Efficacy and safety of macitentan in Fontan-palliated patients: 52-week randomized, placebo-controlled RUBATO phase 3 trial and open-label extension.

OBJECTIVES: The efficacy and safety of macitentan, an endothelin receptor antagonist, were assessed in Fontan-palliated patients over a 52-week, multicenter, randomized, placebo-controlled, double-blind trial (RUBATO-DB) and an open-label extension trial (RUBATO-OL). METHODS: Patients aged ≥12 years, New York Heart Association functional class II/III, underwent total cavopulmonary connection >1 year pre-screening and showed no signs of Fontan failure/clinical deterioration. In RUBATO-DB, primary efficacy endpoint was change in peak oxygen consumption (VO2) from baseline to week 16; secondary endpoints were change from baseline over 52 weeks in peak VO2 and change in mean count/minute of daily physical activity via accelerometer (PA-Ac) from baseline to week 16. Safety was assessed throughout both studies. RESULTS: In RUBATO-DB, 137 patients were randomized to macitentan 10 mg (n=68) or placebo (n=69); 92.7% completed 52-week double-blind treatment. At week 16, mean (SD) change in peak VO2 was -0.16 (2.86) versus -0.67 (2.66) mL/kg/min with macitentan versus placebo (median unbiased treatment difference estimate: 0.62 mL/kg/min [99% repeated confidence interval -0.62; 1.85], p=0.19). No treatment effect was observed in either of the secondary endpoints. During RUBATO-DB, most common adverse events with macitentan were headache, nasopharyngitis and pyrexia. Across RUBATO-DB and RUBATO-OL, most common adverse events were COVID-19, headache and fatigue. RUBATO-OL was prematurely discontinued as RUBATO-DB did not meet its primary or secondary endpoint. CONCLUSIONS: The primary endpoint of RUBATO-DB was not met; macitentan did not improve exercise capacity versus placebo in Fontan-palliated patients. Macitentan was generally well tolerated over long-term treatment.

Protein cross-linkage induced by formaldehyde derived from semicarbazide-sensitive amine oxidase-mediated deamination of methylamine.

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the conversion of methylamine to formaldehyde. This enzyme is located on the surface of the cytoplasmic membrane and in the cytosol of vascular endothelial cells, smooth muscle cells, and adipocytes. Increased SSAO activity has been found in patients with diabetes mellitus, chronic heart failure, and multiple types of cerebral infarcts and is associated with obesity. Increased SSAO-mediated deamination may contribute to protein deposition, the formation of plaques, and inflammation, and thus may be involved in the pathophysiology of chronic vascular and neurological disorders, such as diabetic complications, atherosclerosis, and Alzheimer's disease. In the present study, we demonstrate the induction of cross-linkage of formaldehyde with the lysine moiety of peptides and proteins. Formaldehyde-protein adducts were reduced with sodium cyanoborohydride, hydrolyzed in hydrochloric acid, and the amino acids in the hydrolysates were derivatized with fluorenylmethyl chloroformate and then identified with high-performance liquid chromatography. We further demonstrate that incubation of methylamine in the presence of SSAO-rich tissues, e.g., human brain meninges, results in formaldehyde-protein cross-linkage of particulate bound proteins as well as of soluble proteins. This cross-linkage can be completely blocked by a selective inhibitor of SSAO. Our data support the hypothesis that the SSAO-induced production of formaldehyde may be involved in the alteration of protein structure, which may subsequently cause protein deposition associated with chronic pathological disorders.

Involvement of SSAO-mediated deamination in adipose glucose transport and weight gain in obese diabetic KKAy mice.

Semicarbazide-sensitive amine oxidase (SSAO) is located on outer surfaces of adipocytes and endothelial and vascular smooth muscle cells. This enzyme catalyzes deamination of methylamine and aminoacetone, leading to production of toxic formaldehyde and methylglyoxal, respectively, as well as hydrogen peroxide and ammonium. Several lines of evidence suggest that increased SSAO activity is related to chronic inflammation and vascular disorders related to diabetic complications. We found that a highly potent and selective SSAO inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (FPFA), was capable of reducing numbers of atherosclerotic lesions as well as weight gain in obese KKAy mice fed an atherogenic diet. SSAO inhibitors cause a moderate and long-lasting hyperglycemia. Such an increase in serum glucose is a result of reduction of glucose uptake by adipocytes. SSAO-mediated deamination of endogenous methylamine substrates induces adipocyte glucose uptake and lipogenesis. Highly selective SSAO inhibitors can effectively block induced glucose uptake. The results suggest that increased SSAO-mediated deamination may be concomitantly related to obesity and vascular disorders associated with type 2 diabetes.

A novel sensitive high-performance liquid chromatography/electrochemical procedure for measuring formaldehyde produced from oxidative deamination of methylamine and in biological samples.

Formaldehyde is a well-known environmental toxic hazard. It is also a product of oxidative deamination of methylamine catalyzed by semicarbazide-sensitive amine oxidase (SSAO). Increased SSAO-mediated deamination has been implicated in some pathophysiological conditions, such as diabetic complications. The measurement of formaldehyde in the enzymatic reactions and in vivo production using conventional methods was not straightforward due to limitations of selectivity and sensitivity. A novel high-performance liquid chromatography (HPLC)/electrochemical procedure for the measurement of formaldehyde has been developed. The measurement is based on the formation of adducts between formaldehyde and dopamine. These adducts can be selectively purified and concentrated using a batch method of alumina absorption, separated by HPLC, and electrochemically quantified. The method is highly selective and substantially more sensitive, i.e., detection of picomole levels of formaldehyde, than the conventional methods. The procedure not only facilitates the assessment of SSAO activity in vitro but also is useful for assessing formaldehyde in tissues and biological fluids.

Determination of the norepinephrine level by high-performance liquid chromatography to assess the protective effect of MAO-B inhibitors against DSP-4 toxicity.

Liquid chromatography (LC) combined with electrochemical detection (EC) is suitable for measuring oxidizable biogenic amine levels in small samples of brain tissue. The norepinephrine (NE) content in mouse hippocampus after treatment with various monoamine oxidase-B enzyme (MAO-B) inhibitors ([-]-deprenyl, [+]-rasagiline, and the noradrenergic neurotoxin N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine [DSP-4]) is determined using an LC-EC method. Treatment with a single intraperitoneal dose of (-)-deprenyl (selegiline) before DSP-4 administration markedly reduces the NE depleting effect of the toxin, and (+)-rasagiline does not significantly modify the NE level decreased by the neurotoxin. The MAO-B inhibitory potency of (-)-deprenyl and (+)-rasagiline is also evaluated. Significantly reduced MAO-B enzyme activity in mouse brain and liver is measured 6 h after treatment with their single dose. (+)-Rasagiline is found to be a more potent MAO-B inhibitor than (-)-deprenyl.