Apelin peptides linked to anti‐serum albumin domain antibodies retain affinity in vitro and are efficacious receptor agonists in vivo

Abstract

The apelin receptor is a potential target in the treatment of heart failure and pulmonary arterial hypertension where levels of endogenous apelin peptides are reduced but significant receptor levels remain. Our aim was to characterise the pharmacology of a modified peptide agonist, MM202, designed to have high affinity for the apelin receptor and resistance to peptidase degradation and linked to an anti-serum albumin domain antibody (AlbudAb) to extend half-life in the blood. In competition, binding experiments in human heart MM202-AlbudAb (pK i = 9.39 ± 0.09) bound with similar high affinity as the endogenous peptides [Pyr 1 ]apelin-13 (pK i = 8.83 ± 0.06) and apelin-17 (pK i = 9.57 ± 0.08). [Pyr 1 ]apelin-13 was tenfold more potent in the cAMP (pD 2 = 9.52 ± 0.05) compared to the β-arrestin (pD 2 = 8.53 ± 0.03) assay, whereas apelin-17 (pD 2 = 10.31 ± 0.28; pD 2 = 10.15 ± 0.13, respectively) and MM202-AlbudAb (pD 2 = 9.15 ± 0.12; pD 2 = 9.26 ± 0.03, respectively) were equipotent in both assays, with MM202-AlbudAb tenfold less potent than apelin-17. MM202-AlbudAb bound to immobilised human serum albumin with high affinity (pK D = 9.02). In anaesthetised, male Sprague Dawley rats, MM202-AlbudAb (5 nmol, n = 15) significantly reduced left ventricular systolic pressure by 6.61 ± 1.46 mm Hg and systolic arterial pressure by 14.12 ± 3.35 mm Hg and significantly increased cardiac contractility by 533 ± 170 mm Hg/s, cardiac output by 1277 ± 190 RVU/min, stroke volume by 3.09 ± 0.47 RVU and heart rate by

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.…”

“…3B. Notably, the most abundant fragments were [Pyr 1 ]apelin-13 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) (known to be biologically active 24 ), [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) . Other metabolites identified, although at lower levels (<10% of parent [Pyr 1 ]apelin-13) included [Pyr 1 ]apelin-13 (1)(2)(3)(4)(5)(6)(7)(8) , [Pyr 1 ]apelin-13 (1)(2)(3)(4)(5)(6)(7) and [Pyr 1 ]apelin-13 (1)(2)(3)(4)(5) that are likely to be biologically inactive.…”

“…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.…”

“…We found that [Pyr 1 ]apelin-13 was cleaved into smaller fragments from both termini but that the C-terminal was more susceptible. We identified the biologically active C-terminal cleavage product, [Pyr 1 ]apelin-13 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) , as the most abundant, as well as identifying novel metabolites including [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) .…”