Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications.

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.

Managing complications of collagenase Clostridium histolyticum (CCH) injection.

PURPOSE: The introduction of collagenase Clostridium histolyticum (CCH) has revolutionized the treatment of Peyronie's disease. The efficacy of this therapy has been well demonstrated and the safety profile is favorable. However, post-injection complications are poorly characterized and management of these complications lacks standardization. METHODS: This review includes literature published in English and indexed in the PubMed®, Embase® or Google Scholar™ databases. What follows is a synopsis of relevant articles, including original research studies, in an attempt to better define CCH complications and their respective management strategies. RESULTS: Adverse effects of therapy are common but generally self-limiting. Penile pain and edema are expected events, and most patients experience hematologic sequelae (bleeding, hematoma, ecchymosis, etc.). The intervention rate for penile hematoma is low. Penile fracture is a morbid complication of therapy that is rare and may be challenging to diagnose given the frequency with which pronounced bruising and swelling are encountered. Imaging is a useful adjunct in situations of diagnostic uncertainty. Alternative injection protocols have been evaluated to limit the cost and morbidity of CCH therapy. Clinical efficacy of these protocols is promising, but prospective evaluation is lacking. CONCLUSIONS: No standardized protocols exist for management of post-injection complications of CCH therapy. The majority of these complications are managed conservatively, but suspected penile fracture should be carefully evaluated and imaging employed when needed. Future prospective studies of alternative injection protocols are warranted to decrease morbidity while maintaining efficacy.

A modified technique for intralesional injection of collagenase Clostridium histolyticum for Peyronie's disease results in reduced procedural morbidity using a standardized hematoma classification rubric.

PURPOSE: Early clinical trials of injectable collagenase Clostridium histolyticum (CCh) for Peyronie's disease (PD) demonstrated safety and efficacy. Since then, modified injection protocols have been proposed. Adverse events-such as bruising, swelling, hematoma, and corporal rupture-exceed 50% in many studies, but lack of standardization of hematoma severity limits conclusions about the relative safety of protocols. We propose a modification of the standard injection technique that aims to decrease the rates of adverse events. We further describe a hematoma classification rubric that may standardize safety assessment. METHODS: A modified injection procedure, termed the "fan" technique, was employed in the treatment of PD. All men receiving CCh from January 2016 through January 2019 at a single institution were included in an institutional review board (IRB) approved database. Treatment outcomes and adverse events were retrospectively assessed. A three-tiered hematoma classification rubric was devised to standardize reporting of hematoma, which was defined as concurrent bruising and swelling at the site of injection without loss of erection. RESULTS: Using the fan technique, 152 patients received 1323 injections. Eight hematomas (5.3% of all patients, 0.6% of all injections) were observed. The number of grade I, grade II, and grade III hematomas were 3, 2, and 3, respectively. Bruising or swelling not meeting the definition of hematoma was seen in 54.6% and 27.0% of patients, respectively. There were zero corporal ruptures. CONCLUSION: A modified injection technique results in reduced procedural morbidity. A hematoma classification system provides clarity and standardization to the assessment of safety in PD treatment. Further clinical studies with control arms are required to verify these findings.

Sixty years in the making: collagenase Clostridium histolyticum, from benchtop to FDA approval and beyond.

PURPOSE: The introduction of collagenase Clostridium histolyticum (CCH) as the first and only FDA-approved non-surgical treatment for Peyronie's disease (PD) has been an important step in its management. Our aim is to provide an overview of the historical origins of CCH and its development through FDA approval and beyond for the treatment of PD. METHODS: A PubMed search using the terms Peyronie OR Peyronie's AND collagenase and limited to clinical research studies resulted in 24 articles that were examined for the current review. RESULTS: PD is a connective tissue disorder of the penile tunica albuginea involving fibrotic penile plaques that cause abnormal curvature and, in many cases, erectile pain. Although the exact mechanism and underlying pathophysiology are not well characterized, the known lability of these plaques to exogenous bacterial collagenase combined with a lack of effective medical therapies led to the development of CCH as an evidence-based treatment of PD. The initial discovery of collagenase was followed by in vitro studies on PD plaque tissue and following the phase 3 IMPRESS trial culminated in FDA approval of CCH in 2013. Future directions in CCH therapy include improved patient selection, use in acute phase PD, adjuvant and combination therapies, and novel delivery mechanisms. CONCLUSION: CCH provides an effective non-surgical treatment option for men with PD. We have traced the development of CCH in the treatment of PD from the earliest in vitro investigations to comprehensive multi-study meta-analyses confirming its highly rated efficacy when compared to other historical non-surgical remedies.

Evaluation of caffeine as inhibitor against collagenase, elastase and tyrosinase using in silico and in vitro approach.

Skin ageing results from enhanced activation of intracellular enzymes such as collagenases, elastases and tyrosinase, stimulated by intrinsic ageing and photoageing factors. Recently, caffeine-based cosmetics are introduced that demonstrates to slow down skin photoageing process. However, no attempts have been done so for to understand caffeine functional inhibitory activity against photoageing related enzymes. Hence, this study established the caffeine molecular interaction and inhibition activity profiles against respective enzymes using in silico and in vitro methods, respectively. Results from in silico study indicates that caffeine has comparatively good affinity with collagenase (-4.6 kcal/mol), elastase (-3.36 kcal/mol) and tyrosinase (-2.86 kcal/mol) and formed the stable protein-ligand complex as validated by molecular dynamics simulation (protein-ligand contacts, RMSD, RMSF and secondary structure changes analysis). Moreover, in vitro data showed that caffeine (1000 µg/mL) has statistically significant maximum inhibition activity of 41.86, 36.44 and 13.72% for collagenase, elastase and tyrosinase, respectively.

The Collagenase of the Bacterium Clostridium histolyticum in the Treatment of Irradiation-Induced Capsular Contracture.

BACKGROUND: Irradiation therapy is an important pillar in the treatment of breast cancer. However, it can trigger capsular fibrosis, the most significant complication of implant-based breast reconstruction. As collagen is the main component of fibrotic capsules, the collagenase of the bacterium Clostridium histolyticum poses a potential treatment option for this pathological condition. METHODS: Thirty-six rats received miniature silicone implants on their backs. On day 1, the implant sites of two groups were irradiated with 10 Gy. On day 120, one irradiated group received collagenase injections into the implant pockets (n = 12). Non-irradiated (n = 12) and irradiated capsules (n = 12) were injected with plain solvent solution serving as controls. Data were analyzed by means of in vivo imaging, histology, immunohistochemistry and gene expression analysis. RESULTS: Compared with both controls, the injection of collagenase led to significantly thinner capsules. This was verified by in vivo imaging and histology. Although irradiation provoked alterations in capsule collagen structure and vessel wall thickness, the application of collagenase resulted in a significant reduction of collagen density. This was accompanied by an up-regulation of VEGF-A gene expression. Of note, hematoma formation inside the implant pocket occurred in two cases after collagenase injection. CONCLUSIONS: The collagenase of the bacterium Clostridium histolyticum is effective in degrading irradiation-induced capsular fibrosis around silicone implants. Hematoma formation occurred most likely because of irradiation-induced alterations in vessel wall architecture and capsule vascularization. Further studies need to be performed to address the clinical safety of this novel treatment option.

The Effects of Collagenase Clostridium histolyticum on Plantar Fibromatosis: A Case Study.

Plantar fibromatosis, also known as Ledderhose's disease, is a rare disorder of benign fibroblast proliferation involving the plantar aponeurosis (i.e., plantar fascia). Traditionally, surgical intervention has been the most common treatment for plantar fibromatosis. However, numerous studies have reported high recurrence rates of plantar fibromatosis after surgical intervention, as well as wound healing difficulties and nerve injury. Plantar fibromatosis often coexists with other superficial fibrous diseases such as Dupuytren's contracture and Peyronie's disease; immunohistochemical and ultrastructural analyses suggest a relationship between Ledderhose's disease and Dupuytren's contracture. The US Food and Drug Administration approved collagenase Clostridium histolyticum for the treatment of Dupuytren's contracture in 2010 and Peyronie's disease in 2013. This case study presents the successful treatment of Ledderhose's disease almost 4 years (45.5 months) after off-label use of collagenase C. histolyticum injection in a 22-year-old white female who had recurrent plantar fibromatosis after surgical intervention.

Inhibitory effects of mycosporine-2-glycine isolated from a halotolerant cyanobacterium on protein glycation and collagenase activity.

Mycosporine-2-glycine (M2G), isolated from the halotolerant cyanobacterium Aphanothece halophytica, was purified and characterized in order to determine its utility as a cosmetic and pharmaceutical ingredient. M2G efficiently inhibited protein crosslinking. The inhibitory activity of M2G was significantly greater than that of the well-known Maillard reaction inhibitor aminoguanidine. In addition, M2G and other known mycosporine-like amino acids inhibited bacterial collagenase activity. To the best of our knowledge, this is the first report describing that M2G specifically inhibits the formation of advanced glycation end-products (AGEs), which play a critical role in ageing process and age-related diseases. These observations indicate that M2G may have potential therapeutic applications by suppressing the formation of AGEs and inhibiting excess collagenase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycosporine-like amino acids (MAAs) are known as multifunctional natural compounds. The MAA mycosporine-2-glycine (M2G), isolated from the halotolerant cyanobacterium Aphanothece halophytica, has potential therapeutic applications for the prevention of skin ageing. Purified M2G was endotoxin-free. M2G had greater inhibitory activity of protein cross-linking compared with well-known inhibitor, aminoguanidine and hindered bacterial collagenase activity. The mechanisms for these inhibitory activities of M2G are discussed in this study.

Collagenase clostridium histolyticum in Dupuytren's contracture: a systematic review.

INTRODUCTION: In the last few years, the use of collagenase clostridium histolyticum for management of Dupuytren's contracture has increased. The procedure of enzymatic fasciectomy has become popular because it is non-invasive, safe and fast to perform. SOURCES OF DATA: A systematic search was performed on Medline (PubMed), Web of Science and Scopus databases using the combined keywords 'Dupuytren collagenase' and 'Dupuytren clostridium histolyticum'. Forty-three studies were identified. The quality of the studies was assessed using the Coleman Methodological Score. AREAS OF AGREEMENT: The use of collagenase clostridium histolyticum provides better outcomes in patients with mild-moderate joint contracture, with lower complications and side effects than open fasciectomy. Manipulation can be performed 2-7 days after the injection. The use of collagenase is cost-effective. AREAS OF CONTROVERSY: Most of the studies did not report patient-related outcomes. The role of dynamic splint has to be investigated with randomized clinical trials. GROWING POINTS: The shorter recovery time and the low incidence of serious or major adverse effects are the main advantages of this new technology. AREAS TIMELY FOR DEVELOPING RESEARCH: There is a need to perform studies with longer follow-up because the recurrence rate seems to increase with time. Further investigations are necessary to assess whether it is safe and effective to inject two or more cords at the same time.

CORR® ORS Richard A. Brand Award: Clinical Trials of a New Treatment Method for Adhesive Capsulitis.

BACKGROUND: Conservative and even surgical management of adhesive capsulitis often is prolonged and painful. Management of adhesive capsulitis is lacking evidence-based controlled clinical trials. QUESTIONS/PURPOSES: We asked: (1) Does a collagenase clostridium histolyticum (CCH) injection lyse shoulder capsule collagen in adhesive capsulitis and at what dose? (2) Can a shoulder capsule injection be administered extraarticularly? (3) Do CCH injections result in better scores for pain and function than can be achieved with physical therapy among patients with adhesive capsulitis? METHODS: First, 60 patients with adhesive capsulitis were evaluated by clinical examination. To make the diagnosis of adhesive capsulitis, a patient had to have restricted active ROM of at least 60° in total active ROM in the affected shoulder compared with the unaffected contralateral shoulder; with the scapula stabilized, external rotation with the elbow at the side was a very important determinant. Patients were randomized to receive a single injection of 0.5 mL placebo or 0.145, 0.29, or 0.58 mg CCH. All 60 patients were followed up at 30 days. After that, if patients did not attain treatment thresholds they were eligible for up to five open-label 0.58-mg collagenase injections. For the longer-term followup in the open-label phase, 53 patients (83%) were followed to 12 months, 46 (77%) for 24 months, 36 (60%) for 36 months, 37 (62%) for 48 months, and 25 (42%) for 60 months. The extraarticular injection was directed at the anterior shoulder capsule with the patient in the supine position. To prove that these injections could be delivered reliably to the anterior shoulder capsule extraarticularly, the next study involved volunteers without adhesive capsulitis, in which 10 volunteers received a 10-mL injection of normal saline under ultrasound guidance. Finally, to determine the efficacy and dosing of CCH, four cohorts of 10 patients received up to three ultrasound-guided injections separated by 21 days. These injections were administered at one of four dose-volume levels. A fifth cohort of 10 patients was used as a control group and performed standardized home shoulder exercises only. All patients performed standardized home shoulder exercises three times daily. For Study 3, followup was at 22, 43, 64, and 92 days. No patients were lost to followup. RESULTS: In the first study, a single CCH injection did not provide clinically important improvements from baseline in active ROM, passive ROM, and function and pain scores compared with patients who received placebo. Ultrasound guidance confirmed extraarticular injection of the shoulder capsule in Study 2. The CCH injection was more effective than exercise therapy alone at 0.58 mg/1 mL and 0.58 mg/2 mL compared with exercise only in the primary measure of efficacy (active forward flexion) as shown in Study 3. For active forward flexion the mean in degrees in the 0.58 mg/2 mL group was 38° compared with 12° in the exercise-only group (p = 0.03). For active forward flexion the mean in the 0.58 mg/1mL group was 43° compared with 12° in the exercise-only group (p = 0.01). CONCLUSIONS: Extraarticular injections of CCH for treatment of adhesive capsulitis were well tolerated and seem effective compared with exercise therapy. Future FDA-regulated clinical trials must verify CCH injection therapy for adhesive capsulitis. LEVEL OF EVIDENCE: Level II, therapeutic study.

Structures of three polycystic kidney disease-like domains from Clostridium histolyticum collagenases ColG and ColH.

Clostridium histolyticum collagenases ColG and ColH are segmental enzymes that are thought to be activated by Ca(2+)-triggered domain reorientation to cause extensive tissue destruction. The collagenases consist of a collagenase module (s1), a variable number of polycystic kidney disease-like (PKD-like) domains (s2a and s2b in ColH and s2 in ColG) and a variable number of collagen-binding domains (s3 in ColH and s3a and s3b in ColG). The X-ray crystal structures of Ca(2+)-bound holo s2b (1.4 Šresolution, R = 15.0%, Rfree = 19.1%) and holo s2a (1.9 Šresolution, R = 16.3%, Rfree = 20.7%), as well as of Ca(2+)-free apo s2a (1.8 Šresolution, R = 20.7%, Rfree = 27.2%) and two new forms of N-terminally truncated apo s2 (1.4 Šresolution, R = 16.9%, Rfree = 21.2%; 1.6 Šresolution, R = 16.2%, Rfree = 19.2%), are reported. The structurally similar PKD-like domains resemble the V-set Ig fold. In addition to a conserved ß-bulge, the PKD-like domains feature a second bulge that also changes the allegiance of the subsequent ß-strand. This ß-bulge and the genesis of a Ca(2+) pocket in the archaeal PKD-like domain suggest a close kinship between bacterial and archaeal PKD-like domains. Different surface properties and indications of different dynamics suggest unique roles for the PKD-like domains in ColG and in ColH. Surface aromatic residues found on ColH s2a-s2b, but not on ColG s2, may provide the weak interaction in the biphasic collagen-binding mode previously found in s2b-s3. B-factor analyses suggest that in the presence of Ca(2+) the midsection of s2 becomes more flexible but the midsections of s2a and s2b stay rigid. The different surface properties and dynamics of the domains suggest that the PKD-like domains of M9B bacterial collagenase can be grouped into either a ColG subset or a ColH subset. The conserved properties of PKD-like domains in ColG and in ColH include Ca(2+) binding. Conserved residues not only interact with Ca(2+), but also position the Ca(2+)-interacting water molecule. Ca(2+) aligns the N-terminal linker approximately parallel to the major axis of the domain. Ca(2+) binding also increases stability against heat and guanidine hydrochloride, and may improve the longevity in the extracellular matrix. The results of this study will further assist in developing collagen-targeting vehicles for various signal molecules.

Collagenase Clostridium histolyticum for the Treatment of Peyronie's Disease: The Development of This Novel Pharmacologic Approach.

INTRODUCTION: The conception of collagenase Clostridium histolyticum (CCH) as treatment for Peyronie's disease (PD) was a vital first step in providing a nonsurgical, minimally invasive FDA-approved treatment for men with PD. AIM: To review the origins, clinical research history, and ultimately FDA approval of collagenase as PD treatment. METHODS: A PubMed search using (Peyronie's or Peyronie) AND collagenase, and limited to clinical research studies, returned nine papers that were examined in the current review. RESULTS: Collagenase as a PD treatment arose in response to a lack of effective nonsurgical treatments and the incomplete understanding of underlying PD etiology. Awareness of dense collagen in PD scarring and parallel initial exploration of collagenase to treat herniated lumbar discs coincided with and inspired laboratory-based investigation of collagenase effects on excised PD plaque tissue. The foundational conceptual work and the critical development of purified injectable collagenase allowed the pursuit of clinical studies. Progression of clinical studies into large-scale robust trials culminated in two important outcomes: development of the first validated, PD-specific measure of psychosexual function, the Peyronie's Disease Questionnaire, and the first FDA-approved treatment for PD. CONCLUSIONS: Collagenase therapy began as an attempt to modify the structure of PD-related tunica albuginea scarring, despite the lack of a fundamental understanding of the scar's origin. If we wish to advance PD treatment beyond this first effective step, the future needs to bring us full circle to the starting point: We need a greater understanding of the control of collagen deposition and wound healing in men with PD.

Surgical correction of persistent Peyronie's disease following collagenase clostridium histolyticum treatment.

INTRODUCTION: Collagenase clostridium histolyticum (CCH) is an Food and Drug Administration-approved intralesional injection for treatment of Peyronie's disease (PD) that has been shown to reduce penile curvature deformity and PD symptom bother in phase 2b and phase 3 placebo-controlled clinical trials. For some patients, nonsurgical treatment with CCH may not sufficiently improve penile curvature, and surgical correction may be pursued following CCH therapy. AIM: This study aims to examine intraoperative and postsurgical outcomes of surgical correction of persistent penile curvature in patients with PD who had previously received CCH. METHODS: Retrospective chart review was used to identify patients with PD who had received CCH intralesional injection within either the phase 2b or phase 3 CCH clinical trials and then underwent surgical correction due to remaining penile curvature. Surgical techniques used were partial plaque excision and grafting (PEG) and/or tunica albuginea plication (TAP). MAIN OUTCOME MEASURES: Primary assessments included pre- and postsurgery penile curvature, erectile rigidity, stretched penile length, intraoperative time, and occurrence of adverse events. RESULTS: Seven men were identified who underwent surgical straightening with TAP or PEG following CCH treatment. Mean number of days from the final CCH injection to surgery was 182 (standard deviation 118; median 127 days). Average penile curvature prior to surgical straightening was 58°. No anatomical difficulties or complications secondary to the effects of prior CCH treatment occurred during surgery. Intraoperative time was representative of standard TAP and PEG surgeries (range 88-146 minutes). All men reported penile curvature <20° postsurgery. One patient experienced a postsurgery subgraft hematoma that required aspiration. There were no postsurgery reports of decreased penile sexual sensation and no occurrence of vascular compromise or decreased penile rigidity. CONCLUSION: This initial case series supports the hypothesis that prior CCH treatment is not a contraindication to PEG or TAP surgery in the treatment of penile curvature in patients with PD.

Clinical safety and effectiveness of collagenase clostridium histolyticum injection in patients with Peyronie's disease: a phase 3 open-label study.

INTRODUCTION: Collagenase clostridium histolyticum (CCH; Xiaflex, Auxilium Pharmaceuticals, Inc., Chesterbrook, PA, USA) is a Food and Drug Administration-approved, intralesional treatment for Peyronie's disease (PD). AIM: The aim of this study was to assess the safety and effectiveness of CCH in the treatment of PD. METHODS: This phase 3, open-label study enrolled subjects who were CCH-naïve, were enrolled in a previous pharmacokinetic study, or had received placebo in an earlier phase 2 CCH study. Each treatment cycle included two intralesional injections of CCH 0.58 mg, approximately 24-72 hours apart, and plaque modeling 24-72 hours after the second injection of each cycle. The treatment cycle was repeated after 6 weeks for ≤4 treatment cycles. MAIN OUTCOME MEASURES: The co-primary end points were the mean percent change in penile curvature deformity and the mean improvement in PD bother score (range 0-16) from baseline to week 36. RESULTS: Of the 347 subjects treated with ≥1 injection, 238 had both a penile curvature measurement and a Peyronie's Disease Questionnaire response at baseline and ≥1 subsequent time point. Mean baseline penile curvature deformity was 53.0° and mean PD symptom bother was 7.3. Statistically significant mean improvements from baseline to week 36 were observed in both penile curvature deformity (34.4% [95% confidence interval {CI}, 31.2%, 37.6%]) and PD symptom bother score (3.3 [95% CI, 2.8, 3.7]). Most adverse events (AEs) were mild or moderate in severity and local to the penis. There were three serious treatment-related AEs, two penile hematomas and one corporal rupture; all resolved with treatment. CONCLUSIONS: Potentially clinically meaningful and statistically significant improvements in penile curvature deformity and PD symptom bother scores were observed with intralesional injection of CCH compared with baseline in men with PD. CCH was generally well tolerated, with AEs primarily transient and local to injection site. In conjunction with previous studies, the results of this open-label study support the use of CCH in the treatment of PD.

Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate.

Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients.

Efficacy and safety of collagenase clostridium histolyticum in the treatment of proximal interphalangeal joints in dupuytren contracture: combined analysis of 4 phase 3 clinical trials.

PURPOSE: To examine the results of proximal interphalangeal (PIP) joint contractures from 4 phase 3 clinical trials of collagenase clostridium histolyticum (CCH) injection for Dupuytren contracture. METHODS: Patients enrolled in Collagenase Option for Reduction of Dupuytren I/II and JOINT I/II with one or more PIP joint contractures (20° to 80°) received CCH 0.58 mg/0.20 mL or placebo (Collagenase Option for Reduction of Dupuytren I/II only) injected directly into a palpable cord. The percentage of PIP joints achieving clinical success (0° to 5° of full extension), clinical improvement (50% or more reduction in baseline contracture), and range of motion improvement at 30 days after the first and last CCH injections was assessed. The PIP joint contractures were classified into low (40° or less) and high (more than 40°) baseline severity. Adverse events were recorded. RESULTS: A total of 506 adults (mean age, 63 ± 10 y; 80% male) received 1,165 CCH injections in 644 PIP joint cords (mean, 1.6 injections/cord). Most patients (60%) received 1 injection, with 24%, 16%, and 1% receiving 2, 3, and 4 injections, respectively. Clinical success and clinical improvement occurred in 27% and 49% of PIP joints after one injection and in 34% and 58% after the last injection. Patients with lower baseline severity showed greater improvement and response was comparable between fingers, as were improvements in range of motion. Adverse events occurring in more than 10% of patients were peripheral edema (58%), contusion (38%), injection site hemorrhage (23%), injection site pain (21%), injection site swelling (16%), and tenderness (13%). This incidence was consistent with data reported in phase 3 trials. Two tendon ruptures occurred. No further ruptures occurred after a modified injection technique was adopted. CONCLUSIONS: Collagenase clostridium histolyticum was effective and well tolerated in the short term in patients with Dupuytren PIP joint contractures. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic II.

The Efficacy and Safety of Concurrent Collagenase Clostridium Histolyticum Injections for 2 Dupuytren Contractures in the Same Hand: A Prospective, Multicenter Study.

PURPOSE: To evaluate efficacy and safety of concurrent administration of 2 collagenase clostridium histolyticum (CCH) injections to treat 2 joints in the same hand with Dupuytren fixed flexion contractures (FFCs). METHODS: Patients with 2 or more contractures in the same hand caused by palpable cords participated in a 60-day, multicenter, open-label, phase 3b study. Two 0.58 mg CCH doses were injected into 1 or 2 cords in the same hand (1 injection per affected joint) during the same visit. Finger extension was performed approximately 24, 48, or 72 or more hours later. Changes in FFC and range of motion, incidence of clinical success (FFC ≤ 5°), and adverse events (AEs) were summarized. RESULTS: The study enrolled 715 patients (725 treated joint pairs), and 714 patients (724 joint pairs) were analyzed for efficacy. At day 31, mean total FFC (sum of 2 treated joints) decreased 74%, from 98° to 27°. Mean total range of motion increased from 90° to 156°. The incidence of clinical success was 65% in metacarpophalangeal joints and 29% in proximal interphalangeal joints. Most treatment-related AEs were mild to moderate, resolving without intervention; the most common were swelling of treated extremity, contusion, and pain in extremity. The incidence of skin lacerations was 22% (160 of 715). Efficacy and safety were similar regardless of time to finger extension. CONCLUSIONS: Collagenase clostridium histolyticum can be used to effectively treat 2 affected joints concurrently without a greater risk of AEs than treatment of a single joint, with the exception of skin laceration. The incidence of clinical success in this study after 1 injection per joint was comparable to phase 3 study results after 3 or more injections per joint. Two concurrent CCH injections may allow more rapid overall treatment of multiple affected joints, and the ability to vary the time between CCH injection and finger extension may allow physicians and patients greater flexibility with scheduling treatment.

Collagenase treatment of Dupuytren's contracture using a modified injection method: a prospective cohort study of skin tears in 164 hands, including short-term outcome.

BACKGROUND AND PURPOSE: Treatment of Dupuytren's contracture (DC) with collagenase Clostridium histolyticum (CCH) consists of injection followed by finger manipulation. We used a modified method, injecting a higher dose than recommended on the label into several parts of the cord, which allows treatment of multiple joint contractures in 1 session and may increase efficacy. We studied the occurrence of skin tears and short-term outcome with this procedure. PATIENTS AND METHODS: We studied 164 consecutive hands with DC, palpable cord, and extension deficit of ≥ 20º in the metacarpophalangeal (MCP) and/or proximal interphalangeal (PIP) joint (mean patient age 70 years, 82% men). A hand surgeon injected all the content of 1 CCH vial (approximately 0.80 mg) into multiple spots in the cord and performed finger extension under local anesthesia after 1 or 2 days. A nurse recorded skin tears on a diagram and conducted a standard telephone follow-up within 4 weeks. A hand therapist measured joint contracture before injection and at a median of 23 (IQR: 7-34) days after finger extension. RESULTS: A skin tear occurred in 66 hands (40%). The largest diameter of the tear was ≤ 5 mm in 30 hands and > 10 mm in 14 hands. Hands with skin tear had greater mean pretreatment MCP extension deficit than those without tear: 59º (SD 26) as opposed to 32º (SD 23). Skin tear occurred in 21 of 24 hands with MCP contracture of ≥ 75º. All tears healed with open-wound treatment. No infections occurred. Mean improvement in total (MCP + PIP) extension deficit was 55º (SD 28). INTERPRETATION: Skin tears occurred in 40% of hands treated with collagenase injections, but only a fifth of them were larger than 1 cm. Tears were more likely in hands with severe MCP joint contracture. All tears healed without complications. Short-term contracture reduction was good.

Structural basis for activity regulation and substrate preference of clostridial collagenases G, H, and T.

Clostridial collagenases are among the most efficient enzymes to degrade by far the most predominant protein in the biosphere. Here we present crystal structures of the peptidases of three clostridial collagenase isoforms (ColG, ColH, and ColT). The comparison of unliganded and liganded structures reveals a quaternary subdomain dynamics. In the unliganded ColH structure, this globular dynamics is modulated by an aspartate switch motion that binds to the catalytic zinc. We further identified a calcium binding site in proximity to the catalytic zinc. Both ions are required for full activity, explaining why calcium critically affects the enzymatic activity of clostridial collagenases. Our studies further reveal that loops close to the active site thus serve as characteristic substrate selectivity filter. These elements explain the distinct peptidolytic and collagenolytic activities of these enzymes and provide a rational framework to engineer collagenases with customized substrate specificity as well as for inhibitor design.

Development of a Förster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity.

Due to their efficiency in the hydrolysis of the collagen triple helix, Clostridium histolyticum collagenases are used for isolation of cells from various tissues, including isolation of the human pancreatic islets. However, the instability of clostridial collagenase I (Col G) results in a degraded Col G that has weak collagenolytic activity and an adverse effect on islet isolation and viability. A Förster resonance energy transfer triple-helical peptide substrate (fTHP) has been developed for selective evaluation of bacterial collagenase activity. The fTHP [sequence: Gly-mep-Flp-(Gly-Pro-Hyp)4-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)4-NH2] had a melting temperature (Tm) of 36.2°C and was hydrolyzed efficiently by bacterial collagenase (k(cat)/K(M)=25,000s(-1)M(-1)) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The fTHP bacterial collagenase assay allows for rapid and specific assessment of enzyme activity toward triple helices and, thus, potential application for evaluating the efficiency of cell isolation by collagenases.