55.BJRBJR0010.1302/2046-3758.55.BJR-2016-0072 research-article2016 Freely available online OPEN ACCESS BJR UPPER LIMB A new bromelain-based enzyme for the release of Dupuytren’s contracture DUPUYTREN’S ENZYMATIC BROMELAIN-BASED RELEASE G. Rubin, Objectives M. Rinott, Injectable Bromelain Solution (IBS) is a modified investigational derivate of the medical A. Wolovelsky, grade bromelain-debriding pharmaceutical agent (NexoBrid) studied and approved for a L. Rosenberg, rapid (four-hour single application), eschar-specific, deep burn debridement. We conducted Y. Shoham, an ex vivo study to determine the ability of IBS to dissolve-disrupt (enzymatic fasciotomy) N. Rozen Dupuytren’s cords. Materials and Methods Orthopaedic Specially prepared medical grade IBS was injected into fresh Dupuytren’s cords excised from Department, Central patients undergoing surgical fasciectomy. These cords were tested by tension-loading them Emek Hospital, Afula, to failure with the Zwick 1445 (Zwick GmbH & Co. KG, Ulm, Germany) tension testing system. Israel Results We completed a pilot concept-validation study that proved the efficacy of IBS to induce enzymatic fasciotomy in ten cords compared with control in ten cords. We then completed a dosing study with an additional 71 cords injected with IBS in descending doses from 150 mg/cc to 0.8 mg/cc. The dosing study demonstrated that the minimal effective dose of 0.5 cc of 6.25 mg/cc to 5 mg/cc could achieve cord rupture in more than 80% of cases. Conclusions These preliminary results indicate that IBS may be effective in enzymatic fasciotomy in Dupuytren’s contracture. Cite this article: Bone Joint Res 2016;5:175–177. Keywords: Bromelain; Collagenase; Dupuytren’s contracture; Enzyme; Fasciotomy G. Rubin, MD, Hand Surgeon, M. Rinott, MD, Head of Hand Unit, Article focus Introduction A. Wolovelsky, MD, Hand Surgeon, The aim of this study was to investigate Dupuytren’s contracture (DC) is a connective N. Rozen, MD, PhD, Head of the ability of injectable bromelain solu- tissue disorder characterised by contractile pal- the Orthopaedic Department, tion to dissolve-disrupt Dupuytren’s mar aponeurosis leading to shortening and Orthopaedic Department, Central Emek Hospital, Afula, Israel. cords in an ex vivo study. progressive digital flexion deformity. Open L. Rosenberg, MD, Head of partial fasciectomy has been the mainstay of Plastic and Reconstructive Surgery Key messages treatment for DC. In recent years, there has Department, been an increased interest in less invasive Y. Shoham, MD, Plastic and The study indicates that injectable bromelain Reconstructive Surgeon, solution may be effective in enzymatic fasci- approaches, including sharp needle cordot- Plastic and Reconstructive Surgery otomy in Dupuytren’s contracture. omy/fasciotomy and enzymatic disruption by Department and Hand Unit, 1 Soroka University Medical Center, collagenase injection. Collagenase clostrid- Ben-Gurion University of the Strengths and limitations ium histolyticum is the first non-surgical, phar- Negev, Beer-Sheva, Israel. This is the first preliminary report of a macological treatment for DC with a palpable Correspondence should be sent to new potential use of injectable bromelain cord, approved for use in the United States Dr G. Rubin; email: guytalr@ solution. (2010) and Europe (2011). Clinical trials 2-8 and bezeqint.net A limitation of this study is that it is an post-marketing studies9 have demonstrated doi: 10.1302/2046-3758.55.BJR- 2016-0072 ex vivo model based on human excised the efficacy and favourable safety profiles of Dupuytren’s cords that varied in thickness, collagenase injection for treating DC. Bone Joint Res 2016;5:175–177. Received: 6 March 2016; strength and the presence of nodules, all NexoBrid is a bromelain-derivate enzyme 10,11 Accepted: 14 March 2016 of which may interfere with the results. mixture extracted from pineapple stems. It VOL. 5, NO. 5, MAY 2016 175 176 G. RUBIN, M. RINOTT, A. WOLOVELSKY, L. ROSENBERG, Y. SHOHAM, N. ROZEN Table I. Dose response assessment table. was applied at a constant rate of 20 mm/min displace- ment of the cord ends until cord or suture rupture. % cord disruption Dose (mg/cc) n success Statistical analysis. In the pilot phase, Fisher’s exact test was used to explore the association between the study 0.8 8 37.5 1.7 3 0 groups (IBS injection/control) and cord disruption. In 3.12 7 71.43 the second study, we used the Cochran-Armitage test to 5 5 100 explore the relationship between IBS and the probability of 6.25 19 78.95 9.375 4 50 cord disruption. 12.5 7 57.14 The statistical analyses were performed using SAS 9.2. 25 5 75 Statistical significance was considered when p < 0.05. 37.5 2 100 50 4 75 75 4 50 Results 100 11 100 In the pilot phase, cords had complete disruption in ten of 150 2 100 the ten specimens in the IBS injection group. In many cases, the cords were completely dissolved to the point has been developed, tried and approved as an effective, that, by holding one end, the other end ‘dripped’ down fast, eschar-specific and safe debriding pharmaceutical by its own weight. The thread-tearing threshold of 8.5 kg preparation for deep burn eschar removal.12-15 The infor- (sd 0.5) became the success threshold of the enzymatic mation generated during the NexoBrid development pro- efficacy. In none of the control specimens did the cords/ gramme served to develop an injectable bromelain solution threads break (Fisher’s exact test; p < .0001). The cords (IBS), which is a possible new approach to DC cords. demonstrated a similar stress/elongation graph in each The aim of this study was to investigate the ability of IBS group (Figs 1a and 1b). In the second, multiple-dose to dissolve-disrupt Dupuytren’s cords in an ex vivo study. range study, 71 cords were injected with IBS in descend- ing doses from 150 mg/cc to0.8 mg/cc. The dosing study demonstrated that the minimal effective dose of 0.5 cc of Materials and Methods 6.25 mg/cc to 5 mg/cc can achieve cord rupture in more Material. Lyophilised injectable bromelain-based enzyme than 80% of cases. The Cochran-Armitage test supports (MediWound Ltd, Yavne, Israel) was prepared and the trend hypothesis (p = 0.0021), indicating that the weighed in vials. The enzyme is dissolved in a given vol- probability for cord rupture increases as the dose increases. ume of vehicle to predefined doses. All materials are ster- ile medical grade pharmaceuticals.1 Discussion Tissue preparation. The first phase, concept validation DC develops as a result of increased and uncontrolled study, basically followed an in vitro model that served fibroblast proliferation, as well as due to the deposition of to develop the collagenase DC enzymatic fasciotomy type II and type V collagen and glycosaminoglycans in the studies.16 After receiving Institutional Helsinki Ethical palmar aponeurosis.18 The cause of this fibrosis has not yet Review Board Committee approval and patient informed been elucidated. The treatment is traditionally surgery, consent, ten fresh Dupuytren’s cords were dissected meticulously exposing the fibrotic fascia and excising the from patients undergoing routine fasciectomy. The constricting cords, thereby releasing the flexure- specimens were divided in half across their long axis and contracture. These procedures are demanding of skills and were anchored to one polypropylene (Prolene) suture resources with quite a long convalescence period, mostly (Ethicon, Somerville, New Jersey) by the locking-loop, due to the wide dissection field, surgically-related adverse with the Krackow suture technique at both ends.17 The events and high recurrence rates (12% to 39%, with a mean tensile strength of the suture is 8.5 sd 0.5 kg.16 mean follow-up time of 1.5 to 7.3 years).19 In order to Each half was injected in the middle of the cord either reduce surgical trauma, enzymatic fasciotomy for DC has with 0.5 mL of 100 mg/cc dose of IBS or with a con- previously been tried. It was first introduced by Hueston in trol saline injection of 0.5 mL total volume, by using 1971, using a mixture of trypsin, hyaluronidase, and lido- a 30-gauge needle. The cords were then placed in a caine injected into the cord during surgery.20 McCarthy21 humidifying chamber and incubated at 37° for 24 hours reported long-term results with the same mixture; he before being submitted to tensile strength testing. In the noticed recurrence in seven of nine of his patients in two to second phase, we conducted a dosing study on 71 fresh three years. The options for treating patients with DC have cords excised from Dupuytren’s patients which were broadened with the introduction of collagenase.2,4 injected with descending doses of IBS from 150 mg/cc IBS consists of a sterile, lyophilised, partially purified pro- to 0.8 mg/cc (Table I). teolytic protein mixture with specific enzymatic activity, Mechanical testing. The cords were connected to a derived from specially prepared medical grade bromelain mechanical testing device, Zwick 1445 (Zwick GmbH & raw material extracted from pineapple plant stems. The Co. KG, Ulm, Germany) via the Prolene suture and a load enzyme should be dissolved in a vehicle before injection. BONE & JOINT RESEARCH A NEW BROMELAin-bASED ENZYME FOR THE RELEASE OF Dupuytren’s contrACTURE 177 Stress (N) Stress (N) 100 100 75 75 50 50 25 25 Elongation (mm) Elongation (mm) 0 0 0255075 100 0255075 100 Fig. 1a Fig. 1b Stress/elongation graph of the control group (a) and the study group (b). Bromelain, a clinically used natural pineapple product, has 9. Peimer CA, McGoldrick CA, Fiore GJ. Nonsurgical treatment of Dupuytren’s some reported anti-inflammatory and immunomodulatory contracture: 1-year US post-marketing safety data for collagenase clostridium histolyticum.