Serum MMP-2/MMP-9 activity in healthy and injured dogs.

<p>Box-and-whisker plots summarizing the distribution of MMP 2/9 activity for healthy control dogs (N = 5) and dogs with spinal cord injury (SCI; N = 42) that had serum collected. Values of serum MMP 2/9 activities were significantly (P = 0.0128) greater for dogs with SCI included in the trial than control dogs. The horizontal lines with triangles represent the median value; the horizontal lines at the bottom and top of the boxes represent the 25^th and 75^th percentiles of the data, respectively. The thin vertical lines extending up or down from the boxes to horizontal lines (so-called whiskers) extend to a multiple of 1.75× the distance of the upper and lower quartile, respectively. Horizontal lines with circles represent values outside the limits of the whiskers.</p

<i>In vitro</i> inhibition of MMP-9 by GM6001.

<p>Calibrated MMP-9 activity (Log 10⁶), as measured by a fluorescent electrophoretic technique, was dramatically attenuated by GM6001 <i>in vitro</i> at various concentrations. Plasma concentrations of GM6001, measured 96 hours following a single 100 mg/kg subcutaenous dose (range 0.16−.26 µM or 60–100 ng/mL), approximated those needed <i>in vitro</i> to robustly inhibit MMP-9 (0.2 µM or 77 ng/mL). Groups marked with an asterisk (*) had significantly (P<0.05) different calibrated MMP-9 activity from reference (no GM6001) using a one tailed Student’s t-test.</p

Evaluation of primary outcome in dogs with SCI.

<p>Box-and-whisker plots of TSCIS on day 42 by treatment group, stratified by MFS at admission (MFS = 0, left panel; or MFS >0, right panel). There were no significant differences in TSCIS among dogs with MFS score >0 (right panel), but TSCIS was significantly (P<0.05) greater for the GM6001 and the DMSO group than saline treated dogs with MFS = 0 (left panel). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096408#pone-0096408-g005" target="_blank">Figure 5</a> for a description of box-and-whisker plots. Groups marked with different letters differ significantly (P<0.05).</p

Evaluation of TSCIS motor score at day 42 following SCI.

<p>Box-and-whisker plots of TSCIS motor score on day 42 by treatment group, stratified by MFS at admission (MFS = 0, left panel; or MFS >0, right panel). There were no significant differences in motor score among dogs with MFS >0 (right panel), but motor score was significantly (P<0.05) greater for the GM6001 and the DMSO group than saline treated dogs with MFS = 0 (left panel). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096408#pone-0096408-g005" target="_blank">Figure 5</a> for a description of box-and-whisker plots. Groups marked with different letters differ significantly (P<0.05).</p

T2-weighted magnetic resonance images in dogs with spinal cord injuries from intervertebral disk herniation.

<p>In 1 dog (A, B) that was non-ambulatory with intact pelvic limb movement and sensation, there was focal ventrolateral spinal cord compression at the T12-T13 vertebral articulation without spinal cord signal change. A second dog (C, D) with paraplegia and absent pelvic limb deep nociception had compression at the T12-T13 vertebral articulation. There was extensive spinal cord T2-weighted hyperintensity (white arrows) visible on the sagittal image (C), suggestive of processes seen in contusion injuries such as edema, necrosis, hemorrhage, or cellular infiltrates. The transverse image (D, level of T13 vertebral body) indicated that T2-weighted hyperintensity was predominantly localized to the gray matter.</p

Serum MMP-2/MMP-9 activity pre- and post-drug delivery.

<p>Dogs were first randomized into 3 treatment groups, and serum was obtained prior to administration of saline, DMSO, or GM6001 (panel A). There were no differences in serum MMP-2/MMP-9 activity among treatment groups at the time of admission (panel A); however, at day 3, there was a significant (P = 0.0482) difference in serum activity between GM6001-treated dogs and the other treatment groups (panel B). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096408#pone-0096408-g005" target="_blank">Figure 5</a> for a description of box-and-whisker plots. Groups marked with different letters differ significantly (P<0.05).</p

Values of serum MMP-2/MMP-9 activity were not significantly associated with various clinical variables.

<p>Medians (and ranges) and P values derived from Wilcoxon-rank sum tests are reported for the categorical variables listed above. MMP = matrix metalloproteinase; MFS = Modified Frankel Score.</p

Consolidated Standards of Reporting Trials (CONSORT) Diagram.

<p>Flow diagram depicting progress through different phases of the clinical trial including enrollment, group allocation, and follow-up.</p

Pharmacokinetics of GM6001 in dogs.

<p>Administration of a single 100 mg/kg subcutaneous dose of GM6001 to dogs resulted in the rapid development of peak plasma drug concentrations with drug still detectable 96 hours post-delivery. Administration of a second dose of GM6001 to a sub-group of dogs 12 hours following initial drug delivery resulted in increased plasma drug concentrations at all assessed time points.</p