Indications

Type 2 Diabetes

The increase in Type 2 diabetes (T2D) is strongly linked to the global increase in obesity, caused by physical inactivity and a western diet, as well as an aging population. During development of T2D, insulin resistance, and reduced glucose uptake in skeletal muscle result in an initial combined hyperglycemia and hyperinsulinemia, followed by a decline in β-cell function and insulin secretion. O304 increases glucose uptake in myotubes an insulin-independent mechanism and increases glucose uptake in skeletal muscle in vivo in DIO mice and reduces systemic and age-induced insulin resistance. O304 matches the level of insulin secretion to demand and induces β-cell rest. O304 also prevents accumulation of toxic IAPP aggregates/amyloid in islet cells and reduces plasma glucagon levels. No other anti-diabetic drug can improve glucose homeostasis through this combination of mechanisms.

AMPK and T2D: No currently used anti-glycemic drug increases glucose uptake in skeletal muscle, reduces insulin resistance and preserves β-cell function. Exercise appears more effective than insulin treatment to promote glucose uptake in insulin resistant skeletal muscle of T2D patients, at least in part mediated by activation of AMPK. By increasing glucose uptake in skeletal muscle AMPK indirectly imposes β-cell rest. AMPK appears also to act in β-cells to ensure that insulin secretion matches the demand by supporting normal β-cell glucose sensing and glucose stimulated insulin secretion that promote β-cell rest. AMPK also stimulates macro-autophagy that is important for β-cell function and survival. These combined activities of AMPK activation protect β-cell health and function. PAN-AMPK activator O304 as treatment for T2D

Clinical: In a 28 day phase IIa trial in T2D patients, O304 reduced fasting plasma glucose and insulin resistance/HOMA-IR. Steady-state was reached ~ day 18, and a larger effect of O304 on glucose homeostasis is expected at higher of exposure and by longer duration of treatment.

Diabetic Kidney Disease

The overall increase in the number of people with diabetes has had a major impact on development of diabetic kidney disease (DKD), one of the most frequent complications of both types of diabetes. DKD is the leading cause of end-stage renal disease (ESRD), accounting for approximately 50% of cases in the developed world, and DKD is an area of high unmet medical need. The etiology of the disease progression is complex because of an interplay between systemic metabolic and vascular dysfunctions that involves both tubular and glomerular renal systems. Current therapies slow down but do not prevent the progression of the disease. Recently, SGLT2i/Dapagliflozin that elicits an initial reduction in eGFR, which over time translates to a diminished decline in eGFR compared to placebo, and a significantly reduced risk of ESRD and CV and renal death in both DKD and CKD. Mechanisms of action of SGLT2i in DKD and CKD remains poorly understood but appears to be independent of the hyperemic effect. As an off-target effect SGLT2i inhibits mitochondria and activates AMPK indirectly by lowering energy charge that may contribute to efficacy. Dapagliflozin shows a ~30% reduction in proteinuria in DKD but with no effect in CKD.

AMPK and DKD: A reduced phosphorylation state of T172 AMPK has been correlated with diminished kidney function in both humans and animal models of renal disease. Direct AMPK activators are profoundly reno-protective in a rat model of progressive diabetic nephropathy by reducing proteinuria,  improving glomerular filtration rate and kidney fibrosis, Sallato et. al., J Pharmacol Exp Ther 2017 & Zhou et.al., J Pharmacol Exp Ther 2019

Clinical: In T2D patients, O304 both lowered systemic blood pressure and increased microvascular perfusion. Moreover, in both the phase I and IIa clinical studies O304 potently reduced eGFR by a rapid, stable and reversible haemodynamic effect both in obese non-diabetic subjects and in T2D patients on metformin. O304 also reduced eGFR both in T2D patients with no anti-hypertensive treatment or on top of ACEi/ARB drug treatment (standard of care for DKD). These effects on renal filtration/hyperfiltration are similar to what has been reported for SGLT2 inhibitors. Since O304 also reduces blood glucose and insulin resistance, lowers blood pressure and increases microvascular perfusion. The combination of O304 and SGLT2i as treatment for DKD and CKD is therefore of high interest.

Heart Failure

Patients with heart failure (HF) are commonly divided into those with reduced ejection fraction (EF<40%) and those with preserved ejection fraction (HFpEF; EF>50%). HFpEF without overt coronary arterial disease (CAD) is strongly associated with age and linked to diastolic dysfunction due to myocardial stiffness causing increased left ventricular (LV) end-diastolic pressure pressure and reduced LV filling. For heart failure with reduced EF, a number of therapies have been found to improve patient morbidity and mortality, and treatment is guideline based. However for patients with HFpEF, no treatment has been found to have clinical benefit, and HFpEF is a clinical syndrome associated with poor quality of life, substantial health-care resource utilization, and premature mortality. Thus, the lack of evidence-based treatment guidelines for HFpEF identifies a huge clinical need and an opportunity to develop novel drugs for almost half the population with heart failure. Moreover, improvements in exercise capacity and heart failure symptoms are key to HFpEF patients, and no current treatment can improve working capacity in HFpEF patients.

Microvascular dysfunction: A recent prospective multi-center study has provided evidence that impaired coronary microvascular function is highly prevalent in HFpEF patients and is associated with systemic endothelial dysfunction expressed as increased urinary albumin-to-creatinine ratio (UACR) and lower reactive hyperemic index (RHI). Renal microvascular dysfunction is major cause of CKD, and as coronary microvascular dysfunction is prevalent in HFpEF and associated with increased UACR and RHI, systemic microvascular dysfunction may be causative in both HFpEF and CKD. Thus, microvascular dysfunction may be a promising novel therapeutic target in HFpEF.

O304 and HFpEF: In preclinical species O304 increases LV stroke volume by increasing end-diastolic volume i.e. improves relaxation of the LV both in obese mice after 6 weeks treatment and after 8 month treatment in aged lean mice. O304 also increases peripheral microvascular perfusion and improves exercise endurance in mouse. In addition, a single dose of O304 lowers blood pressure in dog.

O304-Cardiac safety: The recently described AMPK activator by Merck, MK-8722, promoted glycogen accumulation and induced cardiac hypertrophy in preclinical species. O304 increases AMPK activity by a completely different mechanism than MK-8722, and O304 mimics the beneficial metabolic and cardivascular effects of exercise. Consistently, in a 6 month rat tox study no clinical signs and no increase in heart weight was observed. In a 9 month tox study in dog, no clinical sign, no accumulation of glycogen in heart and no ECG adverse effects were observed. Thus, there is no concern for adverse cardiac effects with O304. In contrast, the beneficial cardiorenal and microvascular effects of O304 suggest that O304 could be developed into a long though after treatment for HFpEF.

Clinical: In a 28 day Phase IIa trial in T2D patients on metformin, compared to placebo O304 lowered blood pressure and increased microvascular perfusion in calf muscle. The microvascular effect of O304 was also manifested as an early and reversible reduction in estimated glomerular filtration rate (eGFR), also on top of standard of care (SoC) ACEi/ARB drugs. Thus, the beneficial metabolic, microvascular and cardiorenal effects of O304 indicate that O304 may be suitable as a novel treatment for HFpEF.

Obesity

Obesity is a global epidemic that is strongly associated with the global increase in type 2 diabetes and associated macro- and microvascular complications. Obesity occurs when assimilated energy exceeds energy expenditure and can only be reduced by affecting either side of the energy balance equation. Currently, there are no effective medical treatments for obesity, and current treatments reduce food intake and do not increase energy expenditure (EE). A drug that increases EE for a significant fraction of obese patients, may therefore have profound effects on a myriad of metabolic and cardiovascular complications, and represent a ‘‘first in class’’ pharmaceutical breakthrough.

O304 and Obesity: In DIO mice, O304 both prevents and reduces white adipose tissue (WAT) weight but not lean weight, also at thermoneutrality. Under these conditions O304 increases food intake providing evidence that O304 increases energy expenditure. Consistently, in DIO mice O304 significantly increased oxygen consumption (VO2) during both light and dark period, and decreased significantly respiratory exchange ratio (RER) at day 2 during the light period, and throughout a 3-day measurements during the dark period, providing evidence that mice fed O304-HFD switched their main energy source from carbohydrates to fatty acids. Accordingly, O304 significantly increased EE during both light and dark periods.

To reduce WAT depots, lipolysis needs to be enhanced. Desnutrin/Atgl, which encodes the rate-limiting enzyme catalyzing basal triglyceride (TG) hydrolysis is a direct target of AMPK, and phosphorylation of S406 by AMPK increases ATGL activity, which should increase lipolysis. Accordingly, O304 increased both Atgl mRNA levels and p-S406 ATGL levels inguinal (i)WAT. Moreover, Cpt1 b that increases mitochondrial FA uptake, and PGC-1α, PPARα and Cox8b, which would increase mitochondrial activity/FA oxidation, were also increased by O304. Under these conditions, O304 reduced the low-level UCP1 expression in iWAT, arguing against ectopic expression of UCP1 in WAT as a mechanism for the anti-obesity effect of O304, which mimics the effects of systemic genetic activation of AMPK, which protects against diet induced obesity through Ucp1-independent thermogenesis in WAT. Pollard et al., Nat Metab. 2019.  Importantly, additional mechanisms for activating thermogenesis beyond UCP1 have been identified and characterized to varying extents. Putative futile cycles include, creatine-dependent substrate cycling, calcium-dependent ATP hydrolysis, triacylglycerol/fatty acid (TAG/FA) cycling and mitochondrial H+ permeability through the ADP/ATP carrier mediates UCP-1 independent uncoupling and requires free fatty acids. Whether O304 promotes one or several of these futile cycles to increase energy expenditure will require further work.

O304 increases energy expenditure in dog. Moreover, in 9 month tox study in dogs O304 did not reduce body weight but reduced body weight gain in a potent and dose-dependent manner in dogs that were fed regular diet, which was fully reversible after washout. Under these conditions O304 increased food intake and thus EE. Thus, long term treatment with O304 increased energy expenditure in dog without causing any adverse effects. In summary: By potently increasing energy exposure in both mouse and dog, O304 may be developed into a first-in-class anti-obesity drug.

NAFLD/NASH

Obesity, insulin resistance, and diabetes are strongly linked to the accumulation of excessive lipids in the liver causing nonalcoholic fatty liver disease (NAFLD). Due to the association with obesity and related metabolic diseases, the prevalence of NAFLD has dramatically increased in the past few decades. NAFLD may progress to Nonalcoholic steatohepatitis (NASH) characterized by inflammatory lesions and fibrosis with a greatly increased risk of risk of developing cirrhosis and hepatocellular carcinoma. PNPLA3 is a lipid droplet-associated protein which gene expression is mediated by SREBP-1c in liver.  The PNPLA3(148M) dominant negative variant, is highly prevalent in the general population and causes a large increase in the risk for hepatic triglyceride accumulation (steatosis), inflammation, fibrosis, cirrhosis, and even hepatocellular carcinoma. PNPLA3 (148M) suppresses ATGL activity, the major lipolytic enzyme in liver and fat that is activated AMPK/O304. Currently, there are no approved treatments for NASH including reduction in expression of PNPLA3 (148M), and it remains a significant unmet medical need.

AMPK and NASH: An increasing body of evidence has demonstrated that AMPK activity is repressed during metabolic disorders, including obesity, diabetes and NAFLD. The inhibition of AMPK connects lipid dysregulation to inflammation, liver injury and fibrosis in NAFLD. By suppressing the expression and activity of SREBP-1c AMPK suppress hepatic expression genes encoding enzymes mediating de novo lipogenesis while promoting fatty acid oxidation. Pharmacological activation of AMPK improves NALFD and NASH in both murine and simian models.

O304 and NASH: In diet induced obesity (DIO) in mice, O304 mitigates systemic insulin resistance and reduces fatty liver by reducing hepatic de novo lipogenesis (DNL) and by increasing fatty acid oxidation/plasma b-hydroxybutyrate. Consistently, O304 reduces hepatic mRNA expression of SREBP-1c and of the lipogenic enzymes Acc, Fas and Scd-1 as well as of PNPLA3.  O304 also potently increases the hepatic mRNA expression of the soluble IL-1RN that should mitigate IL-1β mediated inflammation. Moreover, in mice fed “NASH-diets” O304 reduces plasma ALT and AST levels indicative of reduced liver injury. O304 also suppresses an increase in spleen weight, suggesting reduced portal hypertension. Moreover, O304 suppresses DNL in primary human hepatocytes and suppresses IL-1β and TGFβ signaling and markers of inflammation and fibrosis in human cell lines. Taken together, O304 may be developed into novel treatment for NAFLD/NASH.