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Collagenase was injected into the ears of rabbits and the cheek-pouches of hamsters. The acute and long-term microvascular effects were studied by vital microscopy and microangiography. The enzyme was injected at three different concentrations: 120 units/ml, 600 units/ml and 3,000 units/ml. The medium (600 units/ml) and high (3,000 units/ml) concentrations induced effects on the microcirculation such as blood flow impairment and microbleedings. The magnitude of these effects was related to the concentration of the enzyme. Generally, these microvascular effects were of low magnitude as compared with other substances tested using the same experimental models.

Pancreatic distension with collagenase solution followed by stationary in vitro digestion yields large numbers of intact islets. We compared in rats two routes of collagenase injection, pancreatic ductal (PD) and portal venous (PV), for islet yield, in vitro insulin secretory capacities, and in vivo functional viability. The islet yield in the PD method (n = 11) was greater than that in the PV method (n = 8) (682 +/- 27 vs. 417 +/- 39 per pancreas, P less than 0.025). The insulin release from the PD islets in response to 16.7 mM glucose increased gradually following culture, 3.2 +/- 0.8 ng/10 islets/30 min (fresh) to 12.3 +/- 2.1 (24-hr culture). In contrast, insulin release from the PV islets increased during the first 6 hr of culture, but decreased after 24 hr in culture. Under electronmicroscopic examination, the PD islets revealed a well preserved structure with healthy endocrine cells, while the PV islets showed a dilated capillary network and distorted endocrine cell continuity. When 100 PD islets were transplanted into streptozotocin-induced diabetic B6AF1 mice (n = 8), all the recipient mice restored normoglycemia (less than 200 mg/dl) within 1-4 days following transplantation and maintained it until rejection. However, the recipient mice given 100 PV islets showed a significant delay in restoring normoglycemia, and 3 of 8 mice given 100 PV islets were still hyperglycemic on day 4 postgrafting. In summary, pancreatic ductal collagenase injection followed by stationary in vitro digestion reproducibly yields higher numbers of intact and viable islets when compared with portal venous collagenase injection, indicating the superiority of this method to portal venous injection.

Although it has been shown that keratome-sliced skin contains active adenylate cyclase systems which respond to various hormones and drugs, unequivocal proof that the epidermis contains these hormone-responsive systems is still lacking. We demonstrate in this study that "pure" epidermis obtained after either collagenase or trypsin treatment does contain the hormone-sensitive adenylate cyclase systems.

Inhibitory effects of methysergide bimaleate on the collagenase of the Clostridium histolyticum have been established. Having previously shown the inhibitory action of serotonin on this bacterial collagenase, the authors have tested methysergide bimaleate as another inhibitor. After injection in the peritoneum of the rats of methysergide bimaleate and collagenase together, lesions are minimal or absent, in contrast with the dramatic effects of collagenase alone. This shows the antagonist role of methysergide bimaleate in regard to collagenase and suggest that methysergide bimaleate reduce the collagenolysis and may elucidate the possible occurrence of fibrotic lesions after treatment of migraine by methysergide bimaleate in man.

Purified clostridial collagenase was administered intralesionally in 31 men with Peyronie's disease. Within 4 weeks of treatment 20 patients showed an objective improvement. Pain was eliminated in 13 of 14 patients with this complaint at presentation within the same 4-week period. The ability to have intercourse was restored in 3 of 4 patients with this problem. Except for a small corporeal rupture at the site of injection in 1 patient, no significant untoward effects were noted. During the mean 9.8-month followup 1 recurrence of bending was noted.

In the rat peritoneal injections of collagenase or trypsin give rise to severe lesions. In our experience 20% of the animals remain intact. The frequency of lesions increases with older and heavier subjects. Moreover 25% of the rats who remained free of lesions after a first injection of collagenase resist to a second one. This shows that they are strongly protected against the enzyme. The exact nature and location of this protective mechanism are not known.

Six different proteins varying widely in molecular weight, ribonuclease, lysostaphin, ovalbumin, penicillinase, collagenase, and Varidase were tested for their ability to induce circulating antibody formation in rabbits after repeated topical application of the proteins in a water-soluble gel vehicle. After a 12-week exposure period, significant hemagglutinin titers were noted in rabbits treated with ovalbumin, lysostaphin, or ribonuclease; markedly elevated, passive cutaneous anaphylaxis-reacting sera were obtained only from collagenase- or lysostaphin-treated animals. Precipitin antibodies as evidenced by gel diffusion were also found in sera from collagenas- and lysostaphin-treated animals. Topical application of penicillinase was only marginally effective and Varidase was totally ineffective in elicting a positive circulating antibody response. In all cases, topical application of proteins for periods in excess of 3 weeks was required for induction of circulating antibody formation.

An experimental model is described in which collagenase in different concentrations and volumes were applied epidurally or intrathecally in the rabbit lumbar spine. This made it possible to study the tissue effects in a situation similar to that of collagenase leaking from a disc or accidental epidural or intrathecal injection at chemonucleolysis. Epidurally applied collagenase, in higher concentration, caused a local thinning of the dura. This effect was reduced at lower concentrations and volumes. Intrathecally injected collagenase, even in small amounts, caused intrathecal hemorrhage and acute paraplegia in the hind limbs. Therefore, in the clinical situation, intrathecal injection of collagenase must be avoided.

The mixed results of two studies on intradiscal therapy with collagenase versus chymopapain are presented. The first study was performed from January 1983 to March 1984 and consisted of 71 patients treated with collagenase injection (600 ABC units) and 93 patients treated with chymopapain injection (4,000 units) into lower lumbar discs. The second study was started in May 1985 and ended December 1985. The results of 41 patients injected with chymopapain and 45 patients injected with collagenase (400 ABC units) are reported. The overall success rate after 3 months was 69%/63% for chymopapain/high-dose collagenase and 73%/71% for chymopapain/low-dose collagenase and 75%/72% after 6 months for chymopapain/high-dose collagenase. Eighteen percent of the chymopapain-treated patients and 21% of the collagenase-treated patients of the first study had to be operated on within 6 months and 12% of chymopapain patients and 29% of collagenase patients within 3 months in the second study. Six of the 134 patients who had chymopapain treatment had slight allergic reactions. Patients who had collagenase treatment had no allergic reactions under the same regimen of systemic prophylactic measures. Patients who had high-dose collagenase injections suffered significantly more from postinjectional pseudoradicular and low-back pain in the first 3 months. In the first study, no permanent neurologic complications occurred. Two patients in the low-dose collagenase group developed cauda equina syndromes in the 2 weeks after injection because of large extruded disc fragments.

The authors report a consecutive group of 95 patients suffering from chronic ulceration treated by a mixture of Collagenase Clostridiopeptidase A and 1% Chloramphenicol. Some cases involved skin only, some affected skin and muscle, and some also affected bone. The treatment was successful in all cases. The mixture acts by inducing debridement and cleansing of the necrotic areas and stimulating granulation tissue formation. This is followed by epithelialisation. At the same time the vitality of the deep tissues is maintained, even when it involves bone.

Total pancreatectomy and intrasplenic autotransplantation of islet tissue was carried out in dogs. The resected pancreas was prepared by the partial islet isolation technique of gland distention, mincing and collagenase digestion. The influence of variations in technique was studied. Using otherwise identical methods, five collagenase lots were compared. Three lots were effective, resulting in successful transplants in 23 of 26 dogs. Two ineffective lots produced no successes in six dogs (p < 0.001). When minced particle size was varied, particles which would pass freely through a 16 gauge needle resulted in no successes in six dogs. With a larger particle size that would pass freely only through a 15 gauge needle, 15 of 15 transplants were successful (p < 0.001). When tissue was minced by hand, 8 of 11 transplants were successful, compared with 15 of 15 when mechanical mincing was used. With variation in collagenase concentration, success occurred in four of six using 300 units of collagenase/ml of minced tissue, in four of six using 600 units and three of six using 1,200 units. It is concluded that minced particle size and collagenase lot are critical variables in this technique of islet transplantation. Mincing method and concentration of collagenase do not appear to be important variables.

Rheumatoid synovial collagenase was prepared from tissue cultures from rheumatoid arthritis patients, obtained after synovectomy. Alpha 2-macroglobulin was isolated from human plasma and complexed with collagenase or trypsin. Formation of both types of complexes was proven by sodium dodecyl sulfate and rate electrophoresis. Normal rabbits were injected intraarticularly into the right knee, on days 0, 3, and 6, with either alpha 2-macroglobulin-collagenase or alpha 2-macroglobulin-trypsin complexes. Control injections of alpha 2-macroglobulin, trypsin, or rheumatoid synovial collagenase were applied to the left knee joint cavity. Groups of rabbits were killed 18 hours, 1 week, or 3 weeks after the last injection, and cellular exudation into synovial fluid and morphologic alterations of synovium were investigated. Joints injected with alpha 2-macroglobulin showed no synovitis, while joints injected with collagenase showed an experimental synovitis. Alpha 2-macroglobulin-proteinase complexes, however, induced a synovitis, which was more severe than that occurring after injection of proteinases only. In the early stages, synovium showed perivascular accumulation of inflammatory cells, infiltration with neutrophils, proliferation of synovial cells, and exudation of inflammatory cells into synovial fluid. Later stages were characterized by infiltration with mononuclear cells and fibroplasia.

Bone samples from the iliac crest of patients with no signs of bone disorder were treated with collagenase to remove the collagen component and so allow detailed observation of the mineral hydroxyapatite. Both polished and unpolished surfaces were studied in the scanning electron microscope and they showed that the mineral component of bone is composed of small rounded units about 10 nm across which are fused together to form larger spheroidal units roughly 100 nm in diameter. In the unpolished surfaces these 100 nm units are seen to aggregate to form columns approximately parallel to their neighbours and with numerous interconnections forming a continuous mineral phase. The polished sections also show the hydroxyapatite as a continuous phase of contiguous spheroids and the holes from which the collagen fibres were removed are clearly revealed. Lamellations in the surface are interpreted as resulting from adjacent layers of collagen fibres having orientations approximately perpendicular to each other.

An enzymatically induced irritation of the vessel wall by intravenous injection of collagenase (1.5 U per kg body weight) into rats resulted in a transient decrease of the platelet concentration in the flowing blood. This may be caused by an occurrence of inter-endothelial gaps distributed throughout the total area of the circulatory system, as could be observed by electron-microscopic studies. Subendothelial structures, at the sites of those gaps, induce a formation of multilocal microthrombi of platelets. At some gaps within the endothelial layer a deposit of fibrin could also be observed hinting at a participation of the blood coagulation system in the sealing processes.

This pilot study was designed to test the feasibility of using purified clostridial collagenase in the clinical management of Peyronie's disease. The basic properties of this agent are discussed. We studied its effect on Peyronie's plaque tissue by a quantitative in vitro assay utilising the liberation of free alpha-amino groups as an index of enzymatic collagenolysis. Tissue from three patients with Peyronie's disease was used. Tunica albuginea from a second group of three normal patients was studied in the same manner, and no selectivity for the collagen of Peyronie's plaques was identified. Utilising human pericardium as a uniform collagenous substrate, a simple dose-effect relationship was established, and the distribution characteristics of injected collagenase observed. Its effects on blood vessels and nerves in vivo was determined as well as the effects of collagenase on the histology of normal and diseased human tissue in vitro. A tentative dose for use in Peyronie's disease was established, which is discussed in light of existing toxicological data. The study was designed to test the feasibility of purified collagenase in the clinical management of Peyronie's disease. Data included detail plaque digestion and dose-effect relationships in vitro, as well as the histological effects on plaques, blood vessels, and nerves in vivo and in vitro. It is concluded that collagenase may warrant further clinical testing in the treatment of Peyronie's disease.

Twenty-nine patients with persistent, low back and sciatic pain received intradiscal collagenase at a single abnormal disk space after two months of conservative therapy and two weeks of additional bed rest. Complete pain relief was achieved in six patients (21%), notable relief in 12 (42%), moderate in six (21%), and slight in one. Four patients (14%) who obtained no improvement by enzyme injection recovered after extruded disk fragments were removed from the spinal canal during a later operation. Pain relief after collagenase injection took place gradually over a two- to three-week period and was associated with some early backache. Improvement then continued at a slower rate for two to three months. There were no adverse effects of enzyme therapy. Injected disk spaces usually showed radiological narrowing.

Electron microscopic analysis of intravitreal strands produced by the injection of autogenous fibroblasts showed thin, immature collagen after two weeks and the mature banded variety after four weeks. With the use of this intravitreal strand model, it was found that highly purified bacterial collagenase caused extensive digestion of scar tissue after incubation periods of 10, 15, and 30 minutes. There was no morphologic damage to cicatricial cellular elements or to the inner limiting membrane of the retina. A 45-minute exposure of retinas previously injured by photocoagulation to collagenase also did not result in morphologic evidence of damage. The use of collagenase as an adjunct to vitrectomy in cases of extreme vitreal scarring or retinal traction may decrease the complication rate of a procedure that is still extremely hazardous.

The authors report the case of a young female patient submitted to chemonucleolysis at L4-5 with collagenase, in whom severe tissue lesions affecting the cartilaginous end-plate, the vertebral bone, the posterior intervertebral ligament, the epidural fat, and the dura mater were verified at laminectomy, which had become necessary due to functional motor deterioration.

The current management of decubitus ulcers, factors in wound healing and the role of enzymes in treatment are discussed. The therapeutic benefits of collagenase (Santyl) ointment in 21 patients are described, supplemented by serial color photographs. Statistical evidence is provided for the conclusion that collagenase ointment is an excellent adjunct to therapy.

An additional case of epidermolysis bullosa dystrophica inversa is reported. A review of the literature shows that until now reports about probably 15 patients from seven families were published, although the first 13 patients were not investigated by electron microscope [3]. By blistering beneath the basal lamina similar to the Hallopeau-Siemens type these patients develop bullae predominantly at the trunk and the inverse sites of the axillar and genitoanal regions continuously or at close intervals starting shortly after birth. The mucous membranes of the mouth, the esophagus, and the cornea and conjunctiva of the eye are very often involved. By bacterial infection of the skin lesions the patients develop hpochrome anemia. The web-like structures in the esophagus following blistering can be successfully treated by careful dilatation because of the low tendency to scar formation. An autosomal recessive mode of inheritance seems to be most likely.