A novel controlled metabolic accelerator for the treatment of obesity-related heart failure with preserved ejection fraction: Rationale and design of the Phase 2a HuMAIN trial.

AIMS: Compared with those without obesity, patients with obesity-related heart failure with preserved ejection fraction (HFpEF) have worse symptoms, haemodynamics, and outcomes. Current weight loss strategies (diet, drug, and surgical) work through decreased energy intake rather than increased expenditure and cause significant loss of skeletal muscle mass in addition to adipose tissue. This may have adverse implications for patients with HFpEF, who already have reduced skeletal muscle mass and function and high rates of physical frailty. Mitochondrial uncoupling agents may have unique beneficial effects by producing weight loss via increased catabolism rather than reduced caloric intake, thereby causing loss of adipose tissue while sparing skeletal muscle. HU6 is a controlled metabolic accelerator that is metabolized to the mitochondrial uncoupling agent 2,4-dinotrophenol. HU6 selectively increases carbon oxidation from fat and glucose while also decreasing toxic reactive oxygen species (ROS) production. In addition to sparing skeletal muscle loss, HU6 may have other benefits relevant to obesity-related HFpEF, including reduced specific tissue depots contributing to HFpEF; improved glucose utilization; and reduction in systemic inflammation via both decreased ROS production from mitochondria and decreased cytokine elaboration from excess, dysfunctional adipose. METHODS: We describe the rationale and design of HuMAIN-HFpEF, a Phase 2a randomized, double-blind, placebo-controlled, dose-titration, parallel-group trial in patients with obesity-related HFpEF to evaluate the effects of HU6 on weight loss, body composition, exercise capacity, cardiac structure and function, metabolism, and inflammation, and identify optimal dosage for future Phase 3 trials. CONCLUSIONS: HuMAIN will test a promising novel agent for obesity-related HFpEF.

Safety and efficacy of once-daily HU6 versus placebo in people with non-alcoholic fatty liver disease and high BMI: a randomised, double-blind, placebo-controlled, phase 2a trial.

BACKGROUND: HU6 is a controlled metabolic accelerator that is metabolised in the liver to the mitochondrial uncoupler 2,4-dinitrophenol and increases substrate utilisation so that fat and other carbon sources are oxidised in the body rather than accumulated. We aimed to assess the safety and efficacy of HU6 compared with placebo in people with non-alcoholic fatty liver disease (NAFLD) and high BMI. METHODS: This randomised, double-blind, placebo-controlled, phase 2a trial was done at a single community site in the USA. Adults (aged 28-65 years) with a BMI of 28-45 kg/m2, a FibroScan controlled attenuation parameter score of more than 270 decibels per metre, and at least 8% liver fat by MRI-proton density fat fraction (MRI-PDFF) were randomly assigned (1:1:1:1) to receive, under fasting conditions, either once-daily HU6 100 mg, HU6 300 mg, HU6 450 mg, or matching placebo by oral administration for 61 days. Randomisation was blocked (groups of four) and stratified by baseline glycated haemoglobin (<5·7% vs ≥5·7%; 39 mmol/mol). All participants and study personnel involved with outcome assessments were masked to treatment assignment. The primary endpoint was the relative change in liver fat content from baseline to day 61, as assessed by MRI-PDFF, and was analysed in the full analysis set (FAS), which comprised all participants who were randomly assigned, received at least one dose of treatment, and had less than 4·5 kg of weight gain or weight loss from the time of screening to day 1 of treatment. The safety population included all participants who were randomly assigned and received at least one dose of study drug. This study was registered at ClinicalTrials.gov, NCT04874233, and is complete. FINDINGS: Between April 28, 2021, and Nov 29, 2021, 506 participants were assessed for eligibility and 80 adults (39 [49%] women and 41 [51%] men) were enrolled and randomly assigned to placebo (n=20), HU6 150 mg (n=20), HU6 300 mg (n=21), or HU6 450 mg (n=19). One participant in the HU6 450 mg group was excluded from the FAS due to weight gain. Relative mean change in liver fat content from baseline to day 61 was -26·8% (SD 17·4) for the HU6 150 mg group, -35·6% (13·8) for the HU6 300 mg group, -33·0% (18·4) for the HU6 450 mg group, and 5·4% (19·8) for the placebo group. Three people treated with HU6 (two treated with 150 mg and one treated with 300 mg) and two people treated with placebo discontinued treatment due to treatment-emergent adverse events (TEAEs). No serious TEAEs were reported. In those treated with HU6, flushing (19 [32%] participants), diarrhoea (15 [25%] participants), and palpitations (seven [12%] participants) were the most frequently reported TEAEs (in the placebo group, two [10%] participants had flushing, none had diarrhoea, and one [5%] had palpitations). There were no deaths. INTERPRETATION: HU6 could be a promising pharmacological agent for treating patients with obesity and NAFLD and its metabolic complications. FUNDING: Rivus Pharmaceuticals.

Pharmacokinetic and Pharmacodynamic Effects of Oral CXA-10, a Nitro Fatty Acid, After Single and Multiple Ascending Doses in Healthy and Obese Subjects.

10-nitro-9(E)-octadec-9-enoic acid (CXA-10), a novel nitro fatty acid compound, demonstrates potential as a therapeutic agent in multiple disease indications in which oxidative stress, inflammation, fibrosis, and/or direct tissue toxicity play significant roles. Phase I studies were conducted in healthy and obese subjects to evaluate the pharmacokinetics (PK), pharmacodynamics (PD), safety, and tolerability of oral CXA-10 after single and multiple doses in the fed and fasted states that would confirm the mechanisms of action of CXA-10. After single and multiple ascending doses, CXA-10 demonstrated dose-proportional increases in plasma exposure. CXA-10 decreased levels of biomarkers associated with altered inflammation and metabolic stress observed from nonclinical studies. In CXA-10-202, a consistent decrease from baseline was observed with CXA-10 150 mg dose, but not 25 or 450 mg doses, for biomarkers of altered inflammation and metabolic dysfunction, including leptin, triglycerides, cholesterol, MCP-1, and IL-6. In CXA-10-203, after coadministration with CXA-10, geometric mean peak plasma concentration (Cmax ) and area under the plasma concentration-time curve from time point 0 to the end of the dosing interval (AUC0-t ) decreased 20% and 25% for pravastatin, increased 10% and 25% for simvastatin, and decreased 20% and 5% for ezetimibe. These findings are consistent with the pharmacological effects of CXA-10. Adverse events (AEs) were dose-related, and the most frequently reported AEs (>10% of subjects) were diarrhea, abdominal pain, and nausea. CXA-10 was safe and well-tolerated with no clinically significant abnormalities reported on physical examination, vital signs, clinical laboratory evaluations, or electrocardiographic evaluation. Phase II studies are underway in patients with focal segmental glomerulosclerosis and pulmonary arterial hypertension to investigate the efficacy and tolerability of CXA-10 75-300 mg once daily.

CXA-10, a Nitrated Fatty Acid, Is Renoprotective in Deoxycorticosterone Acetate-Salt Nephropathy.

Underlying pathogenic mechanisms in chronic kidney disease (CKD) include chronic inflammation, oxidant stress, and matrix remodeling associated with dysregulated nuclear factor-κ B, nuclear factor-κ B, and SMAD signaling pathways, respectively. Important cytoprotective mechanisms activated by oxidative inflammatory conditions are mediated by nitrated fatty acids that covalently modify proteins to limit inflammation and oxidant stress. In the present study, we evaluated the effects of chronic treatment with CXA-10 (10-nitro-9(E)-octadec-9-enoic acid) in the uninephrectomized deoxycorticosterone acetate-high-salt mouse model of CKD. After 4 weeks of treatment, CXA-10 [2.5 millligrams per kilogram (mpk), p.o.] significantly attenuated increases in plasma cholesterol, heart weight, and kidney weight observed in the model without impacting systemic arterial blood pressure. CXA-10 also reduced albuminuria, nephrinuria, glomerular hypertrophy, and glomerulosclerosis in the model. Inflammatory MCP-1 and fibrosis (collagen, fibronectin, plasminogen activator inhibitor-1, and osteopontin) renal biomarkers were significantly reduced in the CXA-10 (2.5 mpk) group. The anti-inflammatory and antifibrotic effects, as well as glomerular protection, were not observed in the enalapril-treated group. Also, CXA-10 appears to exhibit hormesis as all protective effects observed in the low-dose group were absent in the high-dose group (12.5 mpk). Taken together, these findings demonstrate that, at the appropriate dose, the nitrated fatty acid CXA-10 exhibits anti-inflammatory and antifibrotic effects in the kidney and limits renal injury in a model of CKD.

Nitro-fatty acids: New drug candidates for chronic inflammatory and fibrotic diseases.

Nitrated oleic acid (NO2-OA) was first identified in 2003, and after the characterization of its formation and thiol reactivity, it was used as a prototypical molecule to investigate the physiological actions of endogenous nitrated fatty acids (NO2-FA). Based on in vitro observations showing significant activation of cytoprotective and anti-inflammatory signaling responses by NO2-FA, experiments were designed to determine their pharmacological potential. Supported by strong intellectual protection and favorable pharmacokinetic and pharmacodynamic data, 10-NO2-OA (CXA-10) underwent pharmaceutical development as a drug to treat fibrotic and inflammatory diseases. NO2-FA are at the intersection of three unconventional drug candidate classes that include 1) fatty acids, 2) metabolic intermediates and 3) electrophilic molecules. These three groups use different scaffolds for drug development, are characterized by broad activities and are individually gaining traction as alternatives to mono-target drug therapies. In particular, NO2-FA share key characteristics with currently approved pharmacological agents regarding reactivity, distribution, and mechanism of action. This review first presents the characteristics, liabilities, and opportunities that these different drug candidate classes display, and then discusses these issues in the context of current progress in the preclinical and clinical development of NO2-FA as drugs. Lessons learned from the novel approaches presented herein were considered early on during development to structurally define and improve NO2-FA and their disease targets.

Evaluation of 10-Nitro Oleic Acid Bio-Elimination in Rats and Humans.

Nitrated fatty acids are endogenously present in human and animal tissues, as well as in plant-derived oils. In particular, 10-nitro oleic acid (10-NO2-OA) potently induces Nrf2-dependent antioxidant gene expression and inhibits TLR4/NF-κB signaling, thus promoting an overall cyto-protective and anti-inflammatory response. 10-NO2-OA has been extensively tested in animal models and is currently undergoing clinical evaluation in humans. Bio-elimination pathways for 10-NO2-OA were evaluated in rats (30 mg/kg·day) and in humans (0.34 mg/kg) using samples obtained from a double-blind, dose-rising clinical trial. Quantitative radiochromatographic/MS analysis indicated that the renal and fecal pathways are the main routes for 10-NO2-OA excretion in rats, and allowed the identification of 4-nitro-octanedioic acid (NO2-8:0-diCOOH) as the most abundant metabolite in rat urine. In addition, high resolution LC-MS/MS analysis revealed the presence of a novel series of urinary metabolites including ω-carboxylation and ß-oxidation products, as well as N-acetylcysteine, taurine and sulfo-conjugates in both rats and humans. Overall, the findings reported herein not only provide valuable tools for the experimental evaluation of 10-NO2-OA levels in vivo, but importantly they also set the basis for monitoring its metabolism during potential clinical interventions in humans.

Nitro-fatty acid pharmacokinetics in the adipose tissue compartment.

Electrophilic nitro-FAs (NO2-FAs) promote adaptive and anti-inflammatory cell signaling responses as a result of an electrophilic character that supports posttranslational protein modifications. A unique pharmacokinetic profile is expected for NO2-FAs because of an ability to undergo reversible reactions including Michael addition with cysteine-containing proteins and esterification into complex lipids. Herein, we report via quantitative whole-body autoradiography analysis of rats gavaged with radiolabeled 10-nitro-[14C]oleic acid, preferential accumulation in adipose tissue over 2 weeks. To better define the metabolism and incorporation of NO2-FAs and their metabolites in adipose tissue lipids, adipocyte cultures were supplemented with 10-nitro-oleic acid (10-NO2-OA), nitro-stearic acid, nitro-conjugated linoleic acid, and nitro-linolenic acid. Then, quantitative HPLC-MS/MS analysis was performed on adipocyte neutral and polar lipid fractions, both before and after acid hydrolysis of esterified FAs. NO2-FAs preferentially incorporated in monoacyl- and diacylglycerides, while reduced metabolites were highly enriched in triacylglycerides. This differential distribution profile was confirmed in vivo in the adipose tissue of NO2-OA-treated mice. This pattern of NO2-FA deposition lends new insight into the unique pharmacokinetics and pharmacologic actions that could be expected for this chemically-reactive class of endogenous signaling mediators and synthetic drug candidates.

Man versus machine: is there an optimal method for QT measurements in thorough QT studies?

Electrocardiographic (ECG) recordings from 3 placebo-controlled thorough QT healthy volunteer studies were used to compare QT intervals obtained by manual measurement with those generated by ECG machines. The effect of the positive control was compared to placebo at each time point for data obtained from both sources. Both manual and automated techniques consistently demonstrated statistically significant prolongation of QTcF with the positive controls. The proportion of outlier values was small for both methods. The pairwise comparison between manual and automated uncorrected QT intervals demonstrated clear differences, with intervals derived from one machine on average 16 to 19 milliseconds shorter and from the other 7 milliseconds longer than the manually measured QT intervals, but these differences disappeared when analyzing QT change from baseline. Both manual and automated, commercially available QT algorithms demonstrated small statistically significant effects on the QTc interval induced by positive controls.

Correlation of protein and gene expression profiles of inflammatory proteins after endotoxin challenge in human subjects.

Administration of endotoxin (LPS) in humans results in profound physiological responses, including activation of peripheral blood mononuclear cells and the release of inflammatory factors. The time course of the response of selected inflammatory proteins was examined in healthy subjects (n = 6) administered a single intravenous dose of the purified derivative of endotoxin (3.0 ng/kg). Microarray analysis demonstrated changes in the expression of a number of genes, which were confirmed in separate in vitro endotoxin stimulation experiments. Subsequent TaqMan analysis of genes of interest indicated time-dependent changes in the expression of many of these genes. This included pre-B cell enhancing factor, which was identified on microarray analysis as being markedly upregulated following endotoxin stimulation. Protein expression of the genes examined by TaqMan analysis was measured and demonstrated the appearance of tumor necrosis factor (TNF)-alpha and sTNF-R proteins in the plasma beginning within 1 h after dosing, followed by other cytokines/ inflammatory markers (e.g., IL-1ra, G-CSF, IL-6, IL-8, and IL-10) and suppressors of cytokine signaling (SOCS-1 and SOCS-3). In general, cytokine protein expression correlated well with gene expression; however, the temporal profile of expression of some genes did not correlate well with the protein data. For many of these proteins, the lack of correlation was attributable to alternate tissue sources, which were demonstrated on TaqMan analysis. Principal component analysis indicated that cytokines could be grouped according to their temporal pattern of response, with most transcript levels returning to baseline 24 h following endotoxin administration. The combination of cDNA microarray and TaqMan analysis to identify and quantify changes in gene expression, along with the analysis of protein expression, can be useful in investigating inflammatory and other diseases.