A Novel Long-Acting Human Growth Hormone Fusion Protein (VRS-317): Enhanced In Vivo Potency and Half-Life

ABSTRACT:A novel recombinant human growth hormone (rhGH) fusion protein (VRS-317) was designed to minimize receptor-mediated clearance through a reduction in receptor binding without mutations to rhGH by genetically fusing with XTEN amino acid sequences to the N-terminus and the C-terminus of the native hGH sequence. Although in vitro potency of VRS-317 was reduced approximately 12-fold compared with rhGH, in vivo potency was increased because of the greatly prolonged exposure to the target tissues and organs. VRS-317 was threefold more potent than daily rhGH in hypophysectomized rats and fivefold more potent than daily rhGH in juvenile monkeys. In juvenile monkeys, a monthly dose of 1.4mg/kg VRS-317 (equivalent to 0.26mg/kg rhGH) caused a sustained pharmacodynamic response for 1month equivalent to 0.05mg/kg/day rhGH (1.4mg/kg rhGH total over 28days). In monkeys, VRS-317, having a terminal elimination half-life of approximately 110h, was rapidly and near-completely absorbed, and was well tolerated with no observed adverse effects after every alternate week subcutaneous dosing for 14weeks. VRS-317 also did not cause lipoatrophy in pig and monkey studies. VRS-317 is currently being studied in GH-deficient patients to confirm the observations in these animal studies. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Associatio

Gcg-XTEN: An Improved Glucagon Capable of Preventing Hypoglycemia without Increasing Baseline Blood Glucose

While the majority of current diabetes treatments focus on reducing blood glucose levels, hypoglycemia represents a significant risk associated with insulin treatment. Glucagon plays a major regulatory role in controlling hypoglycemia in vivo, but its short half-life and hyperglycemic effects prevent its therapeutic use for non-acute applications. The goal of this study was to identify a modified form of glucagon suitable for prophylactic treatment of hypoglycemia without increasing baseline blood glucose levels.Through application of the XTEN technology, we report the construction of a glucagon fusion protein with an extended exposure profile (Gcg-XTEN). The in vivo half-life of the construct was tuned to support nightly dosing through design and testing in cynomolgus monkeys. Efficacy of the construct was assessed in beagle dogs using an insulin challenge to induce hypoglycemia. Dose ranging of Gcg-XTEN in fasted beagle dogs demonstrated that the compound was biologically active with a pharmacodynamic profile consistent with the designed half-life. Prophylactic administration of 0.6 nmol/kg Gcg-XTEN to dogs conferred resistance to a hypoglycemic challenge at 6 hours post-dose without affecting baseline blood glucose levels. Consistent with the designed pharmacokinetic profile, hypoglycemia resistance was not observed at 12 hours post-dose. Importantly, the solubility and stability of the glucagon peptide were also significantly improved by fusion to XTEN.The data show that Gcg-XTEN is effective in preventing hypoglycemia without the associated hyperglycemia expected for unmodified glucagon. While the plasma clearance of this Gcg-XTEN has been optimized for overnight dosing, specifically for the treatment of nocturnal hypoglycemia, constructs with significantly longer exposure profiles are feasible. Such constructs may have multiple applications such as allowing for more aggressive insulin treatment regimens, treating hypoglycemia due to insulin-secreting tumors, providing synergistic efficacy in combination therapies with long-acting GLP1 analogs, and as an appetite suppressant for treatment of obesity. The improved physical properties of the Gcg-XTEN molecule may also allow for novel delivery systems not currently possible with native glucagon

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

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

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) Ca²⁺-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