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

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.

Protein trafficking to the cell wall occurs through mechanisms distinguishable from default sorting in tobacco.

The secretory pathway in plants involves sustained traffic to the cell wall, as matrix components, polysaccharides and proteins reach the cell wall through the endomembrane system. We studied the secretion pattern of cell-wall proteins in tobacco protoplasts and leaf epidermal cells using fluorescent forms of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2). The two most representative protein fusions, secGFP-PMEI1 and PGIP2-GFP, reached the cell wall by passing through ER and Golgi stacks but using distinct mechanisms. secGFP-PMEI1 was linked to a glycosylphosphatidylinositol (GPI) anchor and stably accumulated in the cell wall, regulating the activity of the endogenous pectin methylesterases (PMEs) that are constitutively present in this compartment. A mannosamine-induced non-GPI-anchored form of PMEI1 as well as a form (PMEI1-GFP) that was unable to bind membranes failed to reach the cell wall, and accumulated in the Golgi stacks. In contrast, PGIP2-GFP moved as a soluble cargo protein along the secretory pathway, but was not stably retained in the cell wall, due to internalization to an endosomal compartment and eventually the vacuole. Stable localization of PGIP2 in the wall was observed only in the presence of a specific fungal endopolygalacturonase ligand in the cell wall. Both secGFP-PMEI1 and PGIP2-GFP sorting were distinguishable from that of a secreted GFP, suggesting that rigorous and more complex controls than the simple mechanism of bulk flow are the basis of cell-wall growth and differentiation.

A direct HPLC method to estimate streptomycin and its putative ototoxic derivative, streptidine, in blood serum: application to streptomycin-treated humans.

Streptomycin is an aminoglycoside antibiotic with a well-known antituberculosis activity; it is commonly used in clinical practice because it is effective and cheap. However, streptomycin has severe ototoxic effects. The delayed and gradual onset may suggest that a metabolic derivative of the antibiotic could be a potential contributor to ototoxicity. As in a rat experimental model this compound was found to be streptidine, we investigated whether this ototoxic metabolite was also present in the blood of streptomycin-treated patients. To this end, we implemented and optimized a direct reverse-phase HPLC technique to identify and estimate streptomycin and streptidine in serum of streptomycin-treated patients. All criteria for validation of the method were implemented in standard curves in serum of healthy non-treated volunteers by addition of increasing concentration of both compounds and their determination in a trichloroacetic acid deproteinized extract. We found that recovery of streptomycin or streptidine was > or =91.5%. Linearity was r(2)> or =0.99. The intraday and interday precisions were < or =9.7 and < or =10.6%, respectively. The relative intraday and interday error ranged from -9.0 to 8.3% for both compounds in human serum. Studies in patients included five male individuals treated from 35 to 90 days with 1g/day of streptomycin, presenting inner ear malfunction from mild to severe, in whose serum streptidine was always present, and could be successfully separated from streptomycin. Therefore, the validated method used can be a valuable tool to measure and follow these compounds in serum of streptomycin-treated patients.