Safety, pharmacokinetics and sialic acid production after oral administration of N-acetylmannosamine (ManNAc) to subjects with GNE myopathy

Abstract

GNE myopathy is a rare, autosomal recessive, inborn error of sialic acid metabolism, caused by mutations in GNE, the gene encoding UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase. The disease manifests as an adult-onset myopathy characterized by progressive skeletal muscle weakness and atrophy. There is no medical therapy available for this debilitating disease. Hyposialylation of muscle glycoproteins likely contributes to the pathophysiology of this disease. N-acetyl-D-mannosamine (ManNAc), an uncharged monosaccharide and the first committed precursor in the sialic acid biosynthetic pathway, is a therapeutic candidate that prevents muscle weakness in the mouse model of GNE myopathy. We conducted a first-in-human, randomized, placebo-controlled, double-blind, single-ascending dose study to evaluate safety and pharmacokinetics of ManNAc in GNE myopathy subjects. Single doses of 3 and 6g of oral ManNAc were safe and well tolerated; 10g was associated with diarrhea likely due to unabsorbed ManNAc. Oral ManNAc was absorbed rapidly and exhibited a short half-life (~2.4h). Following administration of a single dose of ManNAc, there was a significant and sustained increase in plasma unconjugated free sialic acid (Neu5Ac) (Tmax of 8-11h). Neu5Ac levels remained above baseline 48h post-dose in subjects who received a dose of 6 or 10g. Given that Neu5Ac is known to have a short half-life, the prolonged elevation of Neu5Ac after a single dose of ManNAc suggests that intracellular biosynthesis of sialic acid was restored in subjects with GNE myopathy, including those homozygous for mutations in the kinase domain. Simulated plasma concentration-time profiles support a dosing regimen of 6g twice daily for future clinical trials.

Keywords: First-in-human study; GNE; GlcNAc kinase; N-acetylneuraminic acid; Pharmacokinetics; Sialylation.

Published by Elsevier Inc.

Figures

Figure 1. Sialic Acid Biosynthetic Pathway

The biosynthesis of Neu5Ac (sialic acid) occurs in the cytosol, where glucose undergoes several modifications to become UDP-GlcNAc. The UDP-GlcNAc 2-epimerase activity of the bifunctional GNE enzyme then epimerises UDP-GlcNAc into ManNAc, after which the GNE ManNAc kinase activity further converts this to ManNAc-6-P, which then undergoes two more steps to form cytoplasmic free sialic acid. A nuclear step activates free sialic acid to CMP-sialic acid, which translocates back to the cytosol, where is utilized by the Golgi complex to sialylate glycans (glycoproteins and glycolipids). Cytoplasmic CMP-sialic acid strongly feedback-inhibits the UDP-GlcNAc 2-epimerase enzymatic activity in its allosteric site (dashed line). PEP=phosphoenolpyruvate. Patients with GNE myopathy have deficiency of GNE UDP-GlcNAc 2-epimerase and ManNAc kinase activity, resulting in decreased sialic acid production. Supplementation of ManNAc (uncharged small molecule that crossed membranes readily) circumvents the feedback inhibition step in this pathway and increases sialic acid production. ManNAc can be converted to ManNAc-6-P by the ancillary enzyme GlcNAc kinase, which likely occurs in GNE myopathy patients with severe ManNAc kinase deficiency. See text for further details and references. Figure courtesy of Daryl Leja and Julia Fekecs, NHGRI, NIH.

Figure 2. Plasma Concentration-Time Profiles of ManNAc and Neu5Ac after Oral Administration of a Single Dose of 3, 6 or 10 g of ManNAc to Fasted GNE Myopathy Subjects

Mean cohort and individual subject plasma concentrations (ng/mL) of ManNAc (A) and Neu5Ac (B).

Figure 3. Mean Adjusted Plasma Concentration-Time Profiles

Mean (± SD) plasma concentrations of ManNAc (A) and Neu5Ac (B) after a single oral dose of ManNAc administered to fasted GNE myopathy subjects with baseline adjustment. Baseline adjustment was made by subtracting ManNAc or Neu5Ac concentration at time t=0 h for each subject.

Figure 4. Simulated Concentration-Time Profiles

Simulated mean plasma ManNAc (A) and Neu5Ac (B) concentrations after oral administration of 6 g of ManNAc twice daily to subjects with GNE myopathy [Mean (red line) ±SD (black lines)].