A randomized, placebo-controlled study to evaluate the efficacy and safety of adding omarigliptin to insulin therapy in Japanese patients with type 2 diabetes and inadequate glycaemic control.

AIM: To evaluate the efficacy and safety of adding the once-weekly oral dipeptidyl peptidase-4 inhibitor omarigliptin to treatment of Japanese patients with type 2 diabetes and inadequate glycaemic control on insulin monotherapy. MATERIALS AND METHODS: In a 52-week clinical trial, Japanese patients on insulin monotherapy were randomized to once-weekly omarigliptin 25 mg (N = 123) or placebo (N = 61) for a 16-week, double-blind, placebo-controlled period. After Week 16, patients continued or switched to omarigliptin for a 36-week open-label period. RESULTS: From a mean baseline of approximately 8.8%, the Week 16 least squares mean changes in HbA1c were -0.61% (omarigliptin) and 0.29% (placebo); the between-group difference was -0.90% (p < .001). At Week 52, the mean change from baseline in HbA1c was -0.57% in both the group on omarigliptin for 52 weeks and the group on omarigliptin for 36 weeks (switched from placebo at Week 16). During the first 16 weeks of treatment, the incidences of adverse events (AEs), serious AEs, drug-related AEs and discontinuation from trial medication because of an AE were similar in both groups. A slight increase in incidence of symptomatic hypoglycaemia was observed in the omarigliptin group (n = 13 [10.6%]) compared with placebo (n = 4 [6.6%]). No severe hypoglycaemia was reported during the study. No new safety signals emerged with treatment beyond Week 16 through Week 52. CONCLUSION: The addition of once-weekly omarigliptin to insulin therapy for up to 52 weeks was generally well tolerated and provided clinically meaningful improvement in glycaemic control throughout the trial period. ClinicalTrials.gov: NCT02906709.

Electrochemical sensing system employing fructosamine 6-kinase enables glycated albumin measurement requiring no proteolytic digestion.

Currently available enzymatic methods for the measurement of glycated proteins utilize fructosyl amino acid/peptide oxidases (FAOXs/FPOXs) as sensing elements. FAOXs/FPOXs oxidize glycated amino acids or glycated dipeptides but they are not able to accept longer glycated peptides or intact glycated proteins as substrates. Therefore, pretreatment via proteolytic digestion is unavoidable with the current enzymatic methods, and there remains a need for simpler measurement methods for glycated proteins. In this study, in order to develop a novel sensing system for glycated albumin (GA), a marker for diabetes, with no requirement for proteolytic digestion, we created an electrochemical sensor based on fructosamine 6-kinase (FN6K) from Escherichia coli. Uniquely, FN6K can react directly with intact GA unlike FAOXs/FPOXs. The concentration of GA in samples was measured using a carbon-printed disposable electrode upon which FN6K as well as two additional enzymes, pyruvate kinase and pyruvate dehydrogenase were overlaid. A clear correlation between the response current and the concentration of GA was observed in the range of 20-100 µM GA, which is suitable for measurement of GA in diluted blood samples from both healthy individuals and patients with diabetes. The sensing system reported here could be applied to point-of-care-testing devices for measurement of glycated proteins.

Advancing the development of glycated protein biosensing technology: next-generation sensing molecules.

Research advances in biochemical molecules have led to the development of convenient and reproducible biosensing molecules for glycated proteins, such as those based on the enzymes fructosyl amino acid oxidase (FAOX) or fructosyl peptide oxidase (FPOX). Recently, more attractive biosensing molecules with potential applications in next-generation biosensing of glycated proteins have been aggressively reported. We review 2 such molecules, fructosamine 6-kinase (FN6K) and fructosyl amino acid-binding protein, as well as their recent applications in the development of glycated protein biosensing systems. Research on FN6K and fructosyl amino acid-binding protein has been opening up new possibilities for the development of highly sensitive and proteolytic-digestion-free biosensing systems for glycated proteins.

Cloning and characterization of fructosamine-6-kinase from Arthrobacter aurescens.

Fructosamine-6-kinases (FN6Ks) that catalyze phosphorylation of glycated amino acids, i.e., fructosyl amino acids (FAs), have been shown as a potential recognition element for glycated protein detection. However, there are only two available FN6Ks: those from Escherichia coli which is specific for ε-fructosyl lysine (ε-FK) and Bacillus subtilis which recognizes both ε-FK and α-FA as substrates. In this study, we characterized an FN6K homologue isolated from Arthrobacter, some of whose species are reported to assimilate FA. The BLAST searches of Arthrobacter genomic database, using the bacterial FN6K primary structure information, revealed the presence of an FN6K homologue in Arthrobacter aurescens TC1 strain. Indeed, enzymatic assays confirmed that the putative FN6K from A. aurescens is an FN6K that is specific for ε-FK, although the primary sequence alignments showed similarity of A. aurescens FN6Ks with FN6Ks from B. subtilis and E. coli at the same level. In this study, we describe for the first time the presence of FN6K in Arthrobacter spp. and ε-FK-specific degradation pathway from Gram-positive bacteria, providing important information for the development of FA-recognizing molecules as well as for the FA assimilation system in bacteria.

Substrate specificity engineering of Escherichia coli derived fructosamine 6-kinase.

A three-dimensional structural model of Escherichia coli fructosamine 6-kinase (FN6K), an enzyme that phosphorylates fructosamines at C6 and catalyzes the production of the fructosamine 6-phosphate stable intermediate, was generated using the crystal structure of 2-keto-3-deoxygluconate kinase isolated from Thermus thermophilus as template. The putative active site region was then investigated by site-directed mutagenesis to reveal several amino acid residues that likely play important roles in the enzyme reaction. Met220 was identified as a residue that plays a role in substrate recognition when compared to Bacillus subtilis derived FN6K, which shows different substrate specificity from the E. coli FN6K. Among the various Met220-substituted mutant enzymes, Met220Leu, which corresponded to the B. subtilis residue, resulted in an increased activity of fructosyl-valine and decreased activity of fructosyl-lysine, thus increasing the specificity for fructosyl-valine by 40-fold.