Collagenase‐assisted wound bed preparation: An in vitro comparison betweenVibrio alginolyticusandClostridium histolyticumcollagenases on substrate specificity

Abstract

Bacterial collagenase from the aerobic non-pathogenic Vibrio alginolyticus chemovar iophagus is an extracellular metalloproteinase. This collagenase preparation is obtained through a fermentation process and is purified chromatographically, resulting in a highly purified 82-kDa single-band protein that does not contain non-specific proteases or other microbial impurities. V. alginolyticus collagenase was added to a hyaluronan (HA)-based device to develop a novel debriding agent to improve the treatment of ulcers, necrotic burns, and decubitus in the initial phase of wound bed preparation. In this study, an in vitro biochemical characterisation of V. alginolyticus collagenase versus a commercial preparation from a Clostridium histolyticum strain on various dermal extracellular matrix (ECM) substrates was performed. V. alginolyticus collagenase demonstrated its ability to carry out the enzymatic cleavage of the substrate, allowing a selective removal of necrotic tissues while sparing healthy tissue, as reported in clinical studies and through routine clinical experience. in vitro tests under physiological conditions (pH, presence of Ca++, etc.) have demonstrated that V. alginolyticus collagenase exhibits very poor/limited non-specific proteolytic activity, whereas the collagenase preparation from C. histolyticum is highly active both on collagen and on non-collagenic substrates. This finding implies that while the V. alginolyticus enzyme is fully active on the collagen filaments that anchor the necrotic tissue to the wound bed, it does not degrade other minor, but structurally important, components of the dermal ECM. This feature could explain why collagenase preparation from V. alginolyticus has been reported to be much gentler on perilesional, healthy skin. K E Y W O R D Sbacterial collagenase, clostridium histolyticum, hyaluronic acid, Vibrio alginolyticus, wound

Hyaluronic Acid/Collagenase Ointment in the Treatment of Chronic Hard-to-Heal Wounds: An Observational and Retrospective Study

“…Reports from in vitro investigations have demonstrated that the Vibrio alginolyticus enzyme is as effective as Clostridium histolyticum on many types of collagens with equally active digestion of collagen type I. Conversely, Vibrio alginolyticus collagenase degrades fibronectin and decorin to a lesser extent than Clostridium histolyticum collagenase. This is an important insight, as decorin and fibronectin possess structurally important components of dermal extracellular matrix (ECM), and fibronectin is essential to dermal fibroblast migration within the wound-healing process [42]. The difference may be attributable to a higher selectivity of Vibrio alginolyticus collagenase, in which a completely active enzyme is observed on the collagen filaments binding the necrotic tissue to the wound bed.…”

Engineered PLGA-PVP/VA based formulations to produce electro-drawn fast biodegradable microneedles for labile biomolecule delivery

“…Collagenase is a zinc-dependent metallo-proteinases able to degrade with a high specificity collagen substrates, playing a key role in the modulation of inflammation disorders, chronic wounds, ulcers or removal of cellular debris (Di Pasquale et al 2019). The recent combination of collagen-lytic activity of collagenase with the wound healing properties of hyaluronic acid led to the production of a safe and active pharmaceutical formulation for the topical treatment of wounds, HYALO4 STARTMET (Di Pasquale et al 2019).…”

“…Collagenase is a zinc-dependent metallo-proteinases able to degrade with a high specificity collagen substrates, playing a key role in the modulation of inflammation disorders, chronic wounds, ulcers or removal of cellular debris (Di Pasquale et al 2019). The recent combination of collagen-lytic activity of collagenase with the wound healing properties of hyaluronic acid led to the production of a safe and active pharmaceutical formulation for the topical treatment of wounds, HYALO4 STARTMET (Di Pasquale et al 2019). Unfortunately, being a protease, collagenase suffers of instability in physiological environment; in particular, it has been reported that it has a stability of maximum 8 h when it is stored at 4 °C or 24-48 months when storage temperatures range between − 20 and − 80 °C (Vaccaro et al 2017).…”

Plasma-assisted synthesis of noble nanoparticles coated with hyaluronan: chemico-physical characterization and safety assessment

“…The effect of tested nanoparticles on the enzymatic activity of collagenase was determined spectrophotometrically by quantifying the fragment produced by the enzymatic reaction against an external standard. The assay was performed by modifying the method described by Wunsch-Heidrich et al (1963) [45,46] Briefly, the enzymatic activity of collagenase was detected spectrophotometrically (absorbance of fragment at 320 nm) by measuring the production of the glicil-L-prolil-D-arginine fragment (yellow) produced from the PZ-L-prolil-L-leucyl-glicil-L-prolil-D-arginine (PZ = 4-phenylazobenzyloxycarbonyl) synthetic substrate, after extraction with a solution of ethyl acetate acidified with citric acid. For the assay, a collagenase solution (ranging from 0.35 to 1.2 nkatal/ml) was prepared using a buffer solution of 25 mM TRIS-HCl and 10 mM CaCl2 (pH 7.1 adjusted using HCl).…”