The vascular endothelium normally forms a tight barrier that regulates the entry of fluid, electrolytes and proteins into tissues. Due to the critical need to maintain this barrier function, vascular endothelial cells normally adhere very tightly to one another forming "tight junctions" and they divide only very slowly. When new blood vessel formation is required, for example in response to injury, certain growth factors are released that trigger specific signaling events inside vascular endothelial cells that cause the cells to proliferate and form new blood vessels from pre-existing vessels in a process called angiogenesis. Chief among these angiogenic factors are the fibroblast growth factor (FGF) family and vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF). Inappropriate release of angiogenic factors can occur under certain conditions resulting in compromised vasculostasis associated with disease states including:

1. Edema resulting from loss of tight junction integrity and increased vascular permeability (leakage of fluid, electrolytes and proteins into tissues).
2. Inappropriate vascular cell proliferation and angiogenesis.

Vascular Permeability and Edema
Compromised vasculostasis has serious pathologic consequences. If vascular permeability increases beyond manageable levels, the resulting edema will negatively impact tissue and organ function and ultimately survival. Examples of such life-threatening conditions include pulmonary edema, cerebral edema and cardiac edema.

Edema results from a breakdown of endothelial cell tight junctions coupled to changes in cytoskeletal support elements needed to maintain normal cell-to-cell apposition, thus leading to increased vascular permeability (or vascular leakage). Several vasoactive mediators can cause edema by triggering these events including histamine, bradykinin, thrombin, nitric oxide, eicosanoids (e.g., thromboxanes and leukotrienes), platelet activating factor (PAF), tumor necrosis factor (TNF), interleukins (e.g., IL-1 and IL-6), hepatocyte growth factor (HGF), and VEGF.

Edema and Ischemic Injury
Tissue hypoxia or lack of oxygen (ischemia) is a general cause of edema. Interruption of blood flow (such as when a blood clot forms causing a heart attack or stroke), medical intervention (such as cardioplegia, organ transplantation or angioplasty) or physical trauma can all lead to local ischemia. In turn, hypoxia induces a local increase in VEGF production leading to vascular leakage. This VEGF effect is at the level of the endothelial cell. In other words, VEGF binding to specific VEGF receptors expressed on endothelial cells leads to a cascade of intracellular events culminating in the loss of normal intercellular barrier function.

While hypoxia itself will lead to damage and death of tissue, ischemia-associated edema exacerbates the problem and subjects a much larger tissue area to the risk of permanent injury and death. Vascular leakage directly or indirectly compromises tissue viability by mediating:

1. loss of the normal tissue pH and salt balance,
2. influx of toxic substances including cytokines, pro-inflammatory factors, free radicals, etc. and
3. severe restriction in local tissue perfusion due to microcirculatory collapse as a result of edema ("no reflow" phenomenon).

Once this pathway has been triggered, it cannot be stopped simply by removing the initial cause of ischemia (e.g., removing the blood clot that caused a stroke or heart attack) and the ensuing edema will prevent the restoration of normal blood flow endangering still more tissue. TargeGen® scientists were among the first to show in models of cerebral stroke and myocardial infarct that profound potential therapeutic benefit may be obtained by blocking components of the VEGF signaling cascade that lead to vascular leakage and edema.

Angiogenesis
Excessive endothelial proliferation may also damage tissues (such as the retina in proliferative retinopathies) or fuel unwanted tissue growth (such as with tumor growth). Angiogenesis encompasses both enhanced vascular proliferation and permeability, as newly-formed blood vessels do not generally exhibit the same level of vascular barrier function as do well-established and mature vessels. Examples of such hyper-permeable vasculature can be found in cancers, vasculoproliferative diseases, retinal diseases, and rheumatoid arthritis. TargeGen compounds that simultaneously block multiple angiogenic pathways (e.g., block both VEGF and FGF signaling) are being evaluated for their potential to provide therapeutic benefit in the treatment of such diseases.

Angiogenesis and Vascular Permeability In Serious Diseases of the Eye
The leading cause of adult blindness in the developed world is AMD also know as wet macular degeneration. This disease is characterized by VEGF mediated retinal leakage (an unwanted increase in vascular permeability) and by the abnormal growth of small blood vessels in the back of the eye (angiogenesis). TargeGen® is developing a compound which may be applied topically (eye drops) and is able to penetrate the eye and achieve potentially therapeutic concentration levels in the retina and choroidal regions of the eye. This compound suppresses not only VEGF mediated retinal leakage but also angiogenesis and inflammation. Other debilitating eye conditions such as diabetic macular edema and diabetic retinopathy involve edema, angiogenesis and inflammation and TargeGen® plans to investigate the feasibility to treat these conditions as well as macular degeneration. Currently approved and soon to be approved treatments for macular degeneration require injection into the eye 9-15 times per year. Such injections pose a greater risk of eye infection and retinal detachment. TargeGen’s compound may represent the first meaningful treatment alternative for macular degeneration that does not require repeated injection into the eye.

Myeloproliferative Diseases Driven by the Activation of Individual Kinases
Myeloproliferative diseases are a group of related diseases characterized by the overproduction of certain blood components such as red blood cells. Polycythemia Vera (PV) is a prototypical example of a myeloproliferative disease caused by the activation of a mutant form of a single kinase (Jak2 V617F) that drives the disease. In PV, the Jak-2 kinase mutation (V617F) causes patients to overproduce red blood cells. Over time, they may suffer stroke, other thrombotic events and significant enlargement of the spleen as a consequence. TargeGen® is developing an internally discovered Jak-2 inhibitor (TG101348) which has demonstrated excellent results in published pre-clinical testing. Pending the expected submission of an IND in late 2007, TargeGen® plans to start human clinical trials with TG101348 in early 2008. Other myeloproliferative diseases that involve the Jak2 (V617F) mutation include Essential Thrombocytopenia (ET) and Myelofibrosis with Myeloid Metaplasia (MMM). The prevalence of PV, ET, and MMM collectively is estimated at approximately 200,000 patients. There is no specific therapy currently approved for any of these conditions which are associated with significant morbidity and mortality.

TargeGen's Approach to Drug Discovery
TargeGen's drug discovery approach combines vascular biology with small molecule medicinal chemistry. TargeGen®, to date, has designed, tested and filed patent applications on more than 1500 new chemical entities to date. These molecules are based on advanced rational drug design techniques aided by sophisticated computer modeling and X-ray chrystallography. Within this collection are compounds that inhibit distinct elements of different parts of the VEGF and related signaling pathways that lead to angiogenesis, vascular permeability and inflammation. Additionally, some of these compounds inhibit specific single kinase mutations associated with disease (Jak-2).

Inhibition of an entire pathway can potentially lead to unwanted consequences. VEGF receptor antagonists, for example, may be too "high" on the pathway as their use has been associated with side effects such as hypertension. In contrast, TargeGen's approach is to identify molecules that effectively block angiogenesis and/or vascular permeability by specifically inhibiting downstream elements of the signaling pathways that are associated with these processes. This approach may not only result in blocking inappropriate VEGF and FGF actions but may also block vascular leakage and/or vascular proliferation induced by other factors. An important additional advantage of TargeGen's drug discovery process is the early use of in vivo efficacy data for the selection of initial lead molecules. This approach is unbiased with regard to molecular targets and avoids unnecessary effort on compounds with problematic pharmacodynamic properties or toxicity profiles.