Faculty Bibliography

Background: Diabetic Cardiomyopathy (DbCM) leading to overt heart failure is a common sequalae of both Type 1 and Type 2 Diabetes. Previous attempts to develop treatments for DbCM through inhibition of the enzyme Aldose Reductase (AR) were unsuccessful, due to low AR binding affinity and poor specificity. Off-target inhibition of Aldehyde Reductase (AldR), an enzyme critical for detoxification of aldehydes in the liver and normal hepatocyte physiology, led to common liver-related safety and tolerability issues with first generation ARI compounds. We report on the selectivity and specificity of AT-001, a novel small molecule ARI with optimized affinity and specificity for AR and minimal to zero off-target AldR activity.
Methods and Results: AT-001 was evaluated for AR binding affinity as compared to zopolrestat, a first-generation ARI. AT-001 demonstrated a >300-fold greater affinity for AR versus zopolrestat (IC50 of 30 pmol for AT-001 and ~10 nmol for zopolrestat). In contrast to zopolrestat, AT-001 showed no inhibition of AldR at 50x and 100x EC50 levels in assay medium, while zopolrestat inhibited Aldehyde Reductase by 50% and 60% respectively (spec activity of 2.3 and 1.9 mmol NADPH/min/ mg protein). In addition to these analyses, AT-001 was evaluated in a standard off-target receptor binding analysis of 87 substrates (13 enzyme, 74 binding assays) to characterize pharmacological specificity and selectivity. No significant off-target results (defined as >= 50% inhibitory activity) were observed in this panel.
Conclusion(s): The unique structure and activity of AT-001 provide selectivity for Aldose Reductase and lack of off-target effects. The in-vitro safety of this agent together with the positive safety data from the phase 1/2 program, supports the ongoing pivotal study in DbCM. Keywords: AT-001, Diabetic Cardiomyopathy, Aldol Reductase Inhibitor, Heart Failure Abbreviations: DbCM, CHF, AR, ARI Funding and Conflicts of Interest: Applied Therapeutics Inc (Salary) (Perfetti, Shendelman). Applied Therapeutics Inc (Equity / Shareholder)(Perfetti, Shendelman). Applied Therapeutics Inc (Study Funding) (Ramasamy).
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The role of advanced glycation end products (AGEs) in promoting and/or exacerbating metabolic dysregulation is being increasingly recognized. AGEs are formed when reducing sugars non-enzymatically bind to proteins or lipids, a process that is enhanced by hyperglycemic and hyperlipidemic environments characteristic of numerous metabolic disorders including obesity, diabetes and its complications. In this mini-review, we put forth the notion that AGEs span the spectrum from cause to consequence of insulin resistance (IR) and diabetes, and represent a "common soil" underlying the pathophysiology of these metabolic disorders. Collectively, the surveyed literature suggests that AGEs, both those that form endogenously as well as exogenous AGEs derived from environmental factors such as pollution, smoking and "Western" style diets, contribute to the pathogenesis of obesity and diabetes. Specifically, AGE accumulation in key metabolically-relevant organs induces IR, inflammation and oxidative stress, which in turn provide substrates for excess AGE formation, thus, creating a feed-forward fueled pathological loop mediating metabolic dysfunction.

Obesity and diabetes are leading causes of cardiovascular morbidity and mortality. Although extensive strides have been made in the treatments for non-diabetic atherosclerosis and its complications, for patients with diabetes, these therapies provide less benefit for protection from cardiovascular disease (CVD). These considerations spur the concept that diabetes-specific, disease-modifying therapies are essential to identify, especially as the epidemics of obesity and diabetes continue to expand. Hence, as hyperglycemia is a defining feature of diabetes, it is logical to probe the impact of the specific consequences of hyperglycemia on the vessel wall, immune cell perturbation, and endothelial dysfunction-all harbingers to the development of CVD. In this context, high levels of blood glucose stimulate the formation of the irreversible advanced glycation end products, the products of non-enzymatic glycation and oxidation of proteins and lipids. AGEs accumulate in diabetic circulation and tissues and the interaction of AGEs with their chief cellular receptor, receptor for AGE or RAGE, contributes to vascular and immune cell perturbation. The cytoplasmic domain of RAGE lacks endogenous kinase activity; the discovery that this intracellular domain of RAGE binds to the formin, DIAPH1, and that DIAPH1 is essential for RAGE ligand-mediated signal transduction, identifies the specific cellular means by which RAGE functions and highlights a new target for therapeutic interruption of RAGE signaling. In human subjects, prominent signals for RAGE activity include the presence and levels of two forms of soluble RAGE, sRAGE, and endogenous secretory (es) RAGE. Further, genetic studies have revealed single nucleotide polymorphisms (SNPs) of the AGER gene (AGER is the gene encoding RAGE) and DIAPH1, which display associations with CVD. This Review presents current knowledge regarding the roles for RAGE and DIAPH1 in the causes and consequences of diabetes, from obesity to CVD. Studies both from human subjects and animal models are presented to highlight the breadth of evidence linking RAGE and DIAPH1 to the cardiovascular consequences of these metabolic disorders.

Introduction: Hyperactivation of the polyol pathway (PP) contributes to development of diabetic complications such as diabetic cardiomyopathy (DbCM). Aldose reductase (AR), the rate-controlling enzyme in the PP, catalyzes NADPH-dependent reduction of glucose to sorbitol. Under hyperglycemic and ischemic conditions, PP activation causes intracellular sorbitol accumulation leading to osmotic stress, cell death and diabetic complications. Previous AR inhibitors (ARI) failed due to safety or lack of efficacy. AT-001 is a novel oral ARI with optimized specificity and affinity for AR.
Objective(s): To assess cardioprotective activity of AT-001 in an animal model of DbCM and safety, tolerability and target engagement in a phase 1/2 clinical study. Preclinical
Methods and Results: The cardioprotective effect of AT-100 was studied in transgenic mice expressing human levels of AR (Tg hAR) and rendered diabetic with streptozotocin (55 mg/kg for 5d). Mice were treated with AT-001 40 mg/kg, n=6 or vehicle (V, n=6) for 3d prior to cardiac ischemia reperfusion (IR) injury induced by ligation and reperfusion of the left anterior descending (LAD) coronary artery. Control non-DM Tg hAR mice (non-DM, n=9) were also subjected to IR injury. After 48h recovery mice were euthanized and infarct area and fractional shortening (FS) assessed. Mean (+/-SD) infarct area (ratio of infarcted to total left ventricular area) was significantly lower in AT-001 (35.3+/-2.7) vs. non-DM (43+/-2) and V (59.1+/-0.8); P<0.05 vs non-DM and V). FS 48h post reperfusion, a measure of functional recovery, was greater in AT-001 vs non-DM and V: 38.2+/-2, 24+/-3, 21+/-1.5, respectively; P<0.05 vs. non-DM and V. Clinical methods and Results: The phase 1/2 study consisted of single and multiple oral ascending doses (SAD, n=8 per group) of 5, 10, 20 and 40 mg/kg once daily (QD) and multiple doses (MAD) of placebo (Pbo) or AT-001 5, 20, 40 mg/kg QD or 20 mg/kg twice daily for 7d. Subjects with type 2 diabetes (T2DM), age 18-75 and HbA1c 5.0-8.5% were allocated to AT-001 (n=8) or Pbo (n=2) in each dose cohort. AT-001 was well-tolerated with no drug-related AEs. AR inhibition and target engagement were demonstrated by dose-dependent decreases in sorbitol in AT-001 (up to -50 % change from baseline) vs. Pbo subjects (-3 % change). Maximum inhibition was between 2-4 h and lasted several hours.
Conclusion(s): AT-001 significantly attenuated or prevented cardiac damage in a relevant animal model and was safe, well-tolerated and reduced sorbitol levels in T2DM subjects. These findings support clinical development in DbCM.
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Exquisite regulation of energy homeostasis protects from nutrient deprivation but causes metabolic dysfunction upon nutrient excess. In human and murine adipose tissue, the accumulation of ligands of the receptor for advanced glycation end products (RAGE) accompanies obesity, implicating this receptor in energy metabolism. Here, we demonstrate that mice bearing global- or adipocyte-specific deletion of Ager, the gene encoding RAGE, display superior metabolic recovery after fasting, a cold challenge, or high-fat feeding. The RAGE-dependent mechanisms were traced to suppression of protein kinase A (PKA)-mediated phosphorylation of its key targets, hormone-sensitive lipase and p38 mitogen-activated protein kinase, upon β-adrenergic receptor stimulation-processes that dampen the expression and activity of uncoupling protein 1 (UCP1) and thermogenic programs. This work identifies the innate role of RAGE as a key node in the immunometabolic networks that control responses to nutrient supply and cold challenges, and it unveils opportunities to harness energy expenditure in environmental and metabolic stress.

Incense burning is common worldwide and produces environmental toxicants that may influence health; however, biologic effects have been little studied. In 303 Emirati adults, we tested the hypothesis that incense use is linked to compositional changes in the oral microbiota that can be potentially significant for health. The oral microbiota was assessed by amplification of the bacterial 16S rRNA gene from mouthwash samples. Frequency of incense use was ascertained through a questionnaire and examined in relation to overall oral microbiota composition (PERMANOVA analysis), and to specific taxon abundances, by negative binomial generalized linear models. We found that exposure to incense burning was associated with higher microbial diversity (p < 0.013) and overall microbial compositional changes (PERMANOVA, p = 0.003). Our study also revealed that incense use was associated with significant changes in bacterial abundances (i.e. depletion of the dominant taxon Streptococcus), even in occasional users (once/week or less) implying that incense use impacts the oral microbiota even at low exposure levels. In summary, this first study suggests that incense burning alters the oral microbiota, potentially serving as an early biomarker of incense-related toxicities and related health consequences. Although a common indoor air pollutant, guidelines for control of incense use have yet to be developed.

Background/UNASSIGNED:(the gene encoding RAGE), have been reported to be associated with T2D and its complications, we tested for potential relationships between these factors and T2D status in Emirati subjects. Methods/UNASSIGNED:In a case-control study, we recruited Emirati subjects with T2D and controls from the Sheikh Khalifa Medical City in Abu Dhabi. Anthropomorphic characteristics, levels of plasma sRAGE and esRAGE, and routine chemistry variables were measured. Results/UNASSIGNED: = 0.01, after adjustment for age and sex). Conclusion/discussion/UNASSIGNED:Levels of sRAGE, but not esRAGE, were associated with T2D status in Abu Dhabi, but not after correction for eGFR. Elevated levels of plasma insulin in both control and T2D subjects suggests the presence of metabolic dysfunction, even in subjects without diabetes.