A novel cyclic biased agonist of the apelin receptor, MM07, is disease modifying in the rat monocrotaline model of pulmonary arterial hypertension

Abstract

Background and Purpose Apelin is an endogenous vasodilatory and inotropic peptide that is down‐regulated in human pulmonary arterial hypertension, although the density of the apelin receptor is not significantly attenuated. We hypothesised that a G protein‐biased apelin analogue MM07, which is more stable than the endogenous apelin peptide, may be beneficial in this condition with the advantage of reduced β‐arrestin‐mediated receptor internalisation with chronic use. Experimental Approach Male Sprague–Dawley rats received either monocrotaline to induce pulmonary arterial hypertension or saline and then daily i.p. injections of either MM07 or saline for 21 days. The extent of disease was assessed by right ventricular catheterisation, cardiac MRI, and histological analysis of the pulmonary vasculature. The effect of MM07 on signalling, proliferation, and apoptosis of human pulmonary artery endothelial cells was investigated. Key Results MM07 significantly reduced the elevation of right ventricular systolic pressure and hypertrophy induced by monocrotaline. Monocrotaline‐induced changes in cardiac structure and function, including right ventricular end‐systolic and end‐diastolic volumes, ejection fraction, and left ventricular end‐diastolic volume, were attenuated by MM07. MM07 also significantly reduced monocrotaline‐induced muscularisation of small pulmonary blood vessels. MM07 stimulated endothelial NOS phosphorylation and expression, promoted proliferation, and attenuated apoptosis of human pulmonary arterial endothelial cells in vitro. Conclusion and Implications Our findings suggest that chronic treatment with MM07 is beneficial in this animal model of pulmonary arterial hypertension by addressing disease aetiology. These data support the development of G protein‐biased apelin receptor agonists with improved pharmacokinetic profiles for use in human disease.

Development and validation of an LC-MS/MS method for detection and quantification of in vivo derived metabolites of [Pyr1]apelin-13 in humans

“…A previous study showed in vitro that neprilysin cleaves [Pyr 1 ] apelin-13 between Arg 4 and Leu 5 and between Leu 5 and Ser 6 amino acids 25 , thereby making neprilysin the first enzyme identified to date that completely inactivates the peptide. Importantly, we have now shown in this study the presence of one of these proposed neprilysin cleavage products, [Pyr1]apelin-13 (6)(7)(8)(9)(10)(11)(12)(13) , in humans in vivo apelin-13 (1)(2)(3)(4)(5) with 5.88 minutes retention time. These data were acquired using Orbitrap Mass spectrometer used for metabolite identification.…”

“…[Pyr 1 ]apelin-13 was also cleaved between Pro 10 and Met 11 and between Ser 6 and His 7 resulting in generation of [Pyr 1 ]apelin-13 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) and [Pyr 1 ]apelin-13 (1)(2)(3)(4)(5)(6) but the enzyme responsible for producing these metabolites remains unknown. The corresponding N-terminal fragments of these C-terminal metabolites [Pyr 1 ] www.nature.com/scientificreports www.nature.com/scientificreports/ apelin-13 (11)(12)(13) and [Pyr 1 ]apelin-13 (7)(8)(9)(10)(11)(12)(13) , were also identified. These data were consistent with a previous in vivo study in male rats which also identified the C-terminal metabolites 22 .…”

“…In this study, we have developed a highly sensitive method for detection and quantification of [Pyr1]apelin-13 in human plasma. For the first time in humans in vivo we have identified as the most abundant metabolite of [Pyr 1 ]apelin-13, the ACE2 cleavage product, [Pyr 1 ] apelin-13 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) that we have previously demonstrated retains significant biological activity in addition to the putative neprilysin metabolites [Pyr 1 ]apelin-13 (4)(5)(6)(7)(8)(9)(10)(11)(12)(13) and [Pyr 1 ]apelin-13 (6)(7)(8)(9)(10)(11)(12)(13) . Combined inhibition of ACE2 and neprilysin may be a novel strategy to enhance endogenous apelin levels in conditions in which the peptide is downregulated.…”

“…Of note, the peak areas of these fragments were lower compared to those observed for the C-terminal fragments. The most abundant N-terminal fragments observed were [Pyr 1 ]apelin-13 (6)(7)(8)(9)(10)(11)(12)(13) , [Pyr 1 ]apelin-13 (11)(12)(13) , [Pyr 1 ]apelin-13 (7)(8)(9)(10)(11)(12)(13) and [Pyr 1 ]apelin-13 (10)(11)(12)(13) (Fig. 7A,B).…”

“…7A,B). Other fragments present but low in abundance include [Pyr 1 ]apelin-13 (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) , [Pyr 1 ]apelin-13 (4)(5)(6)(7)(8)(9)(10)(11)(12)(13) and [Pyr 1 ]apelin-13 (8)(9)(10)(11)(12)(13) . The mass accuracy of the experimentally acquired monoisotopic m/z for these metabolites are shown in Fig.…”