Asunto(s)
Bolsa Sinovial/efectos de los fármacos , Bursitis/tratamiento farmacológico , Clostridium histolyticum/enzimología , Colagenasa Microbiana/administración & dosificación , Articulación del Hombro/efectos de los fármacos , Dolor de Hombro/tratamiento farmacológico , Distinciones y Premios , Fenómenos Biomecánicos , Bolsa Sinovial/fisiopatología , Bursitis/diagnóstico , Bursitis/fisiopatología , Humanos , Inyecciones Intralesiones , Colagenasa Microbiana/efectos adversos , Colagenasa Microbiana/aislamiento & purificación , Ensayos Clínicos Controlados Aleatorios como Asunto , Recuperación de la Función , Articulación del Hombro/fisiopatología , Dolor de Hombro/diagnóstico , Dolor de Hombro/fisiopatología , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Distinciones y Premios , Bolsa Sinovial/efectos de los fármacos , Bursitis/tratamiento farmacológico , Clostridium histolyticum/enzimología , Colagenasa Microbiana/administración & dosificación , Articulación del Hombro/efectos de los fármacos , Dolor de Hombro/tratamiento farmacológico , Adulto , Fenómenos Biomecánicos , Bolsa Sinovial/diagnóstico por imagen , Bolsa Sinovial/fisiopatología , Bursitis/diagnóstico , Bursitis/fisiopatología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inyecciones Intralesiones , Masculino , Colagenasa Microbiana/efectos adversos , Colagenasa Microbiana/aislamiento & purificación , Persona de Mediana Edad , Dimensión del Dolor , Rango del Movimiento Articular , Recuperación de la Función , Articulación del Hombro/diagnóstico por imagen , Articulación del Hombro/fisiopatología , Dolor de Hombro/diagnóstico , Dolor de Hombro/fisiopatología , Factores de Tiempo , Resultado del Tratamiento , Ultrasonografía Intervencional , Estados UnidosRESUMEN
Collagenase Clostridium Histolyticum (CCH) (Xiaflex(®), Xiapex(®)) intralesional injection is a mixture of class I (AUX-I) and class II (AUX-II) clostridial collagenases. It is indicated for the treatment of adult men with Peyronie's disease with a palpable plaque and curvature deformity of ≥ 30° at the start of therapy. This article reviews the efficacy and tolerability of CCH in this indication and briefly summarizes its pharmacology. CCH treatment significantly improved penile curvature deformity and reduced patient-reported bother associated with Peyronie's disease in the 52-week, double-blind, phase III IMPRESS I and II studies. Treatment benefit with CCH was also seen in 36-week, open-label studies, providing further support for its efficacy. CCH was generally well tolerated in patients with Peyronie's disease, with most treatment-related adverse events being of mild or moderate severity. Serious treatment-related adverse events (penile haematoma or corporal ruptures) were reported in <1% of CCH recipients in clinical studies. Although further studies assessing the long-term effects of CCH intralesional injection are needed, current evidence indicates that this is a minimally invasive, effective and generally well tolerated treatment option for patients with Peyronie's disease.
Asunto(s)
Clostridium histolyticum/enzimología , Colagenasa Microbiana/uso terapéutico , Induración Peniana/tratamiento farmacológico , Pene/efectos de los fármacos , Animales , Colágeno/metabolismo , Interacciones Farmacológicas , Humanos , Hidrólisis , Inyecciones Intralesiones , Masculino , Colagenasa Microbiana/administración & dosificación , Colagenasa Microbiana/efectos adversos , Colagenasa Microbiana/aislamiento & purificación , Induración Peniana/diagnóstico , Induración Peniana/metabolismo , Pene/metabolismo , Pene/patología , Resultado del TratamientoRESUMEN
Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen) on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.
Asunto(s)
Alanina/química , Colágeno Tipo I/química , Colagenasa Microbiana/química , Péptidos/química , Ingeniería de Proteínas , Secuencia de Aminoácidos , Clostridium histolyticum/química , Clostridium histolyticum/enzimología , Colágeno Tipo I/síntesis química , Hidrólisis , Colagenasa Microbiana/antagonistas & inhibidores , Colagenasa Microbiana/aislamiento & purificación , Datos de Secuencia Molecular , Péptidos/síntesis química , Estabilidad Proteica , Estructura Secundaria de Proteína , EstereoisomerismoRESUMEN
BACKGROUND: Islet isolation currently requires collagenase, neutral protease and other components. Thermolysin (TL) from Bacillus thermoproteolyticus is the gold standard neutral protease. However, we speculated that neutral protease derived from Clostridium histolyticum (Ch; ChNP) would be biologically superior for islet isolation. Tryptic-like activity has also been reported to be important. Therefore, we focused on clostripain (CP), since it is one of the main proteases in Clostridium histolyticum which possesses tryptic-like activity. We then examined the synergistic effects of highly purified ChNP and CP on rat islet isolation. METHODS: The same amount of collagenase was used in all four groups (TL, ChNP, TL+CP and ChNP+CP; n = 12/group). The efficiency was evaluated by the islet yield and function. An immunohistochemical analysis, in vitro digestion assay for each enzyme component and evaluation of the activation of endogenous exocrine proteases during islet isolation were also performed. RESULTS: The islet yield of the TL group was significantly higher than that of the ChNP group (P < 0.01). The islet yield was dose dependently increased in the ChNP+CP group, but was decreased in the TL + CP group. The islet yield in the ChNP + CP group was significantly higher than that in the TL group, but their islet function was similar. Different specificities for laminin, especially laminin-511, were observed in the TL, ChNP, and CP groups. CONCLUSIONS: Clostripain had a strong synergistic effect with ChNP, but not with TL. Therefore, ChNP and CP, in combination with collagenase derived from the same bacteria, may effectively increase the isolation efficiency without affecting the quality of islets.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridium histolyticum/enzimología , Cisteína Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Islotes Pancreáticos/enzimología , Recolección de Tejidos y Órganos/métodos , Animales , Proteínas Bacterianas/genética , Clostridium histolyticum/genética , Cisteína Endopeptidasas/genética , Endopeptidasas/genética , Colagenasa Microbiana/aislamiento & purificación , Colagenasa Microbiana/metabolismo , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/metabolismo , Termolisina/metabolismo , Factores de TiempoRESUMEN
Purified Clostridium histolyticum collagenase (CHC), an Food and Drug Administration-approved drug that does not affect nerves or blood vessels, was assessed as a potential treatment for fibroids in this proof-of-principle study. Fibroids (1-4 cm, capsules intact) and myometrial specimens from 5 patients were injected posthysterectomy with CHC or vehicle containing methylene blue and incubated for 24 hours. Percentage of collagen-stained area was estimated using Masson-Trichrome-stained slides. Collagen fibers were observed with picrosirius staining. Tissue stiffness was objectively measured by rheometry (complex shear modulus [Pa]). Injected materials spread within and beyond fibroids as visualized by methylene blue. Of the 8 treated fibroids, 7 were softened and some contained liquefied centers. Relative percentage of collagen-stained area (mean ± standard deviation) in treated fibroids (38 ± 12%; n = 7) was less than that in control fibroids (66 ± 17%; n = 5). Treated myometrium (40 ± 30% collagen; n = 3) was similar to control myometrium (53 ± 8%; n = 2). Picrosirius staining demonstrated loss of collagen fibers in treated fibroids. Treated fibroids were less stiff (3630 ± 2410 Pa; n = 4) than controls (5930 ± 830 Pa; n = 4). Treated and control myometrium had similar stiffness (2149 ± 927 Pa; n = 3 and 3314 ± 494 Pa; n = 2, respectively) and were never liquefied. In conclusion, injections of CHC into encapsulated fibroids are feasible and effective. Heterogeneity of collagen types and quantities within individual fibroids may contribute to varied responses and need additional investigation. Further study of collateral effects on myometrium is indicated. Injected CHC has potential for treatment of fibroids.
Asunto(s)
Clostridium histolyticum/enzimología , Leiomioma/tratamiento farmacológico , Colagenasa Microbiana/administración & dosificación , Miometrio/efectos de los fármacos , Neoplasias Uterinas/tratamiento farmacológico , Módulo de Elasticidad , Estudios de Factibilidad , Femenino , Colágenos Fibrilares/metabolismo , Humanos , Inyecciones Intralesiones , Leiomioma/metabolismo , Leiomioma/patología , Colagenasa Microbiana/aislamiento & purificación , Miometrio/metabolismo , Miometrio/patología , Técnicas de Cultivo de Tejidos , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
BACKGROUND: Our purpose was to analyze and compare the use of direct health resources and costs generated in the treatment of Dupuytren's contracture using two different techniques: subtotal fasciectomy and infiltration with Collagenase Clostridium Histolyticum (CCH) in regular clinical practice at the Orthopedic and Traumatology Surgery (OTS) Department at the Hospital de Denia (Spain). METHODS: Observational, retrospective study based on data from the computerized clinical histories of two groups of patients- those treated surgically using a one or two digit subtotal fasciectomy technique (FSC) and those treated with CCH infiltration, monitored in regular clinical practice from February, 2009 to May, 2012. Demographic (age, sex), clinical (number of digits affected and which ones) and use of resources (hospitalizations, medical visits, tests and drugs) data were collected. Resource use and associated costs, according to the hospital's accounting department, were compared based on the type of treatment from Spain's National Health Service. RESULTS: 91 patients (48 (52.8%) in the FSC group) were identified. The average age and number of digits affected was 65.9 (9.2) years and 1.33 (0.48) digits affected in the FSC group, and 65.1 (9.7) years and 1.16 (0.4) digits in the CCH group.Overall, the costs of treating Dupuytren's disease with subtotal FSC amount to 1,814 for major ambulatory surgery and 1,961 with hospital stay including admission, surgical intervention (904), examinations, dressings and physiotherapy. As to collagenase infiltration, costs amount to 952 (including minor surgery admission, vial with product, office examination and dressings). Finally, comparing total costs for treatments, a savings of 388 is estimated in favor of CCH treatment in the best-case scenario (patient under MAS system with no need for physiotherapy) and 1,008 in the worst-case scenario (patient admitted to hospital needing subsequent physiotherapy), implying a savings of 29% and 51%, respectively. CONCLUSIONS: This study demonstrates that treating patients with DC by injection with CCH at the OTS department of the Hospital de Denia generates a total savings of 29% and 51% (388 and 1008) compared with fasciectomy at the time of treatment. Long term evolution of CCH treatment is uncertain and the recurrence rate unknown.
Asunto(s)
Clostridium histolyticum/enzimología , Costos de los Medicamentos , Contractura de Dupuytren/economía , Contractura de Dupuytren/terapia , Fasciotomía , Recursos en Salud/economía , Costos de Hospital , Unidades Hospitalarias/economía , Colagenasa Microbiana/economía , Colagenasa Microbiana/uso terapéutico , Procedimientos Ortopédicos/economía , Ortopedia/economía , Centros Traumatológicos/economía , Anciano , Ahorro de Costo , Análisis Costo-Beneficio , Contractura de Dupuytren/diagnóstico , Femenino , Recursos en Salud/estadística & datos numéricos , Humanos , Tiempo de Internación/economía , Masculino , Colagenasa Microbiana/aislamiento & purificación , Persona de Mediana Edad , Modelos Económicos , Programas Nacionales de Salud/economía , Visita a Consultorio Médico/economía , Modalidades de Fisioterapia/economía , Estudios Retrospectivos , España , Factores de Tiempo , Resultado del TratamientoRESUMEN
BACKGROUND: The optimal enzyme blend that maximizes human islet yield for transplantation remains to be determined. In this study, we evaluated eight different enzyme combinations (ECs) in an attempt to improve islet yield. The ECs consisted of purified, intact or truncated class 1 (C1) and class 2 (C2) collagenases from Clostridium histolyticum (Ch), and neutral protease (NP) from Bacillus thermoproteolyticus rokko (thermolysin) or Ch (ChNP). METHODS: We report the results of 249 human islet isolations, including 99 deceased donors (research n=57, clinical n=42) and 150 chronic pancreatitis pancreases. We prepared a new enzyme mixture (NEM) composed of intact C1 and C2 collagenases and ChNP in place of thermolysin. The NEM was first tested in split pancreas (n=5) experiments and then used for islet autologous (n=21) and allogeneic transplantation (n=10). Islet isolation outcomes from eight different ECs were statistically compared using multivariate analysis. RESULTS: The NEM consistently achieved higher islet yields from pancreatitis (P<0.003) and deceased donor pancreases (P<0.001) than other standard ECs. Using the NEM, islet products met release criteria for transplantation from 8 of 10 consecutive pancreases, averaging 6510 ± 2150 islet equivalent number/gram (IEQ/g) pancreas and 694,681 ± 147,356 total IEQ/transplantation. In autologous isolation, the NEM yielded more than 200,000 IEQ from 19 of 21 pancreases (averaging 422,893 ± 181,329 total IEQ and 5979 ± 1469 IEQ/kg recipient body weight) regardless of the severity of fibrosis. CONCLUSIONS: A NEM composed of ChNP with CIzyme high intact C1 collagenase recovers higher islet yield from deceased and pancreatitis pancreases while retaining islet quality and function.
Asunto(s)
Bacillaceae/enzimología , Clostridium histolyticum/enzimología , Endopeptidasas/metabolismo , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/metabolismo , Colagenasa Microbiana/metabolismo , Termolisina/metabolismo , Recolección de Tejidos y Órganos/métodos , Adulto , Distribución de Chi-Cuadrado , Endopeptidasas/aislamiento & purificación , Femenino , Humanos , Masculino , Colagenasa Microbiana/aislamiento & purificación , Persona de Mediana Edad , Minnesota , Análisis Multivariante , Análisis de Regresión , Termolisina/aislamiento & purificación , Supervivencia Tisular , Trasplante Autólogo , Trasplante Homólogo , Resultado del Tratamiento , Adulto JovenRESUMEN
Elevated interstitial fluid pressure (IFP) in a tumor is a barrier to tumor accumulation of systemic delivery of nanocarriers. In this study, we investigated whether intravenous injection of type I collagenase (collagenase-1) reduced IFP in tumors and increased the accumulation and gene expression of cationic liposome/plasmid DNA complex (lipoplex) in tumors after intravenous injection into mice bearing mouse lung carcinoma LLC tumors. Collagenase-1 reduced the amount of type I collagen in the tumor, and significantly decreased IFP by 65% at 1h after injection. Therefore, collagenase-1 induced 1.5-fold higher accumulation and 2-fold higher gene expression of lipoplex in tumors after intravenous injection. These findings indicated that intravenous injection of collagenase-1 improved the accumulation of lipoplex by decreasing IFP in tumors. These results support the potential use of collagen digestion as a strategy to improve systemic gene delivery into tumors.
Asunto(s)
Carcinoma Pulmonar de Lewis/metabolismo , Clostridium histolyticum/enzimología , Colágeno Tipo I/metabolismo , Colagenasas/administración & dosificación , ADN/metabolismo , Líquido Extracelular/metabolismo , Colagenasa Microbiana/administración & dosificación , Transfección/métodos , Animales , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/terapia , Línea Celular Tumoral , Colagenasas/aislamiento & purificación , ADN/química , Femenino , Terapia Genética , Inyecciones Intravenosas , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Ratones , Ratones Endogámicos C57BL , Colagenasa Microbiana/aislamiento & purificación , Presión , Factores de Tiempo , Distribución TisularRESUMEN
Collagenase clostridium histolyticum is a novel treatment for Dupuytren's contracture, approved by the U.S. Food and Drug Administration in February 2010. Prior to its availability, surgery was the only treatment for contracture related to this disorder. Dupuytren's disease is a benign, progressive fibroproliferative disorder affecting the palms of the hands. It is characterized by the formation of collagen- rich nodules and cords, which gradually shorten by the action of myofibroblasts, resulting in finger contractures. Intralesional use of clostridial collagenase has been evaluated in a total of 1,082 patients receiving 2,630 injections during its clinical development, including 2 large prospective, randomized, double-blind, placebo-controlled trials: Collagenase Option for Reduction of Dupuytren's I (CORD I) and CORD II. Both studies showed a statistically significant reduction in contracture compared to placebo and treatment was well-tolerated with the majority of adverse events self-limited. Serious adverse events related to collagenase activity were rare. Maximal improvement was seen in patients with less severe contractures and with contractures of the metacarpophalangeal joint. This first-in-class biologic, injectable clostridial collagenase histolyticum, provides a safe, effective alternative to surgery for patients with Dupuytren's contracture.
Asunto(s)
Clostridium histolyticum/enzimología , Contractura de Dupuytren/tratamiento farmacológico , Colagenasa Microbiana/administración & dosificación , Animales , Interacciones Farmacológicas , Humanos , Inyecciones Intralesiones , Colagenasa Microbiana/efectos adversos , Colagenasa Microbiana/aislamiento & purificación , Colagenasa Microbiana/farmacocinética , Resultado del TratamientoAsunto(s)
Síndrome de Creutzfeldt-Jakob/etiología , Trasplante de Islotes Pancreáticos/efectos adversos , Animales , Bovinos , Separación Celular/métodos , Clostridium histolyticum/enzimología , Clostridium histolyticum/crecimiento & desarrollo , Síndrome de Creutzfeldt-Jakob/transmisión , Medios de Cultivo/efectos adversos , Encefalopatía Espongiforme Bovina/etiología , Encefalopatía Espongiforme Bovina/transmisión , Humanos , Colagenasa Microbiana/aislamiento & purificación , Modelos Estadísticos , Método de Montecarlo , Medición de Riesgo/estadística & datos numéricosAsunto(s)
Clostridium histolyticum/enzimología , Colagenasas/metabolismo , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Colagenasa Microbiana/metabolismo , Termolisina/metabolismo , Recolección de Tejidos y Órganos/métodos , Colagenasas/aislamiento & purificación , Femenino , Humanos , Masculino , Colagenasa Microbiana/aislamiento & purificación , Persona de Mediana Edad , Termolisina/aislamiento & purificación , Supervivencia TisularAsunto(s)
Clostridium histolyticum/enzimología , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Colagenasa Microbiana/metabolismo , Recolección de Tejidos y Órganos/métodos , Humanos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Isomerismo , Colagenasa Microbiana/aislamiento & purificación , Persona de Mediana Edad , Termolisina/metabolismo , Técnicas de Cultivo de Tejidos , Supervivencia TisularRESUMEN
IMPORTANCE OF THE FIELD: Dupuytren's disease is a non-malignant, progressive disorder of the hands that can severely limit hand function and diminish overall quality of life. With global life expectancy increasing, the prevalence of this disease appears to be increasing amongst all ethnic groups. Treatment has traditionally remained surgical with few effective, nonsurgical options. However, with the introduction of collagenase clostridium histolyticum to treat Dupuytren's contractures, physicians and surgeons may be provided with a new, office-based, non-surgical option to treat this disease. AREAS COVERED IN THIS REVIEW: The literature behind the use of collagenase to treat Dupuytren's disease; including its mechanism of action, safety, efficacy and clinical evidence behind its recent FDA approval. WHAT THE READER WILL GAIN: The latest information available on collagenase through a comprehensive review of PubMed and the websites of licensing organizations for medicinal products. TAKE HOME MESSAGE: Phase III, clinical trials on collagenase for treatment of Dupuytren's contractures have recently been completed. Meeting primary and secondary objectives, collagenase has obtained FDA approval for clinical use. Collagenase now provides a non-operative option for Dupuytren's disease. Although short-term results show that collagenase is safe and efficacious, long-term effects of repeat injections and contracture recurrence rates have yet to be examined.
Asunto(s)
Clostridium histolyticum/enzimología , Contractura de Dupuytren/tratamiento farmacológico , Colagenasa Microbiana/uso terapéutico , Humanos , Colagenasa Microbiana/efectos adversos , Colagenasa Microbiana/química , Colagenasa Microbiana/aislamiento & purificación , Resultado del TratamientoRESUMEN
BACKGROUND: After the discontinuation of the manufacturing Liberase HI because of a small potential for prion disease transmission, Roche Diagnostics (Indianapolis, IN) developed a new enzyme product (Liberase MTF [mammalian tissue free]), which is similar to Liberase HI with the exception that no mammalian tissue is used in the manufacture of the collagenase component. We report our experience using the MTF enzyme in clinical islet isolations compared with Serva NB-1 with modified enzyme delivery method. METHODS: Islets were isolated from 41 pancreata using MTF enzyme (n=17) or NB-1 enzyme (n=24). NB-1 enzymes were delivered using a modified (nonsimultaneous) enzyme delivery method whereas isolations using MTF used the standard method of simultaneous collagenase and thermolysin perfusion. Islets were purified on a COBE 2991 Cell Blood Processor and subsequently cultured. RESULTS: The average islet mass after purification was 392+/-36 x 10 islet equivalent (IE) for MTF versus 371+/-40 x 10 IE for Serva NB-1 (P=0.63). Post-IE/cm of tissue was 110+/-9 x 10 IE/cm and 91+/-11 x 10 IE/cm for MTF and NB-1, respectively (P=0.07). The isolation success rate (>400,000 IE) for MTF was 53% compared with 33% for Serva (P=0.33). CONCLUSION: We conclude that MTF may be successfully used for high-yield human islet isolation and clinical transplantation and provides similar quality islets to those derived using NB-1.
Asunto(s)
Clostridium histolyticum/enzimología , Colagenasas/metabolismo , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Colagenasa Microbiana/metabolismo , Termolisina/metabolismo , Recolección de Tejidos y Órganos/métodos , Glucemia/metabolismo , Péptido C/sangre , Colagenasas/aislamiento & purificación , Humanos , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Colagenasa Microbiana/aislamiento & purificación , Persona de Mediana Edad , Termolisina/aislamiento & purificación , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Supervivencia Tisular , Resultado del TratamientoRESUMEN
BACKGROUND: Purified tissue dissociation enzymes (TDEs) are critical to successful human islet isolation required for clinical transplantation, but little is known about the characteristics of the key enzymes-class I (C1) and class II (C2) collagenase from Clostridium histolyticum-used in these procedures. Here, we show the differences between the C1 collagenase found in purified collagenase products manufactured by three suppliers and the impact of differences in C1 between two suppliers on human islet yield. METHODS: Collagenase from Roche, Serva/Nordmark (Uetersen, Germany), and VitaCyte (Indianapolis, IN) were analyzed by analytical high-performance liquid chromatography and collagen degradation activity (CDA), an assay that preferentially detects intact C1 collagenase. Human islet isolations were performed using current standard practices. RESULTS: These studies showed that the highest amount of intact C1 that correlated with a high specific CDA (CDA unit per milligram of protein). The highest specific CDA was found in VitaCyte product followed by the Roche and Serva/Nordmark products. The products of VitaCyte were used successfully for human islet isolation (n=14) with an average final islet yield obtained was 419,100+/-150,900 islet equivalent number (IEQ) (4147+/-1759 IEQ/g pancreas). Four of these preparations were used successfully in clinical transplantation procedures. These TDEs gave significantly better results when compared with earlier data where 27 isolations were performed using Serva NB1 collagenase and NB neutral protease where the final islet yield was 217,500+/-152,400 IEQ (2134+/-1524 IEQ/g pancreas). CONCLUSIONS: These data indicate the importance of intact C1 and the use of the appropriate analytical assays to correlate biochemical characteristics of TDEs to islet quality and yield.
Asunto(s)
Clostridium histolyticum/enzimología , Colágeno/metabolismo , Colagenasas/metabolismo , Diabetes Mellitus Tipo 1/cirugía , Trasplante de Islotes Pancreáticos/métodos , Colagenasa Microbiana/metabolismo , Recolección de Tejidos y Órganos , Adulto , Animales , Resinas de Intercambio Aniónico , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Colagenasas/aislamiento & purificación , Modelos Animales de Enfermedad , Femenino , Humanos , Trasplante de Islotes Pancreáticos/normas , Masculino , Ratones , Ratones Desnudos , Colagenasa Microbiana/aislamiento & purificación , Persona de Mediana Edad , Estudios Retrospectivos , Técnicas de Cultivo de Tejidos , Recolección de Tejidos y Órganos/normasRESUMEN
The paper determines the action specificity concerning the bonding type of collagenases of Strepomyces sp. 1349 and keratinases of Streptomyces sp. 1382. Experimental data obtained evidence for the wide specificity of the obtained enzymatic drugs. It has been established that both collagenases and keratinases display high specificity in respect of the bonds made by the residues of hydrophobic amino acids. Collagenases of Streptomyces sp. 1349, as to their wide action specificity, differed from collagenase of Clostridium histolyticum described in literature, that was confirmed by the results of double immunodiffusion in agar by Ouchterloni. Preparations of streptomycete collagenase obtained by the authors did not interact with serum obtained for highly purified collagenase of C. histolyticum of the firm "Merck". Investigation of the feather keratin lysates by the fractions of keratinases 1 and 2 have shown the difference in the content of amino acids released after hydrolysis that may be determined by different specificity of the enzymes action. Cysteine in the amount of 3.8% was also found in keratin lysate of the enzymatic fraction 1, that may evidence for the capacity of the fraction 1 to break the disulphide bonds in keratin molecule.
Asunto(s)
Colagenasa Microbiana/metabolismo , Péptido Hidrolasas/metabolismo , Streptomyces/enzimología , Aminoácidos/química , Cisteína/química , Disulfuros , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Inmunodifusión , Colagenasa Microbiana/química , Colagenasa Microbiana/aislamiento & purificación , Péptido Hidrolasas/química , Péptido Hidrolasas/aislamiento & purificación , Streptomyces/genética , Especificidad por SustratoRESUMEN
The schemes of isolation and purification of collagenolytic enzymes of Streptomyces sp. 1349 and keratinolyte enzymes of Streptomyces sp. 1382, which include fractionation by ammonium sulphate separation on TSK-gels: ion-exchange chromatography on Toyopearl DEAE-650(M) and gel-filtration on Toyopearl HW-50, as well as highly efficient liquid chromatography. The purified enzyme preparations proved to be proteases of serine type (collagenase 2 and keratinases) as well as metalloproteases (collagenases 1 and 3). It has seen established that collagenases are enzymes of broad specificity, which are active in respect of proteins of both globular and fibrillar nature. And vice versa, keratinases are proteolytic enzymes of narrow specificity which hydrolyze native keratin. Molecular masses of purified enzyme preparations, from the data of SDS-PAAG are approximately 30-40 kDa (collagenases 1-3) and about 15-20 kDa (keratinases 1 and 2). It is shown that the charged aminoacid residues (about 85%) prevail in enzyme molecules. The enzymes are distinguished by pH- and thermooptima.
Asunto(s)
Colagenasa Microbiana/aislamiento & purificación , Péptido Hidrolasas/aislamiento & purificación , Streptomyces/enzimología , Sulfato de Amonio , Resinas de Intercambio Aniónico , Cromatografía Liquida , Concentración de Iones de Hidrógeno , Colagenasa Microbiana/química , Colagenasa Microbiana/metabolismo , Peso Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Polímeros , Resinas Sintéticas , Especificidad por Sustrato , TemperaturaRESUMEN
Human 92-kDa type IV collagenase/gelatinase (MMP9) has been expressed in insect cells and secreted into the cell medium via a baculovirus expression system. The expression level of the proenzyme from Trichoplusia ni cells was estimated to be > = 300 mg/L of cell medium. The recombinant protein was purified in a single step using heparin-affinity chromatography with an overall yield of ca. 70%. The purified zymogen could be activated in vitro using 4-aminophenylmercuric acetate to yield an active protease. Kinetic analysis of the activated recombinant enzyme demonstrates that this material is comparable to activated MMP9 from natural human sources. The recombinant enzyme provides a useful source of protein for a variety of biochemical and biophysical studies aimed at elucidating the structure and function of human MMP9.
Asunto(s)
Colagenasas/genética , Precursores Enzimáticos/genética , Colagenasa Microbiana/genética , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Clonación Molecular , Colagenasas/biosíntesis , Colagenasas/aislamiento & purificación , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/aislamiento & purificación , Precursores Enzimáticos/metabolismo , Vectores Genéticos , Humanos , Cinética , Metaloproteinasa 9 de la Matriz , Colagenasa Microbiana/aislamiento & purificación , Colagenasa Microbiana/metabolismo , Datos de Secuencia Molecular , Mariposas Nocturnas/citología , Mariposas Nocturnas/metabolismo , Nucleopoliedrovirus/genética , Péptidos/metabolismo , Acetato Fenilmercúrico/análogos & derivados , Acetato Fenilmercúrico/farmacología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie , Spodoptera/citología , Spodoptera/metabolismoRESUMEN
Crude Clostridium histolyticum collagenase was purified by gel filtration and fractionated by anion exchange chromatography into class I with high collagen digestion activity (CDA) and low FALGPA (2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine) hydrolysis activity (FHA), class II with low CDA and high FHA, and a fraction called class I/II with intermediate activities. The roles of these collagenase classes in rat pancreatic islet isolation were investigated. Dissociations were carried out with 360 mg of pancreatic tissue in 10 ml of buffer containing 10% (wt/vol) albumin to suppress endogenous proteolytic activity, 100 U of C. histolyticum neutral protease, and one or two purified collagenase(s). For purified nonfractionated (PNF) collagenase, 2.6 mg of enzyme containing 2.4 U CDA and 38.0 U FHA was used, and for the separate classes, comparable amounts of activity were added. PNF collagenase dissociated the tissue completely in 32 min and yielded 5.0 +/- 0.4 microliters islet tissue/g pancreas. Class I collagenase alone dissociated pancreatic tissue extremely slowly and incompletely; only a few islets were released (0.7 +/- 0.2 microliters/g pancreas). Class II collagenase alone dissociated the tissue adequately in 50 min, and a high islet yield of 5.7 +/- 0.6 microliters/g was obtained. With class I/II, a similar dissociation time (47 min) and islet yield (5.5 +/- 0.3 microliters/g) were obtained. Combining class I and class II collagenase resulted in a more rapid dissociation (32 min) and a higher islet yield (7.1 +/- 0.8 microliters/g) than that obtained with PNF collagenase (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)