical consortium, an international patient registry with over 15,700 members that is expanding rapidly, and an open access genetic testing program that provides no cost comprehensive genetic testing to people clinically diagnosed with an IRD living in the United States. These programs are described with particular focus on the challenges and outcomes of establishing the registry and genetic testing program.

Lipophilic modifications to dinucleoside polyphosphates and nucleotides that confer antagonist properties at the platelet P2Y12 receptor.

Platelet P2Y12 receptors play a central role in the regulation of platelet function and inhibition of this receptor by treatment with drugs such as clopidogrel results in a reduction of atherothrombotic events. We discovered that modification of natural and synthetic dinucleoside polyphosphates and nucleotides with lipophilic substituents on the ribose and base conferred P2Y12 receptor antagonist properties to these molecules producing potent inhibitors of ADP-mediated platelet aggregation. We describe methods for the preparation of these functionalized dinucleoside polyphosphates and nucleotides and report their associated activities. By analysis of these results and by deconstruction of the necessary structural elements through selected syntheses, we prepared a series of highly functionalized nucleotides, resulting in the selection of an adenosine monophosphate derivative (62) for further clinical development.

Structure-activity relationships of dinucleotides: Potent and selective agonists of P2Y receptors.

Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y(1) while uridine containing dinucleotides have some level of agonist response on P2Y(2) and P2Y(6). The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.

Effect of 5-MCA-NAT, a putative melatonin MT3 receptor agonist, on intraocular pressure in glaucomatous monkey eyes.

PURPOSE: 5-MCA-NAT, a putative melatonin MT3 receptor agonist, reduced intraocular pressure (IOP) in ocular normotensive rabbit eyes. This study evaluates the effect of topical application of 5-MCA-NAT on IOP in monkey eyes with laser-induced unilateral glaucoma. METHODS: A multiple-dose study was performed in 8 glaucomatous monkey eyes. One 25-microL drop of 5-MCA-NAT (2%) was applied topically to the glaucomatous eye at 9:30 am and 3:30 pm for 5 consecutive days. IOP was measured hourly for 6 hours beginning at 9:30 am for one baseline day, one vehicle-treated day, and treatment days 1, 3, and 5 with 5-MCA-NAT. RESULTS: Compared with vehicle treatment, twice daily administration of 5-MCA-NAT for 5 days reduced (P < 0.05) IOP from 1 hour to 5 hours after the first dose, and the IOP-lowering effects were shown to last at least 18 hours following administration, based on IOP measurements made after the fourth and eighth doses. The ocular hypotensive effect of 5-MCA-NAT was enhanced with repeated dosing. The maximum reduction (P < 0.001) of IOP occurred at 3 hours after each morning dose, and was 4.0 +/- 0.5 (mean +/- SEM) mm Hg (10%) on day 1, 5.6 +/- 0.8 mm Hg (15%) on day 3, and 7.0 +/- 1.1 mm Hg (19%) on day 5. Adverse ocular or systemic side effects were not observed during the 5 days of treatment. CONCLUSIONS: 5-MCA-NAT, a putative melatonin MT3 receptor agonist, reduces IOP in glaucomatous monkey eyes. Melatonin agonists with activity on the putative MT3 receptor may have clinical potential for treating elevated IOP.

Localization of ocular P2Y2 receptor gene expression by in situ hybridization.

The objective of this study was to determine the cellular localization of P2Y(2) receptor gene expression in rabbit and primate ocular tissues using the technique of non-isotopic in situ hybridization. Fresh frozen whole eye from a New Zealand White rabbit and whole eye and eyelid from a rhesus macaque were cut into 5 microm thick sections and mounted onto glass slides. In situ hybridization was performed on ocular cryosections using digoxigenin-labeled P2Y(2) receptor riboprobes. Alkaline phosphatase-conjugated anti-digoxigenin antibody was used to localize riboprobe hybridization, which was subsequently visualized by staining with a precipitating alkaline phosphatase substrate. Cytoplasmic staining indicative of antisense riboprobe hybridization to P2Y(2) receptor mRNA was observed in the palpebral and bulbar conjunctival epithelium, including goblet cells, the corneal epithelium, and in meibomian gland sebaceous and ductal cells. Staining was also observed in both layers of the ciliary body epithelium, subcapsular epithelium of the lens, and corneal endothelium. In the posterior eye, staining was observed in various layers of the retina, including ganglion cell, inner nuclear, inner segment and retinal pigment epithelium layers, in the optic nerve head, and in a variety of structures within the choroid. No specific staining of sense riboprobe was seen on any of the ocular structures. These results demonstrate that the P2Y(2) receptor gene is expressed in a variety of ocular cells types and suggest that P2Y(2) receptors are associated with diverse physiological functions throughout the eye.

Established and emerging fluorescence-based assays for G-protein function: heterotrimeric G-protein alpha subunits and regulator of G-protein signaling (RGS) proteins.

Heterotrimeric G-proteins are molecular switches that couple serpentine receptors to intracellular effector pathways and the regulation of cell physiology. Ligand-bound receptors cause G-protein alpha subunits to bind guanosine 5'-triphosphate (GTP) and activate effector pathways. Signal termination is facilitated by the intrinsic GTPase activity of G-protein alpha subunits. Regulators of G-protein signaling (RGS) proteins accelerate the GTPase activity of the G-protein alpha subunit, and thus negatively regulate G-protein-mediated signal transduction. In vitro biochemical assays of heterotrimeric G-proteins commonly include measurements of nucleotide binding, GTPase activity, and interaction with RGS proteins. However, the conventional assays for most of these processes involve radiolabeled guanine nucleotide analogues and scintillation counting. In this article, we focus on fluorescence-based methodologies to study heterotrimeric G-protein alpha subunit regulation in vitro. Furthermore, we consider the potential of such techniques in high-throughput screening and drug discovery.

Selective P2Y2 receptor agonists stimulate vaginal moisture in ovariectomized rabbits.

OBJECTIVE: To determine the expression of P2Y(2) receptors in vaginal and cervical tissues and the effects of P2Y(2) receptor agonists INS45973 and INS365 on vaginal moisture. DESIGN: Pilot in vivo and histological study using animal subjects. SETTING: Experimental laboratory research. ANIMAL(S): Female New Zealand White rabbits were used for in vivo studies and female cynomolgus monkey (Macaca fascicularis) was used for in situ hybridization. INTERVENTION(S): Rabbits were kept intact or ovariectomized. Two weeks after ovariectomy, animals received daily vaginal instillation of vehicle or drugs for 16 days. MAIN OUTCOME MEASURE(S): Vaginal moisture was assessed in rabbits on 4 separate days during the treatment period. The P2Y(2) receptor mRNA distribution was assessed by in situ hybridization of monkey vagina and cervix. RESULT(S): Compared to control, vaginal moisture was significantly diminished in ovariectomized animals treated with vehicle. INS365 (8.1%) and INS45973 (0.9%) increased vaginal moisture in ovariectomized animals to levels that were comparable to or significantly higher than control animals, respectively. In situ hybridization studies indicated that P2Y(2) receptor mRNA was localized to endocervical and cervical gland, epithelium, and stratified squamous epithelium of the vagina. CONCLUSION(S): INS45973 and INS365 may interact with P2Y(2) receptors in the cervix and vagina to stimulate vaginal moisture in the estrogen (E)-deprived state. The P2Y(2) receptor agonists provide a potential nonhormonal alternative for treating vaginal dryness in postmenopausal women.

The P2Y(2) receptor agonist INS37217 stimulates RPE fluid transport in vitro and retinal reattachment in rat.

PURPOSE: To investigate the effects of INS37217, a synthetic P2Y(2) receptor agonist, on intracellular calcium signaling, electrophysiology, and fluid transport in vitro and on experimentally induced retinal detachment in rat eyes in vivo. METHODS: Freshly isolated monolayers of bovine and human fetal RPE were mounted in Ussing chambers for measurements of cytosolic calcium levels ([Ca(2+)](i)), membrane voltages and resistances, and transepithelial fluid transport. Retinal detachments were experimentally produced in Long-Evans rats by injecting modified phosphate-buffered saline into the subretinal space (SRS). Experimental or vehicle solutions were injected into the vitreous, and the size of blebs in the SRS was scored under masked conditions. RESULTS: Addition of INS37217 to Ringer's solution bathing the apical membrane transiently increased [Ca(2+)](i), altered membrane voltages and resistances and generally produced responses that were similar in magnitude to those of uridine triphosphate (UTP). In fluid transport experiments performed with the capacitance probe technique, INS37217 significantly increased fluid absorption across freshly isolated bovine and fetal human RPE monolayers. All in vitro results were blocked by apical 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), which has been shown to block P2Y(2) receptors in the RPE. Intravitreal administration of INS37217, but not UTP, in the rat model of retinal detachment enhanced the removal of SRS fluid and facilitated retinal reattachment when compared with vehicle control. CONCLUSIONS: These findings indicate that INS37217 stimulates the RPE fluid "pump" function in vitro by activating P2Y(2) receptors at the apical membrane. In vivo INS37217 enhances the rates of subretinal fluid reabsorption in experimentally induced retinal detachments in rats and may be therapeutically useful for treating a variety of retinal diseases that result in fluid accumulation in the subretinal space.