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.

Phosphonate as a Stable Zinc-Binding Group for "Pathoblocker" Inhibitors of Clostridial Collagenase H (ColH).

Microbial infections are a significant threat to public health, and resistance is on the rise, so new antibiotics with novel modes of action are urgently needed. The extracellular zinc metalloprotease collagenase H (ColH) from Clostridium histolyticum is a virulence factor that catalyses tissue damage, leading to improved host invasion and colonisation. Besides the major role of ColH in pathogenicity, its extracellular localisation makes it a highly attractive target for the development of new antivirulence agents. Previously, we had found that a highly selective and potent thiol prodrug (with a hydrolytically cleavable thiocarbamate unit) provided efficient ColH inhibition. We now report the synthesis and biological evaluation of a range of zinc-binding group (ZBG) variants of this thiol-derived inhibitor, with the mercapto unit being replaced by other zinc ligands. Among these, an analogue with a phosphonate motif as ZBG showed promising activity against ColH, an improved selectivity profile, and significantly higher stability than the thiol reference compound, thus making it an attractive candidate for future drug development.

Clostridium histolyticum (AA4500) for the Treatment of Adhesive Capsulitis of the Shoulder: A Randomised Double-Blind, Placebo-Controlled Study for the Safety and Efficacy of Collagenase - Single Site Report.

BACKGROUND/HYPOTHESIS: Adhesive capsulitis of the shoulder results in pain and restricted movement of the glenohumeral joint. Hypothesis: There would be a difference in active range of movement in the affected shoulder of patients with adhesive capsulitis after receiving a series of injections of collagenase Clostridium histolyticum (CCH) compared to placebo. METHODS: This study reports the results from a single site that was part of a 321-participant, multicenter, double-blind, prospective parallel-group, randomized controlled clinical trial. Inclusion criteria: over 18 years of age, unilateral idiopathic adhesive capsulitis for >3 months, but <12 months. Exclusion criteria: recent physical therapy, injections, subacromial impingement, calcific tendonitis or glenohumeral joint arthritis in the affected shoulder. Subjects were randomized 3:1 to receive CCH 0.58 mg or placebo under ultrasound guidance. Injections were on days 1, 22, and 43. The primary outcome measure was a functional assessment of active range of movement. RESULTS: Overall, 37 patients were screened, 26 subjects were excluded, and 11 subjects were randomly assigned to the treatment group (n=9) or the control group (n=2). Both control and treatment groups showed improvement in ROM between baseline and day 95. In the treatment group, AROM improved from the baseline of 272.89° (SD 86.25) to 462.11° (SD 96.89) and the control group from 246.00° (SD 5.66) to 451.50° (SD 50.20) at day 95 with no statistical difference between groups p=0.78. Site data were in line with the whole study findings. Treatment-related adverse events at the injection site, including haematoma (bruising) and localised pain and swelling, were common. CONCLUSION: Although the participants showed improvement in function, statistical significance was neither reached in the site nor the overall study cohort. Given the adverse events and the potential risks of the procedure, we would not recommend this drug for the treatment of adhesive capsulitis of the shoulder. LEVEL OF EVIDENCE: 2, cohort from one site of RCT.

Premna odorata: Seasonal Metabolic Variation in the Essential Oil Composition of Its Leaf and Verification of Its Anti-Ageing Potential via In Vitro Assays and Molecular Modelling.

The metabolic variation in the essential oil composition of Premna odorata leaves obtained from different seasons was quantitatively and qualitatively determined employing GC/MS (Gas Chromatography coupled with Mass Spectrometry) and GC/FID (Gas chromatography equipped with flame ionization detector) techniques. It displayed the existence of 97 constituents accounting for 94.19%, 92.27%, 91.95% and 92.63% for POS (spring), POM (summer), POA (autumn) and POW (winter) whole essential oils. ß-Caryophyllene constituting the main metabolite in the oil in the different seasons. To better visualize the differences between them, GC data were exposed to chemometric analysis. A PCA (principal component analysis) score plot revealed the closeness of POS and POW. Molecular modelling on collagenase, elastase and hyaluronidase enzymes active centres shows that different compounds existing in the essential oil of Premna odorata leaves shows binding to the active sites with variable degrees that suggested its anti-ageing potential. Palmitic acid displayed the highest fitting for both the collagenase and elastase active centres in both pH-based and rule-based ionization methods with ∆G equals -78.27 and -44.77 kcal/mol, respectively; meanwhile, heptacosane showed the highest fitting score in the hyaluronidase centre with ∆G = -43.78 kcal/mol. In vitro assays consolidates the obtained modelling studies in which essential oil shows considerable anti-elastase and anti-hyaluronidase potential as evidenced by their IC50 values being 49.3 and 37.7 µg/mL, respectively; meanwhile, the essential oil of Premna odorata leaves displayed mild anti-collagenase potential. Thus, it can be concluded that Premna odorata could serve as a promising anti-ageing naturally occurring drug that could be effectively incorporated by pharmaceutical industries in cosmetics combating ageing and skin wrinkling.

N-Aryl-3-mercaptosuccinimides as Antivirulence Agents Targeting Pseudomonas aeruginosa Elastase and Clostridium Collagenases.

In light of the global antimicrobial-resistance crisis, there is an urgent need for novel bacterial targets and antibiotics with novel modes of action. It has been shown that Pseudomonas aeruginosa elastase (LasB) and Clostridium histolyticum (Hathewaya histolytica) collagenase (ColH) play a significant role in the infection process and thereby represent promising antivirulence targets. Here, we report novel N-aryl-3-mercaptosuccinimide inhibitors that target both LasB and ColH, displaying potent activities in vitro and high selectivity for the bacterial over human metalloproteases. Additionally, the inhibitors demonstrate no signs of cytotoxicity against selected human cell lines and in a zebrafish embryo toxicity model. Furthermore, the most active ColH inhibitor shows a significant reduction of collagen degradation in an ex vivo pig-skin model.

Efficacy of Collagenase Clostridium histolyticum (Xiapex®) in Patients with the Acute Phase of Peyronie's Disease.

BACKGROUND AND OBJECTIVE: Plaque formation ordinarily takes place in the acute phase of Peyronie's disease. There is no unanimous consent regarding the management of the acute phase of Peyronie's disease. The objective of this study was to evaluate the advantages of using a single intralesional injection of collagenase Clostridium histolyticum in patients with the active phase of Peyronie's disease and to assess its effect on disease progression by reducing penile curvature and ameliorating pain during sexual intercourse. METHODS: Sexually active men aged older than 18 years with the acute phase of Peyronie's disease were enrolled. All patients received treatment with a single intralesional injection of collagenase Clostridium histolyticum. The primary outcome of the study was the change in penile curvature after treatment while secondary outcomes were the change in sexual function (International Index of Erectile Function [IIEF-5]) and in the Peyronie Disease Questionnaire (PDQ) and its sub-scores, PDQ-PS (psychological symptoms), PDQ-PP (penile pain) and PDQ-BD (bother disease). RESULTS: Overall, 74 patients were enrolled. Mean penile curvature at baseline was 41.1° ± 12.2°. The mean changes before and at the 3-month evaluation in terms of penile curvature, Visual Analog Scale score at rest, and Visual Analog Scale score during intercourse were - 19.3 ± 8.4 (p < 0.0001), - 0.8 ± 1.1 (p < 0.0001) and - 3.8 ± 0.9 (p < 0.0001) with the benefit persisting also after 6 months. Moreover, improvements of mean IIEF-5 score (1.1 ± 0.9, p = 0.03; 0.9 ± 0.5, p = 0.02), PDQ-PS (- 2.7 ± 2.2; - 2.5 ± 2.0, p = 0.01), PDQ-PP (- 1.2 ± 1.6; - 1.1 ± 1.2, p = 0.02) and PDQ-BD (- 3.8 ± 3.4; - 3.5 ± 3.1, p = 0.001) were observed 3 and 6 months after the end of treatment, respectively. At the multivariable regression analysis, the time since disease onset (modelled with non-linear terms) and baseline curvature were independently associated with the degree of curvature improvement (coefficient: 0.30; 95% confidence interval 0.16-0.44) after a single intralesional injection (all p < 0.03). CONCLUSIONS: Although intralesional therapy with collagenase Clostridium histolyticum is not yet indicated for the acute phase of Peyronie's disease, these preliminary results suggest the effectiveness of this minimally invasive option by improving penile curvature and IIEF-5 and PDQ scores.

A state-of-art review on collagenase Clostridium Histolyticum and Peyronie's disease: drug profile, clinical evidence and safety outcomes.

Introduction: Collagenase clostridium histolyticum (CCH) is the first and only licensed medical treatment for men with Peyronie's disease (PD). Published literature shows intralesional CCH injection as an effective and safe minimally invasive treatment in a specific subgroup of PD patients.Areas covered: The authors discuss pharmacodynamics and pharmacokinetics as well as clinical outcomes and safety profile from major CCH studies in PD. All relevant CCH studies published in PubMed and EMBASE databases up to June 2019 were included.Expert opinion: Given the variability in treatment schedule and drug access coupled with the potential need for further treatment, strict patient selection and the use of adjunctive strategies are key determinants to maximize clinical efficacy of intralesional CCH. Furthermore, longer-term follow-up data on the clinical outcomes, safety and durability of CCH in larger multi-center studies and post-marketing surveillance data are necessary to provide a comparison to other standard PD treatment options.

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.

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.

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.

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.

Using Tongue Depressors as a Technique to Aid Cord Localization During Collagenase Injection For Dupuytren Disease.

Collagenase injections are used as a minimally invasive treatment for Dupuytren contracture of the palm and fingers, with good published success. Most complications are related to injection-site swelling, pain, and ecchymosis. However, the risk of flexor tendon and digital nerve injury can occur when injecting cords that cause proximal interphalangeal joint contractures. In addition, poor localization and incorrect injection of the solution into cords may result in an incomplete correction. We describe a technique to improve visualization and isolation of Dupuytren cords using a tongue depressor.

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.

Adverse effects associated with collagenase clostridium histolyticum in Dupuytren disease: A prospective study.

BACKGROUND: Collagenase clostridium histolyticum is now recognized as a viable treatment for Dupuytren disease. The high rate of adverse effects reported in patients continues to spark debate and raise questions about the true frequency of effects and their associated mechanisms of action. HYPOTHESIS: To investigate whether outcomes of CCH treatment are related to the number of adverse effects experienced. To evaluate short-term clinical outcomes in a series of patients. MATERIAL AND METHODS: Prospective single-center cohort study. The Primary End Point for effectiveness at 30 days was deficit of 0°-5°. Adverse effects were evaluated during CCH injection, removal of the dressing prior to finger extension, and finger extension. To investigate the relationship between adverse effects and treatment effectiveness, we analyzed the association between number of effects and clinical outcome at 30 days. RESULTS: A total of 208 injections were evaluated. The mean baseline contracture was 32.11°. Ninety-four patients (45.2%) had a mild contracture. Treatment was effective at 30 days in 194 of the injections (93.3%). The rate of effectiveness per joint was 93.5% for metacarpophalangeal joints (n=129) and 92.9% for proximal-interphalangeal joints (n=65). In total, 734 adverse effects were reported (mean, 3.53). No statistically significant associations were identified between disease severity and secondary effects. Variance analysis showed statistically significant differences in patients with severe contractures (mean, 3.91; 95% CI 3.57-4.25), and in patients with proximal-interphalangeal contractures (mean, 4.17; 95% CI 3.76-4.59). CONCLUSIONS: We found no relationship between number of adverse effects and treatment effectiveness at one month following CCH injection. LEVEL OF PROOF: IV, cohort prospective study.

Development of Collagenase Treatment for Dupuytren Disease.

Proof-of-principle, basic-science studies, using a rat-tail tendon model and surgically removed Dupuytren cords, began collagenase Clostridium histolyticum (CCH) development. Clinical studies in humans were then conducted, where the primary endpoint was reduction in contracture to within 0° to 5° of extension. Phase 2 studies, which confirmed the optimal dose of collagenase as 0.58 mg, showed injectable CCH reduced contractures in MP and PIP joints to within 0° to 5° in many joints and was well tolerated. Clinical results from phase 3 studies confirmed the efficacy and safety of injectable CCH as a viable nonsurgical intervention.

Multi-institutional Prospective Analysis of Intralesional Injection of Collagenase Clostridium Histolyticum, Tunical Plication, and Partial Plaque Excision and Grafting for the Management of Peyronie's Disease.

OBJECTIVE: To compare in a prospective nonrandomized fashion, the outcomes of collagenase clostridium histolyticum (CCH) and surgical interventions (tunical plication [TP] and partial plaque excision and grafting [PEG]) in patients with Peyronie's disease (PD). Intralesional injection of CCH is the only Food and Drug Administration-approved nonsurgical intervention for PD. MATERIALS AND METHODS: Between June 2015 and January 2017, 57 patients with PD and dorsal, dorsolateral, and/or lateral penile curvatures 30°-90° were included in this study. Patients with ventral curvature, hourglass deformity, and previous surgery for PD were excluded. All patients completed questionnaires and underwent penile measurements and penile vascular studies prior to and after completion of treatment, and were followed for at least 6 months. RESULTS: CCH was administered to 18 patients, TP performed in 14, and PEG in 25. The median follow-up for the whole cohort was 12 months (6-28). Mean changes in curvature after treatment were 23.3° (34.4%) for CCH, 72.0° (92.2%) for TP, and 71.8 ° (94.9%) for PEG, P<.001. Mean changes in International Index of Erectile Function-5 scores were +5.7 for CCH, +4.9 for TP, and +2.2 for PEG, P = .395. Mean penile length changes were -0.2 cm for CCH, -1.0 cm for TP, and +0.9 cm for PEG, P<.001. Three patients in the CCH group had bothersome residual curvature and underwent uneventful TP. CONCLUSION: Surgical options are associated with superior curvature correction. CCH is an effective and safe option for motivated patients who prefer to avoid surgery. PEG is associated with a small stretched penile length gain.

Comparative Outcomes of Dupuytren Disease Treatment.

Despite more than a hundred years of publications on Dupuytren disease, there has been a lack of consensus on definitions and outcomes until recently. Staging and classifications systems have an important historical context; however, more recently, outcomes rely on patient-reported outcomes, angular correction, and definitions of recurrence. This article reviews commonly used assessments, classifications, and staging systems for Dupuytren disease.

Complications of Treatment for Dupuytren Disease.

Dupuytren contracture is a progressive disease involving collagen within the palmar fascia. When the contracture progresses to meet specific parameters, intervention is considered and includes collagenase injection, percutaneous or open fasciotomy, or palmar fasciectomy. Complications after treatment include contracture recurrence, digital nerve injury or postoperative neurapraxia, flexor tendon injury/rupture, delayed wound healing or skin necrosis, dysvascular digit/arterial injury, hematoma, and infection. Patients with severe or recurrent Dupuytren contracture are more likely to experience complications. Patient education is paramount; one must consider the patient's goals for treatment, functional requirements, time frame for recovery, and tolerance for complications when deciding about treatment.

Bringing It All Together: A Practical Approach to the Treatment of Dupuytren Disease.

As minimally invasive options for treatment of Dupuytren contractures become increasingly widespread, it is important that the evidence is carefully evaluated and patients are informed of the risks and benefits of the options available. The authors advocate a shared decision-making process, using evidence-based medicine, to guide patients in their treatment choices. In this article, the authors present their thoughtful approach to selecting the appropriate Dupuytren treatment of patients, along with detailed, practical technical tips to avoid complications during the execution of these interventions; both collagenase injection and limited fasciectomy techniques are described in detail.