Oxidative Deamination of Emixustat by Human Vascular Adhesion Protein-1/Semicarbazide-Sensitive Amine Oxidase

Abstract

Emixustat potently inhibits the visual cycle isomerase retinal pigment epithelium protein 65 (RPE65) to reduce the accumulation of toxic bisretinoid by-products that lead to various retinopathies. Orally administered emixustat is cleared rapidly from the plasma, with little excreted unchanged. The hydroxypropylamine moiety that is critical in emixustat's inhibition of RPE65 is oxidatively deaminated to three major carboxylic acid metabolites that appear rapidly in plasma. These metabolites greatly exceed the plasma concentrations of emixustat and demonstrate formation-rate-limited metabolite kinetics. This study investigated in vitro deamination of emixustat in human vascular membrane fractions, plasma, and recombinant human vascular adhesion protein-1 (VAP-1), demonstrating single-enzyme kinetics for the formation of a stable aldehyde intermediate in all in vitro systems. The in vitro systems used herein established sequential formation of the major metabolites with addition of assay components for aldehyde dehydrogenase and cytochrome P450. Reaction phenotyping experiments using selective chemical inhibitors and recombinant enzymes of monoamine oxidase, VAP-1, and lysyl oxidase showed that only VAP-1 deaminated emixustat. In individually derived human vascular membranes from umbilical cord and aorta, rates of emixustat deamination were highly correlated to VAP-1 marker substrate activity (benzylamine) and VAP-1 levels measured by enzyme-linked immunosorbent assay. In donormatched plasma samples, soluble VAP-1 activity and levels were lower than in aorta membranes. A variety of potential comedications did not strongly inhibit emixustat deamination in vitro. This work was supported by Acucela Inc., and has not been previously presented. https://doi.org/10.1124/dmd.118.085811. s This article has supplemental material available at dmd.aspetjournals.org. human MAO; rhVAP-1, recombinant human VAP-1; RPE65, retinal pigment epithelium protein 65; SSAO, semicarbazide-sensitive amine oxidase; VAP-1, vascular adhesion protein-1. 504

Comparison of Inhibitor and Substrate Selectivity between Rodent and Human Vascular Adhesion Protein-1

“…The histidine residues that coordinate the copper are highlighted in green. 4 Mediators of Inflammation [32]. These values were in close agreement with the 7.75 μM value reported here.…”

Rational Alteration of Pharmacokinetics of Chiral Fluorinated and Deuterated Derivatives of Emixustat for Retinal Therapy

“…VAP-1 is a lesser known phase-1 metabolic pathway 53 important for the primary amine oxidation of clinically used drugs including primaquine 54 and tresperimus 55 in addition to emixustat. 15 Our results here suggest that the deuteration of primary amines susceptible to VAP-1 oxidation could be a generally effective approach to prolonging their in vivo activity. The impact of alpha deuterium substitution of amines on VAP-1 metabolic susceptibility was previously studied in vitro using benzylamine and various phenylethylamines as test substrates.…”

“…VAP-1 is a lesser known phase-1 metabolic pathway important for the primary amine oxidation of clinically used drugs including primaquine and tresperimus in addition to emixustat . Our results here suggest that the deuteration of primary amines susceptible to VAP-1 oxidation could be a generally effective approach to prolonging their in vivo activity.…”

“…( R )-Emixustat, derived from retinylamine (Figure B), is currently undergoing clinical development as a potent RPE65 inhibitor. − However, the complete suppression of RPE65 activity has undesirable blinding consequences, thus only a narrow concentration range of the drug is tolerable. Effective use of emixustat is further complicated because it is rapidly metabolized, − limiting the duration of its pharmacological effects. A second strategy is to lower the toxic effects of unbound all- trans -retinal and temporarily sequester it by condensation with a primary amine. , Because most RPE65 inhibitors contain an amino group, they can play a dual role of slowing down the metabolism of all- trans -retinal as well as sequestering it …”

The novel visual cycle inhibitor (±)-RPE65-61 protects retinal photoreceptors from light-induced degeneration

“…No detectable plasma levels were seen after administration of the 2 mg dose [ 44 ], and systemic exposure was very low even at the highest dose (40 mg) [ 43 ]. Similarly, emixustat metabolites are cleared from the circulation very quickly [ 48 ]. This prolonged PD response may indicate drug accumulation in the retina.…”