Peripheral Arterial Disease (PAD)
PAD is an atherosclerotic condition that is caused by reduced blood flow to the legs which can provoke symptoms of leg pain (“intermittent claudication”) during exercise. PAD is often observed with comorbid conditions such hypertension, dyslipidemia, diabetes, cigarette smoking, and/or physical inactivity, and involves multiple organ systems. Patients with PAD are at high risk for myocardial infarction, stroke, and all-cause mortality. Globally, >200 million people were living with PAD 2010, an almost 24% increase in 10 years. PAD is under-diagnosed and under-treated, there are limited therapeutic options. Thus, PAD represents a large clinical need.

Our solution to PAD: O304

Background
AMPK has been shown to exhibit multiple beneficial effects on the cardiovascular system, acting both on endothelial and vascular smooth muscle cells. AMPK activation enhances endothelial cell survival, increases angiogenesis and reduces vasoconstriction, platelet activation and smooth muscle cell proliferation. AMPK activation also reduces ischemia reperfusion damage in multiple organs. All these processes preserve and protect vascular function, an important precondition to provide tissue and cells with nutrients through an intact vasculature to maintain cellular energy levels. AMPK also exhibits anti-inflammatory activity, and appears to suppress atherosclerosis.

O304 regulates the expression of vascular effector molecules ex vivo
AMPK promotes angiogenesis by regulating the expression of vascular endothelial growth factor (Vegf) in an autoregulatory loop. O304 induces the expression of Vegf in both Human Umbilical Vein Endothelial Cells (HUVEC) and in rat L6 myotubes. Thrombospondin-1 (TSP-1) has been identified as a key negative regulator of nitric oxide (NO) signaling and TSP-1 limits the angiogenic activity of NO in endothelial cells. Blood vessel diameter and blood flow are also limited by TSP-1 through its ability to block responses to NO. TSP-1 limits both the vasodilator activity of NO in vascular smooth muscle and NOs antithrombotic activity in platelets. O304 reduces the expression of TSP-1 in dose-dependent manner in HUVEC.
Endothelin-1 (ET-1) is also a very potent vasoconstrictor which also acts to limit nitric oxide (NO) signaling. O304 reduces the expression of ET-1 in HUVEC.
Moreover, skeletal muscle cells express neural nitric oxide synthase (nNOS), and O304 induces the expression of nNOS in rat L6 myotubes. Thus, by suppressing TSP-1 and ET-1 and inducing Vegf and nNOS expression, O304 may exhibit multiple beneficial effects on the cardiovascular system.

O304 increases endurance in sedentary mice
Physical training increases endurance by enhancing the performance of muscle and the cardiovascular system. Genetic loss of AMPK activity in muscle causes severe reduction of endurance due to reduction in capillary density in nonpostural muscles. Chronic treatment with the AMPK activator AICAR increases endurance in sedentary normal mice, mimicking some aspects of physical exercise. Thus increased endurance in sedentary mice, which may reflect effects on the vascular system, can be used to test in vivo efficacy of putative AMPK activators.

Long-term treatment with O304 significantly increases treadmill running distance to exhaustion in aged normal mice and in genetically obese/diabetic Ob/Ob mice. In normal mice, the effect of O304 on endurance was maintained after 1 but not 2 weeks of wash-out. Moreover, compared to vehicle O304-treated mice showed lower blood lactate levels at exhaustion. Thus, the effect of O304 is long-lasting and involves increased clearance of lactate indicating that O304, at least in part, increases endurance through effects on the cardiovascular system.

O304 enhances the function of the cardiovascular system in sedentary mice
O304 increases blood flow in hind legs of sedentary mice monitored by Laser Doppler Imaging and the number of α-smooth muscle actin expressing blood vessels in red soleus muscle. O304 also generates a small but significant reduction in blood pressure and appears to increase left ventricle stroke volume.

O304 also reduces plasma levels of TSP-1 and ET-1 both in mouse and rat, indicating that O304 may increase NO signaling, which is consistent with reduced vasoconstriction, blood pressure and platelet activation and increased peripheral blood flow.

Thus, O304 exhibits multiple beneficial effects on the cardiovascular system by increasing the formation arterioles and capillaries, by increasing vasodilation and peripheral blood flow and by reducing blood pressure.