Validation of Matrix Metalloproteinase-9 (MMP-9) as a Novel Target for Treatment of Diabetic Foot Ulcers in Humans and Discovery of a Potent and Selective Small-Molecule MMP-9 Inhibitor That Accelerates Healing

Abstract

Diabetic foot ulcers (DFUs) are a significant health problem. A single existing FDA-approved drug for this ailment, becaplermin, is not standard-of-care. We previously demonstrated that upregulation of active matrix metalloproteinase (MMP)-9 is the reason that the diabetic wound in mice is recalcitrant to healing and that MMP-8 participates in wound repair. In the present study, we validate the target MMP-9 by identifying and quantifying active MMP-8 and MMP-9 in human diabetic wounds using an affinity resin that binds exclusively to the active forms of MMPs coupled with proteomics. Furthermore, we synthesize and evaluate enantiomerically pure ( R)- and ( S)-ND-336, as inhibitors of the detrimental MMP-9, and show that the ( R)-enantiomer has superior efficacy in wound healing over becaplermin. Our results reveal that the mechanisms of pathology and repair are similar in diabetic mice and diabetic humans and that ( R)-ND-336 holds promise for the treatment of DFUs as a first-in-class therapeutic.

Hyperbaric oxygen therapy accelerates wound healing in diabetic mice by decreasing active matrix metalloproteinase‐9

“…We have previously reported the detrimental role of MMP‐9 and the beneficial role of MMP‐8 in wound healing of diabetic mice . We also identified active MMP‐8 and MMP‐9 in human DFUs, with >30‐fold higher active MMP‐9 levels in diabetic patients with more severe and infected ulcers . In support of the detrimental role of MMP‐9, we found that aclerastide, an angiotensin II analog that stimulates NADPH oxidase to mediate cell growth, increases active MMP‐9 levels that may have played a role in aclerastide's clinical failure .…”

“…We have previously reported the detrimental role of MMP‐9 and the beneficial role of MMP‐8 in wound healing of diabetic mice . We also identified active MMP‐8 and MMP‐9 in human DFUs, with >30‐fold higher active MMP‐9 levels in diabetic patients with more severe and infected ulcers .…”

“…However, dysregulation of ROS can be detrimental in hindering the repair process by causing tissue destruction . In addition, ROS upregulate MMP‐9 in vascular smooth muscle cells, and we have shown previously that both ROS and active MMP‐9 are increased in untreated diabetic mice after wounding . The latter is detrimental to diabetic wound healing .…”

“…The affinity resin was synthesized in 14 synthetic steps using a previously reported method . A 10‐mg aliquot of tissue homogenate was incubated with 100 μL of the affinity resin at 4 °C for 2 hours and processed as previously described …”

“…We applied this methodology to wounds of diabetic mice and identified active MMP‐8 and MMP‐9, and further determined that MMP‐8 plays a repair role whereas the function of MMP‐9 contributes to the recalcitrance in diabetic wound healing . Recently, we analyzed debridement tissue from DFU patients with the affinity resin coupled with mass spectrometry; the exact same active MMP‐8 and MMP‐9 were identified in diabetic humans as seen in diabetic mice . The more severe and infected human DFUs have >30‐fold higher levels of active MMP‐9 than non‐diabetic controls .…”