Safety, tolerability, pharmacokinetics, and pharmacodynamics of novel glucokinase activator HMS5552: results from a first-in-human single ascending dose study.

BACKGROUND: HMS5552, a novel fourth-generation glucokinase (GK) activator, has demonstrated promising effects on glycemic control in preclinical models of type 2 diabetes. This single ascending dose study was conducted to investigate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of HMS5552 during its first-in-human exposure. METHODS: Sixty healthy subjects were enrolled. In each of six dose-cohorts (5, 10, 15, 25, 35, and 50 mg), ten subjects were randomized with eight subjects receiving the same cohort-dose of HMS5552 and two receiving placebo. Plasma HMS5552 exposure, glucose, and insulin were measured repeatedly during fasting and after a standardized meal. Assessment included safety, PK, and PD endpoints. RESULTS: HMS5552 showed dose-proportional increases in area under the curve 0 to the last quantifiable concentration (AUC0-t) and maximum plasma concentration (Cmax). Slopes estimated by linear regression for AUC0-t and Cmax were ~1.0 (0.932 and 0.933, respectively). Geometric mean elimination half-life ranged from 4.48 to 7.51 hours and apparent clearance ranged from 11.5 to 13.1 L/h across all doses. No significant sex effect was observed in PK parameters. HMS5552 also demonstrated dose-related PD responses in terms of maximum glucose change from baseline (%) and mean glucose area under effect curve 0-4 hours change from baseline (%) (P<0.001). Fifteen adverse events were reported by nine subjects (ten with HMS5552 and five with the placebo). All adverse events were mild in intensity and resolved without any treatment. CONCLUSION: This first-in-human single ascending dose study provided predicted PK of HMS5552 with dose-proportional increases in AUC0-t and Cmax, as well as dose-related glucose-lowering effects over the range of 5-50 mg in healthy subjects. HMS5552 at doses up to 50 mg in healthy subjects was safe and well-tolerated.

Conformational selection or induced fit? A critical appraisal of the kinetic mechanism.

For almost five decades, two competing mechanisms of ligand recognition, conformational selection and induced fit, have dominated our interpretation of ligand binding in biological macromolecules. When binding-dissociation events are fast compared to conformational transitions, the rate of approach to equilibrium, k(obs), becomes diagnostic of conformational selection or induced fit based on whether it decreases or increases, respectively, with the ligand concentration, [L]. However, this simple conclusion based on the rapid equilibrium approximation is not valid in general. Here we show that conformational selection is associated with a rich repertoire of kinetic properties, with k(obs) decreasing or increasing with [L] depending on the relative magnitude of the rate of ligand dissociation, k(off), and the rate of conformational isomerization, k(r). We prove that, even for the simplest two-step mechanism of ligand binding, a decrease in k(obs) with [L] is unequivocal evidence of conformational selection, but an increase in k(obs) with [L] is not unequivocal evidence of induced fit. Ligand binding to glucokinase, thrombin, and its precursor prethrombin-2 are used as relevant examples. We conclude that conformational selection as a mechanism for a ligand binding to its target may be far more common than currently believed.

Activating mutations in the human glucokinase gene revealed by genetic selection.

We describe the discovery of 11 new activating mutations in the human glk gene associated with the disease persistent hyperinsulinemic hypoglycemia of infancy (PHHI). Three of the newly identified substitutions colocalize to a region of the glucokinase polypeptide where a synthetic allosteric activator binds. Of these substitutions, I211F is the most active variant identified to date, with a k(cat)/K(0.5,glucose) value (6.6 x 10(4) M(-1) s(-1)) that is 12-fold higher than that of wild-type glucokinase. The stimulatory mutations described herein represent surreptitious genetic determinants of PHHI. They also identify novel features of the glucokinase scaffold that could be targeted during the development of diabetes therapeutics.

Cell-specific roles of glucokinase in glucose homeostasis.

Mutations in the glucokinase (GK) gene cause two different diseases of blood glucose regulation: maturity onset diabetes of the young, type 2 (MODY-2) and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). To gain further understanding of the pathophysiology of these disorders, we have used both transgenic and gene-targeting strategies to explore the relationship between GK gene expression in specific tissues and the blood glucose concentration. These studies, which have included the use of aCre/loxP gene-targeting strategy to perform both pancreatic beta-cell- and hepatocyte-specific knockouts of GK, clearly demonstrate multiple, cell-specific roles for this hexokinase that, together, contribute to the maintainance of euglycemia. In the pancreatic beta cell, GK functions as the glucose sensor, determining the threshold for insulin secretion. Mice lacking GK in the pancreatic beta cell die within 3 days of birth of profound hyperglycemia. In the liver, GK facilitates hepatic glucose uptake during hyperglycemia and is essential for the appropriate regulation of a network of glucose-responsive genes. While mice lacking hepatic GK are viable, and are only mildly hyperglycemic when fasted, they also have impaired insulin secretion in response to hyperglycemia. The mechanisms that enable hepatic GK to affect beta-cell function are not yet understood. Thus, the hyperglycemia that occurs in MODY-2 is due to impaired GK function in both the liver and pancreatic beta cell, although the defect in beta-cell function is clearly more dominant. Whether defects in GK gene expression also impair glucose sensing by neurons in the brain or enteroendocrine cells in gut, two other sites known to express GK, remains to be determined. Moreover, whether the pathophysiology of PHHI also involves multitissue dysfunction remains to be explored.

Impairment of glucokinase translocation in cultured hepatocytes from OLETF and GK rats, animal models of type 2 diabetes.

We examined sugar-induced translocation of glucokinase in cultured hepatocytes from Otsuka Long-Evans Tokushima Fatty and Goto-Kakizaki rats, animal models of type 2 diabetes, and compared this with that in Long-Evans Tokushima Otsuka and Wistar rats, respectively, as control strains. When hepatocytes from the four strains were incubated with 5 mM glucose, glucokinase was present predominantly in the nuclei. Higher concentrations of glucose, 5 mM glucose plus 1 mM fructose, and 5 mM glucose plus 1 mM sorbitol all induced the translocation of glucokinase from the nucleus to the cytoplasm in hepatocytes from these rats. The extent of glucokinase translocation under these conditions, however, was less marked in both diabetic rat types than in the control rats. The extent of the phosphorylation of glucose as estimated by the release of 3H2O from [2- 3H] glucose is significantly lower in Goto-Kakizaki rats than in Wistar rats. The results indicate that the translocation of glucokinase is impaired in the hepatocytes of diabetic rats. They also suggest that the impaired translocation of glucokinase is associated with abnormal hepatic glucose metabolism in type 2 diabetes.

Rabbit optic nerve phosphorylates glucose through a glucokinase-like enzyme: studies in normal and spontaneously hyperglycemic animals.

We investigated glucose phosphorylation at various concentrations of glucose (1, 5, 10, 25, 50, 100 mmol/liter) in rabbit optic nerve. In the 3000 g supernatant of whole rabbit optic nerve homogenates from female albino rabbits (n = 10, 1.8-2.0 kg body weight, mean +/- SEM morning glycemia: 8.25 +/- 0.29 mmol/liter), the glucose phosphorylating activity (NADP reduction measured as change in optical density at 366 nm at pH 7.5) increased progressively with the increase in glucose concentration (r = 0.89; P < 0.05) and approached the maximum at a very high glucose level (100 mmol/liter), with values (mean +/- SEM) of 8.75 +/- 0.97 nanomol/min/mg protein and 11.57 +/- 1.15 at 1 and 100 mmol/liter glucose, respectively (+32.23%; P < 0.01). At a more alkaline pH (8.2; n = 5, mean +/- SEM morning glycemia: 8.83 +/- 0.07 mmol/liter) glucose phosphorylation was higher than at pH 7.5 and retained the glucose concentration dependence (r = 0.95, P < 0.01). These kinetic characteristics are reminiscent of those of the low-affinity enzyme glucokinase, which is typically present in the liver. By subtracting the activity at 1 mmol/liter glucose from that at higher glucose concentrations, we calculated the "glucokinase component," forms the "total" glucose phosphorylating activity. In five rabbits (of similar age and weight) with spontaneous hyperglycemia (mean +/- SEM: 11.71 +/- 0.60 mmol/liter), the optic nerve glucose phosphorylating activity was lower (value at 1 mmol/liter glucose: 5.42 +/- 1.31, -38.06%, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)