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 6 g of oral ManNAc were safe and well tolerated; 10 g was associated with diarrhea likely due to unabsorbed ManNAc. Oral ManNAc was absorbed rapidly and exhibited a short half-life (~2.4 h). Following administration of a single dose of ManNAc, there was a significant and sustained increase in plasma free sialic acid (Neu5Ac) (Tmax of 8-11 h). Neu5Ac levels remained above baseline 48 h post-dose in subjects who received a dose of 6 or 10 g. 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 6 g twice daily for future clinical trials.

No abstract

No abstract

N-Acetylneuraminic acid (Neu5Ac or NANA) is the most predominant sialic acid in mammals. As a terminal component in many glycoproteins and glycolipids, sialic acid is believed to be an important biomarker related to various diseases. Its precursor, N-acetylmannosamine (ManNAc), is being investigated as a potential treatment for GNE myopathy. In this work, we developed two highly sensitive and selective liquid chromatography–tandem mass spectrometry (LC-MS/MS) methods for the quantitation of ManNAc and free Neu5Ac in human plasma. A fit-for-purpose approach was adopted during method validation and sample analysis. To measure the endogenous compounds and overcome the interference from plasma samples, a surrogate matrix that contained 5% bovine serum albumin (BSA) was used for the preparation of calibration standards and certain levels of quality control (QC) samples. QC samples at higher concentrations were prepared in the authentic matrix (human plasma) to best mimic incurred samples. For both methods, an Ostro 96-well phospholipid removal plate was used for sample extraction, which efficiently removed the phospholipids from the plasma samples prior to LC injection, eliminated matrix effect, and improved sensitivity. Chromatographic separation was achieved using hydrophilic interaction chromatography (HILIC) and gradient elution in order to retain the two polar compounds. The lower limit of quantitation (LLOQ) for ManNAc and Neu5Ac was 10.0 and 25.0 ng/mL, respectively. The overall accuracy of the two assays was no less than 91.7% based on three levels of QC samples. Inter- and intra-run precision (coefficient of variation [%CV]) across three analytical runs was less than 6.7% for ManNAc and less than 10.8% for Neu5Ac. These methods have been validated to support clinical studies.

Two 2-alkenyl-3-hydroxyaIkyl-l,4-dimethoxynaphthalenes are cyclised with potassium t-butoxide in dimethylformamide to give 3-alkyl-3,4-dihydro-5,1 O-dimethoxynaphtho[2,3-c]pyrans under anaerobic conditions. One of these products is treated with the same solvent and base, but in air, to give the two possible 4-hydroxy derivatives.We recently reported the oxidative cyclisation of the naphthalene dimethyl ether (1) with cerium(1v) ammonium nitrate to give a mixture of the isomeric quinones (2) and (3), the latter predominating. These quinones are related to the aphid pigments, e.g. protoaphin fb and protoaphin sl. The trans-prop-1-enyl substituent of (1) was derived from propyl by bromination-dehydrobromination but, for this particular system, certain difficulties were encountered which prompted us to seek alternative routes to (1) by conjugation of an allyl group. This has been achieved and, in the course of this investigation, novel cyclisations of compounds related to (1) were discovered which are useful in the synthesis of naturally occurring quinones. The work leading to these cyclisations is described in this paper. Results and Discussion2-Acetyl-l,4-naphthoquinone (4) was allylated with commercially available vinylacetic acid in the presence of silver nitrate and potassium peroxodisulphate to give the quinone (5) in 43% yield. Small quantities of a second yellow product, isomeric with (5) and difficult to separate from it, were observed, but this compound proved not to be quinonoid. The 'H n.m.r. spectrum showed, inter aka, a two-proton doublet of doublets at 6 4.80 ( J 1 and 4 Hz), a one-proton doublet of triplets at 6 5.91 ( J 4 and 10 Hz), a one-proton doublet of doublets at 6 6.78 (J 1 and 10 Hz), and a very-low-field oneproton singlet at 6 13.86. This enabled the assignment of structure (6) to the compound, which presumably arises through acid-catalysed enolisation of quinone (3, followed by cyclisation, as shown. Because of the difficulty of separating this compound from the quinone (9, it was characterised as