Targeting retinal and choroid neovascularization using the small molecule inhibitor carboxyamidotriazole.

Neovascular ocular diseases as exemplified by proliferative diabetic retinopathy (PDR), exudative age-related macular degeneration (AMD), and retinopathy of prematurity (ROP) are severe diseases affecting all age groups in the US. We asked whether a small molecule, carboxyamidotriazole (CAI) known for its anti-angiogenic and anti-tumor effects and its ability to be administered orally in humans, could have anti-angiogenic effects in ocular in vitro and in vivo angiogenesis models. The anti-proliferative effects of CAI were examined by BrdU incorporation using human retinal and dermal endothelial cells and human pigment epithelial cells. The effect of CAI was determined using the Matrigel tube formation assay. The mouse model of choroidal neovascularization (CNV) initiated by laser rupture of Bruch's membrane was used to quantify in vivo effects of aqueous beta-hydroxypropyl cyclodextrin (bHPCD) formulations of CAI on neovascularization. The pharmacokinetics (PK) of CAI after intravitreal administration of bHPCD-CAI was studied in rabbit. The intravitreal toxicology of bHPCD-CAI was also examined in rat ocular tissue. We observed that CAI treatment of human endothelial cells decreased cell proliferation in a dose-dependent manner. In the in vivo tests bHPCD-CAI treatment reduced choroidal neovascular lesion volume, also in a dose-dependent manner. The intravitreal PK of bHPCD-CAI demonstrated that highly efficacious concentrations of CAI are reached in the vitreous compartment. No ocular toxicology was observed with intravitreous injection of CAI. These studies support the potential of developing intravitreal CAI in an bHPCD ocular formulation for treatment of proliferative retinopathies in humans.

Nonpeptide somatostatin receptor agonists specifically target ocular neovascularization via the somatostatin type 2 receptor.

PURPOSE: To define the molecular pharmacology underlying the antiangiogenic effects of nonpeptide imidazolidine-2,4-dione somatostatin receptor agonists (NISAs) and evaluate the efficacy of NISA in ocular versus systemic delivery routes in ocular disease models. METHODS: Functional inhibitory effects of the NISAs and the somatostatin peptide analogue octreotide were evaluated in vitro by chemotaxis, proliferation, and tube-formation assays. The oxygen-induced retinopathy (OIR) model and the laser model of choroidal neovascularization (CNV) were used to test the in vivo efficacy of NISAs. Transscleral permeability of a candidate NISA was also measured. RESULTS: NISAs inhibited growth factor-induced HREC proliferation, migration and tube formation with submicromolar potencies (IC(50), 0.1-1.0 microM) comparable to octreotide. In the OIR model, systemic administration of the NISAs RFE-007 and RFE-011 inhibited retinal neovascularization in a dose-dependent manner, comparable to octreotide. In the CNV model, intravitreal RFE-011 resulted in a 56% reduction (P < 0.01) in CNV lesion area, whereas systemic administration resulted in a 35% reduction (P < 0.05) in lesion area. RFE-011 demonstrated transscleral penetration. CONCLUSIONS: Micromolar concentrations of octreotide and NISAs are necessary for antiangiogenic effects, whereas nanomolar concentrations are effective for endocrine inhibition. This suggests that the antiangiogenic activity of NISAs and octreotide is mediated by an overall much less efficient downstream coupling mechanism than is growth hormone release. As a result, the intravitreal or transscleral route of administration should be seriously considered for future clinical studies of SSTR2 agonists used for treatment of ocular neovascularization to ensure efficacious concentrations in the target retinal and choroidal tissue.

Analgesic activity of a non-peptide imidazolidinedione somatostatin agonist: in vitro and in vivo studies in rat.

Several lines of evidence support an important role for somatostatin receptors (SSTRs) in pain modulation. The therapeutic use of established SSTR peptide agonists for this indication is limited by their broad range of effects, need for intrathecal delivery, and short half-life. Therefore, the goal of the present study was to investigate the analgesic effect of SCR007, a new, highly selective SSTR2 non-peptide agonist. Behavioral studies demonstrated that paw withdrawal latencies to heat were significantly increased following intraplantar SCR007. Furthermore, both intraperitoneal and intraplantar injection of SCR007 significantly reduced formalin- and capsaicin-induced flinching and lifting/licking nociceptive behaviors. Recordings from nociceptors using an in vitro glabrous skin-nerve preparation showed that SCR007 reduced heat responses in a dose-dependent fashion, bradykinin-induced excitation, heat sensitization and capsaicin-induced excitation. In both the behavioral and single fiber studies, the SCR007 effects were reversed by the SSTR antagonist cyclo-somatostatin, demonstrating receptor specificity. In the single fiber studies, the opioid antagonist naloxone did not reverse SCR007-induced anti-nociception suggesting that SCR007 did not exert its effects through activation of opioid receptors. Analysis of cAMP/protein kinase A (PKA) involvement demonstrated that SCR007 prevented forskolin- and Sp-8-Br-cAMPS (a PKA activator)-induced heat sensitization, supporting the hypothesis that SCR007-induced inhibition could involve a down-regulation of the cAMP/PKA pathway. These data provide several lines of evidence that the non-peptide imidazolidinedione SSTR2 agonist SCR007 is a promising anti-nociceptive and analgesic agent for the treatment of pain of peripheral and/or central origin.

Nucleic acid analysis using an expanded genetic alphabet to quench fluorescence.

Organic chemistry has made possible the synthesis of molecules that expand on Nature's genetic alphabet. Using the previously described nonstandard DNA base pair constructed from isoguanine and 5-methylisocytosine, we report a highly specific and sensitive method that allows for the fast and specific quantitation of genetic sequences in a closed tube format. During PCR amplification, enzymatic site-specific incorporation of a quencher covalently linked to isoguanine allows for the simultaneous detection and identification of multiple targets. The specificity of method is then established by analysis of thermal denaturation or melting of the amplicons. The appropriate functions of all reactions are further verified by incorporation of an independent target into the reaction mixture. We report that the method is sensitive down to the single copy level, and specificity is demonstrated by multiplexed end-point genotypic analysis of four targets simultaneously using four separate fluorescent reporters. The method is general enough for quantitative and qualitative analysis of both RNA and DNA using previously developed primer sets. Though the method described employs the commonly used PCR, the enzymatic incorporation of reporter groups into DNA site-specifically should find broad utility throughout molecular biology.