8 research outputs found
Construct 1 inhibits increase in blood glucose after end of fasting in cynomolgus monkeys.
<p>The effect of long-lived construct 1 on appetite suppression was tested in normal cynomolgus monkeys. Panels A–C show overlaid plots of blood glucose profiles after placebo or construct 1 administration for each individual animal. Solid arrows mark the time when food was returned to the animals (t = 6 hours).</p
Gcg-XTEN confers temporally-controlled resistance to insulin-induced hypoglycemia in dogs.
<p>Beagle dogs were fed three hours prior to the start of the experiment and fasted thereafter. At time = 0, animals received either a dose of 0.6 nmol/kg Gcg-XTEN or placebo (open arrows). Animals (n = 4 per group) received a challenge of 0.05 U/kg insulin to induce hypoglycemia at either 6 hr (A) or 12 hr (B) after initial dose, indicated by solid arrows. Values shown are the average blood glucose plus or minus the standard deviation. (C) A hypothetical timeline for human administration. Assuming Gcg-XTEN dosing at 21:00, 6 hr post dose corresponds to 03:00 (during sleep) where protection of hypoglycemia is desired, and 12 hr post dose corresponds to 09:00 where the pharmacodynamic effect should have expired to allow for a morning meal.</p
Chronic dosing of Construct 1 in Diet-Induced Obese Mice: Weight Loss.
<p>Change in body weight in Diet-Induced Obese mice over the course of 28 days continuous drug administration. Values shown are the average +/− SEM of 10 animals per group. Groups were found to be significantly different (p<0.05) by repeated measures ANOVA.</p
Insulin Levels in Gcg-XTEN or placebo treated dogs.
a<p>Concentration values are reported as µIU/mL ± standard deviation.</p
Chronic dosing of Construct 1 in Diet-Induced Obese Mice: Fasting Blood Glucose.
<p>Change in fasting blood glucose in Diet-Induced Obese mice after 28 days continuous drug administration. Values shown are the average +/− SEM of 10 animals per group. Groups were not found to be significantly different by t test.</p
Biophysical Characterization and Stability of Gcg-XTEN.
<p>Gcg-XTEN was produced recombinantly in <i>E. coli</i> and purified to homogeneity using three column steps (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010175#s4" target="_blank">methods</a>). (A) SDS-PAGE analysis of the purified protein product (lane 2). Molecular weight markers are shown in lane 1 with relevant size markers labeled at the left. Note that the true molecular weight of the molecule is 16305 daltons (confirmed by mass spectrometry; not shown). Slow migration in SDS-PAGE relative to globular protein standards is typical of XTEN fusion proteins due to differences in primary amino acid composition. (B) Glucagon receptor (GcgR) Ca<sup>2+</sup>-flux assay comparing the efficacy of Gcg-XTEN to unmodified glucagon. Calculated EC50 values for each curve fit are shown. (C) Reverse phase C18 HPLC analysis and (D) Size exclusion chromatography HPLC analysis of the purified Gcg-XTEN construct at the time of production. (E) Reverse phase C18 HPLC analysis and (F) Size exclusion chromatography HPLC analysis of Gcg-XTEN after 6 months storage at either −80°C (black), 2–8°C (blue), or 25°C (red). Note the scale is expanded in panel E to better illustrate the appearance of minor peaks at 25°C.</p
Optimizing the pharmacokinetic profile of Gcg-XTEN.
<p>(A) Schematic representations of four glucagon constructs. The length of attached XTEN in number of amino acids is indicated on each construct. (B) Size exclusion chromatography of the four purified constructs. Chromatograms are labeled and colored for each construct as in panel A. The dashed chromatogram shows the profile of a mixed reference size standard (BioRad Laboratories). The molecular weight of each reference peak is noted. A large increase in apparent molecular weight due to an increased hydrodynamic radius is typically observed for XTEN fusion proteins in this assay. (C) Pharmacokinetic profile of constructs 1–2 over 24 hours in cynomolgus monkeys. Curves are labeled and colored as in panel A. For construct 2, serum concentration at 24 hours was below limit of detection (approximately 0.1 nM), hence the dashed line approximates the slowest terminal half-life consistent with this observation. Construct 3 was also tested in parallel, but was below limit of detection at all time points, suggesting that it has a very short plasma half-life. Based on the rapid clearance of construct 3, construct 4 was not tested in animals.</p
Gcg-XTEN shows extended pharmacodynamics in dogs.
<p>Glucagon (A) or Gcg-XTEN (B) was injected at 14 or 12 nmol/kg, respectively, into fasted beagle dogs (n = 4 per group) and blood glucose levels were monitored in comparison to placebo injection. (C) The difference in blood glucose area under the curve for the first hour after injection of placebo, Gcg-XTEN, or Glucagon (Gcg) relative to pre-injection baseline is shown (n = 4–8 animals per group). The dose level for each group is indicated. Values for all panels are the average values plus or minus the standard deviation.</p