Date of Award

8-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Pharmacometrics

Research Advisor

Charles R. Yates, Pharm.D./Ph.D.

Committee

Jerome Baudry, Ph.D. Bernd Meibohm, Ph.D., FCP Duane D. Miller, Ph.D. Frank Park, Ph.D. Phillip D. Rogers, Pharm.D./Ph.D.

Keywords

Angiogenesis, BXD, Focal Adhesion, Neovascularization, Paxillin, Retinopathy

Abstract

Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are among the most common causes of blindness in adults. Vision loss can occur during the advanced stages of DR and AMD as a consequence of unregulated and dysfunctional growth of new blood vessels, or neovascularization (NV) in the retina or choroid. NV can also be triggered by numerous other ocular insults and diseases including radiation retinopathy (RR) and retinal vein occlusion. These latter cases are generally less common but, like DR and AMD, they are characterized by an initial injury, chronic inflammation, and ischemia which perpetuates episodes of retinal neovascularization (RNV).

Current targets for RNV include vascular endothelial growth factor, VEGF which is achieved through anti-VEGF protein therapeutics aimed at sequestering the growth factor and preventing the activation of its receptor. However, prospective studies show that anti-VEGF resistance has become a major clinical concern in patients receiving long-term therapy. Thus, targeting downstream signaling proteins linked to pathological RNV represents an alternative or adjunctive approach to approved anti-VEGF treatments, which may provide better patient outcomes through enhanced efficacy of antiangiogenic therapy.

Our first goal was to understand how RNV progresses from early stage injury to proliferative ischemic retinopathy, in order to justify protein targets for drug discovery. We first began with an investigation into the causality of radiation injury itself to identify mechanisms of radiation sensitivity and/or resistance in the genetically diverse, murine BXD strains using a total-body irradiation (TBI) model. Our studies suggested mean survival time (MST) over 30 days may in fact be related to genetic variation in genes associated with endothelial progenitor cells (EPC) localization, wound healing, and focal adhesion (FA) dynamics involving both the hematopoietic and gastrointestinal systems. We targeted these mechanisms of tissue repair by blocking the homing of hematopoietic-derived cells to sites of irradiation (IR) injury which proved fatal to mice treated with an integrin-paxillin inhibitor, 6-B345TTQ. In a physiological flow-based assay, we inhibited circulating leukocytes from interacting with an inflamed endothelium, in vitro. These results suggested that the reparative/inflammatory angiogenic response triggered by radiation could be blocked by targeting FA signaling, a central process of RNV progression in ischemic retinopathies.

Findings in BXD studies linked tissue reparative processes involving ischemia- induced angiogenesis with mortality. We hypothesized that by targeting early injury in retinal endothelial cells (REC), we could prevent late-stage RNV. Thus, we first explored how RECs respond to radiation injury at levels high enough to cause significant vision impairments in RR. Previously identified radioprotectant, KZ-41, was used in these studies to ameliorate IR-induced injury to RECs through decreased inflammatory stress kinase activation, cell death, and subsequent IR-induced proliferation, in vitro. FA activation through paxillin was found to be a crucial mechanism by which KZ-41 inhibited ischemia-induced RNV in the murine oxygen-induced retinopathy (OIR) model.

Targeting stress kinase activation of FA signaling post-IR injury served as a way to prevent the pathological progression of RNV, in vivo. However, it is difficult to predict when or how to treat the inflammation early in ischemic retinopathy, especially in chronic conditions such as diabetes, when the injury has already occurred. Therefore, we sought to target the common focal point of ischemic disease by focusing on drivers of late stage RNV, the focal adhesion signaling complex. Using VEGF as the driver of in vitro angiogenesis, we explored growth factor-induced FA signaling in RECs to validate target proteins Src, focal adhesion kinase (FAK), and paxillin as crucial to RNV progression. Our work helped to identify a novel paxillin modulator, JP-153 which afforded excellent antiangiogenic activity, in vitro. JP-153 achieved potent inhibition of RNV in the OIR model through topical application by disrupting paxillin activation. Together, these data suggested paxillin is a key driver of RNV and may serve as a viable target for the treatment of neovascular eye disease.

In Chapter 6, we characterized the pharmacokinetic profile of JP-153 with regard to its absorption, distribution, metabolism, and elimination (ADME) after both oral and intravenous administration. We found that JP-153 exhibited rapid metabolism in rats with an oral bioavailability of approximately 30%. During these studies, we successfully developed a sensitive and selective analytical method using mass spectrometry in order to detect JP-153 concentrations in rat plasma. JP-153 possessed a relatively rapid clearance profile, which is an ideal characteristic for ocular therapeutics. Lower systemic exposures decrease the risk of cardiovascular side effects, a common concern with antiangiogenic therapies. Though, further work to characterize its ocular pharmacokinetic profile is needed to identify the proper dosing regimen in future studies. Thus, these data herein have served as a basis for further development of JP-153 series analogs, used either as a topical or systemic therapeutic for in vivo efficacy studies and pre-clinical work.

In conclusion, our work has successfully provided rationales for new drug targets and clinically relevant pharmacological agents to halt RNV. The following chapters describe and discuss novel ways in which we target inflammatory signaling and protein-protein interactions related to FA protein paxillin to effectively stop angiogenesis in the retina. Importantly, targeting paxillin has much broader implications in treating angiogenesis in general, and work studying paxillin modulation in cancer cells represents interesting hypotheses for future work in our laboratory.

ORCID

http://orcid.org/0000-0003-3075-7680

DOI

10.21007/etd.cghs.2016.0409

Comments

Two year embargo expires August 2018.

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