In Vitro Study of Novel Collagenase (XIAFLEX®) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

Abstract

Dupuytren's disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH) - Xiaflex® - is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex® against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex® exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex® did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex® significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex® enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex® on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.

Ex vivo evaluation of antifibrotic compounds in skin scarring: EGCG and silencing of PAI-1 independently inhibit growth and induce keloid shrinkage

“…RNA extraction was performed using RNeasy mini kit (Qiagen, Crawley, UK). 15 Subsequently, 1 mg mRNA was converted to cDNA using qScript cDNA Synthesis kit (Quanta BioSciences, Gaithersburg, MD, USA) 15 Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was carried out with standard protocol 15 using a number of gene-specific primers (Supplementary Table S3). …”

Effect of Short-Term Enzymatic Treatment on Cell Migration and Cartilage Regeneration:In VitroOrgan Culture of Bovine Articular Cartilage

“…Collagenase isolated from Clostridium histolyticum was clinically proven for treatment of Depuytren's disease. 18 It acts as a collagenolytic enzyme, which digests triple-helical collagens into small peptides. Since the collagenase contains both type I and II, it is also applicable to digest the extracellular matrix of articular cartilage, which has mainly type II collagen.…”

An overview on therapeutic potential and various applications of microbial collagenases

“…Injectible collagenase is more efficient than surgical fasciotomy and has milder side effects, which leads to higher patient satisfaction [97,98]. Studies on the influence of CHCs on Dupuytren's cords and on Dupuytren's disease fibroblasts have demonstrated that clostridial collagenases can efficiently digest ECM of Dupuytren's disease cords without inducing significant cytotoxicity or structural damage to non-collagenous tissue elements [99,100]. In the last decade translational research has introduced the non-surgical technique of enzymatic fasciotomy with collagenase injections and able to increase quality of life [96,101].…”