HK1076037B - Use of collagenase to facilitate guide wire crossing in total arterial occlusions - Google Patents

Description

Use of collagenase to facilitate passage of a guidewire through a total arterial occlusion

The present invention relates to the field of percutaneous interventions of arterial occlusions using local infusion of collagenase or a combination of enzymes including collagenase.

Background

Problem scope:

chronic Total Occlusion (CTO) is extremely common in patients undergoing diagnostic catheterization. It has been reported that up to 20% of patients undergoing angiography have one or more chronic total coronary occlusions¹. Balloon angioplasty is a therapeutic approach to CTO and the first success reported in 1982². In 1998, Percutaneous Coronary Intervention (PCI) has been increasing at a rate of over one million cases per year³And currently CTO accounts for approximately 10% -15% of PCI^4-7. However, due to the severe limitations of PCI in such patients, clinicians often decide to have these patients undergo bypass surgery or adhere to medical treatment (often ineffective). The appearance of one or more CTOs in vessels supplying viable areas of the myocardium was the most prominent with bypass surgery rather than PCICommon causes.

Limitation of PCI

The major limitations of PCI in CTO are the decreased success rate of treatment compared to arterial stenosis (but not complete occlusion), and the high rate of restenosis. By using stents, the restenosis problem has been ameliorated^8-10. However, during the past 20 years, even due to certain improvements in angioplasty devices such as hydrophilic guidewires^16，17The success rate of treatment has also only shown limited improvement, i.e. 50-60% from the eighties of the twentieth century^11，12The improvement is 60 to 70 percent of the nineties of the twentieth century^5，13-15. However, in stenotic but unoccluded arteries, PCI has achieved over 95% success. Indeed, for CTO, a 70% success rate may overestimate PCI, as such attempts are typically only made if a reasonable chance of success for the lesion is felt. Several lesion signatures have been identified that are predictive of the success of the procedure and influence the decision to perform angioplasty. The duration of the occlusion, which is often difficult to determine, is a primary predictor. When a reliable estimate of this could be made, it has been reported in two trials that newly occurring coronary occlusion (i.e. duration < 3 months) was successfully dilated in a proportion of 74% and 89% (of the cases tested)^5，13. However, if the duration of occlusion exceeds 3 months, the success rate drops to 59% and 45%, respectively. Other variables that may be predictive of surgical failure include long occlusive segment lesion length (> 15mm)^1，18，19The presence of bridging side tissue (collatoral), the conical funnel not introduced into the occlusion segment, and possibly smaller vessel size²⁰. Failure rates in absolute occlusion (no distal opacity) were higher compared to functional total occlusion (minor total occlusion with a weak late-stage advancing opacity of the distal segment, no apparent continuity)^6，19，21。

Why does PCT fail in chronic total occlusion?

Failure to pass wires through the CTO is the leading cause of PCI failure, accounting for failureOver 75% of^5，19. Compared with the conventional wire technology^28，29With new type of conductors specially designed for total occlusion, e.g. Magnum^TMConducting wire²²Low speed rotational angioplasty device^23，24And excimer laser powder wire (Prima)^TM Total Occlusion Device)^25-27Recent technological innovations have failed to improve success rates. Therefore, purely mechanical methods of designing stiffer and more powerful leads to attempt to completely occlude through fibrosis have had only limited effectiveness. Although thrombolytic therapy is effective for acute coronary occlusion, only a small number of natural chronic coronary occlusions have been treated by long-term thrombolytic infusion and have limited effectiveness^30，31This strategy has been largely abandoned. There are no other published reports of drug treatment of chronically occluded arteries to improve angioplasty outcomes.

Why should CTO be turned on?

The region of the myocardium fed by a chronic occluded artery may still be viable, particularly for the slowly-developing occlusions associated with extensive lateral organization. Myocardial ischemia is a common secondary symptom of CTO because for increased myocardial demand situations (exercise, postprandial, stress), blood flow through the lateral tissues is insufficient. Thus, for CTO, severe angina is the most common cause of attempts to adopt PCI. Insufficient blood supply to viable myocardium (referred to as "hibernating myocardium") is also a major cause of possible reversible myocardial dysfunction leading to heart failure. Furthermore, there is increasing data indicating that CTO is a precursor to poor prognosis. The two-year corrected mortality rate is reported to be higher for patients with total occlusion than for patients with secondary total occlusion³². For patients with a single vascular disease lasting an average of 4 years, sudden death rate (15%) was significantly higher in patients with CTO than in patients with advanced stenosis (3%)³³. Recent data indicate that revascularization of CTO improves left ventricular function (the major determinant of heart failure) and possible long-term mortality^34-38. Suero and colleagues confirmed, with and withoutCompared to successful CTO treatment, the 10-year survival rate was significantly improved (73.5% vs 65.1%)⁷. The survival benefit of the open artery may be due to improved electrical stability of the myocardium, reduced ventricular tachyrhythms, no late phase possibility and protection of vagal tone^53-55。

Matrix metalloproteinases were used for the experiments:

collagenase preparations have been used for in vitro cell culture experiments for a long time. These preparations separate cells from tissue by degrading the surrounding matrix, and these cells are subsequently used in cell culture. Very few reports have been made on the use of collagenase preparations for in vivo experiments. Experimental models for intracerebral hemorrhage in rats have been developed by direct systemic infusion of bacterial collagenases (types XI and VII) or a combination of collagenases and heparin into the caudate nucleus^48-50. In this model, erythrocytes aggregate near caudate nuclear vessels 10 minutes after injection, and extensive bleeding occurred 4 hours after injection, estimated to be due to degradation of parenchymal and basement membrane collagen of thin walled intracerebral vessels⁴⁹。

Kerenyi and coworkers⁵¹Several different enzymes, including collagenase, have been reported for use in rabbit atherosclerosis models. The enzymes are delivered via a dual balloon catheter, in which the two balloons are inflated and the enzymes are injected into the space between the two inflated balloons. These enzymes were left for up to 30 minutes and then the arteries were immediately removed. In this model, rabbits were fed a high cholesterol diet, resulting in the development of moderate atherosclerotic plaques that were minimally stenotic (about 30%) and thus not occluding or becoming a barrier to passage of a guidewire or angioplasty balloon catheter. The frequent release of multiple enzymes (trypsin or papain alone or in combination with collagenase) not only results in the dissolution of atherosclerotic plaques, but also causes extensive damage to the media of the arterial vasculature. Collagenase itself has little effect. These studies support the following theory: collagenase is used to degrade the extracellular matrix in the vessel wall, but care is taken to increase the doseThe possible limitation of this treatment to be performed, in particular in the thin-walled arteries.

Thus, while there is some experimental basis that supports the use of the matrix-degrading properties of collagenase to address the common atherosclerotic plaque, chronic total arterial occlusion is a unique manifestation of atherosclerotic disease. Furthermore, none of these studies addresses the unique clinical condition of chronically occluded arteries in which long segments of the artery are completely occluded and do not allow the passage of a guidewire, which is absolutely required for balloon angioplasty and stenting. In addition, in this particular condition (chronic total occlusion), the parameters of a successful treatment method such as exact enzyme composition and amount, local administration strategy, and appropriate incubation time of the enzyme before attempting to pass the guidewire are unknown. Clinical experimental studies of occluded arteries have been limited by the lack of suitable animal models. Hyo-Chun Yoon et al, Journal of interfacial Radiology January-February1996, p.65-74, discloses a porcine animal model of chronic peripheral arterial occlusion. The model presents occlusions that persist for 1 month and 3 months. There was a significant difference in the degree of internalization within the occlusion. Treatment with collagenase and urokinase infusion was attempted, but in neither animal patency was restored and attempts to pass the lead through the occlusion were unsuccessful. The role of collagenases used after urokinase treatment in degrading native blood vessels and organized thrombi is uncertain. Yoon et al summarized the results that neither urokinase nor collagenase appeared to be effective against chronic hemagglutination during the dose and time course with their porcine model.

Several patents have been published including claims for the use of collagenase or other matrix degrading enzymes to reduce the amount of atherosclerotic plaque within a blood vessel. In U.S. patent 6,025,477, Calendoff proposes targeting of enzymes to atherosclerotic plaques by combining a proenzyme [ fibroblast collagenase, gelatinase, polymorphonuclear collagenase, granulocyte collagenase, stromelysin I, stromelysin II or elastase ] with an agent (preferably a bifunctional antibody) capable of specifically binding to atherosclerotic plaques to form an agent-atherosclerotic plaque complex (column 16; lines 61-66). The zymogen also bound to the agent is then activated by cleavage and conversion to an enzyme capable of solubilizing atherosclerotic plaque components (column 42, columns 19-32).

Similar methods for targeted delivery of matrix degrading enzymes to atherosclerotic plaques are proposed in U.S. patent 5,811,248(Ditlow) and U.S. patent 6,020,181 (Bini). Ditlow proposes a method using an agent comprising a CDR-grafted antibody or fragment thereof conjugated to an enzyme capable of degrading atherosclerotic plaques (column 5, lines 11-15). Bini proposed a method in which a fibrinolytic matrix metalloproteinase was conjugated to a moiety specific for a biological targeting molecule such as an antibody, and such biological targeting molecule would be directed preferentially to the fibrinogen matrix to increase the fibrinolytic potency (column 14, lines 1-10). However, these enzyme delivery methods may only be associated with non-occluded arteries, particularly atherosclerotic disease in general. These teachings are not suitable for the particular application where angioplasty is to be performed in an occluded artery which receives only a very small amount of circulating blood flow due to total occlusion. In such cases, very high concentrations of enzyme are required, which are only possible by local delivery systems. In addition, the exact delivery parameters and amounts of these enzymes must be optimized to ensure adequate changes in the composition and material of the occlusive plaque without damaging the outer layers of the arterial wall (media and adventitia). In U.S. patent 6,020,181, Bini proposed a method for inducing degradation of fibrin (ogen) by using a fibrinolytic matrix metalloproteinase, preferably MMP-3 or MMP-7. This patent is concerned with acute arterial occlusion which contains a large amount of thrombus and fibrin and is responsible for acute myocardial infarction and sudden death. The method may be carried out in vivo as a thrombolytic therapy, wherein a fibrinolytic matrix metalloproteinase is administered to the individual to degrade the thrombus in situ. However, the use of this fibrinolytic matrix metalloproteinase is not a problem with angioplasty in chronically occluded arteries which contain large amounts of collagen and other extracellular matrix components, and very small amounts of fibrin or fibrinogen, and furthermore, as noted above, the systemic administration method does not involve local delivery into the arterial occlusion.

To sum up, CTO remains an important class of PCI damage with very limited success rate, mainly due to the inability to pass wires through occlusions. The fibrotic collagen-rich nature of these occlusive plaques is responsible for the obstruction of the passage of the guidewire. The vast majority of patients with chronic total occlusion symptoms are treated by drug therapy, which is often of limited effectiveness, or invasive bypass surgery. In addition to causing significant angina, there is strong evidence that CTO is also associated with poor left ventricular function and survival rates are lower than for stenotic (but not occluded) lesions or successfully dilated chronic occlusions. Stenting treatment of CTO has significantly improved long-term patency, which is another limitation of angioplasty. Thus, current evidence suggests that opening a total occlusion by percutaneous intervention is underutilized and new approaches are needed.

The treatment of occlusive plaques is needed to facilitate the passage of a guidewire through an occlusion — a prerequisite for successful angioplasty. More specifically, there is a need to chemically alter the collagen content and structure in these occlusive fibrous plaques to facilitate the passage of conventional guidewires.

There is also a need for animal models of chronic total arterial occlusion to aid in the study and development of methods for treating chronic arterial occlusions that cannot be passed by conventional angioplasty guidewires.

Summary of The Invention

The present invention provides a method of developing an in vivo animal model of chronic arterial occlusion, said method comprising the steps of: isolating an arterial segment of the animal, stopping blood flow of the isolated arterial segment of the animal artery with an occlusive ligature, injecting thrombin locally into the arterial segment to form an acute thrombotic occlusion, waiting to allow the acute thrombotic occlusion to convert to a chronic fibrotic occlusion.

The present invention provides methods for treating chronically occluded animal tubes and lumens. The first step of the method is to administer a therapeutically effective amount of a proteolytic enzyme containing formulation to the vicinity of the occlusive atherosclerotic plaque. Followed by a pre-angioplasty waiting period before the angioplasty wire passes over the plaque (plaque). After this waiting period, the angioplasty guidewire is passed through the occlusive plaque.

Brief Description of Drawings

The preferred embodiments of the present invention have been chosen for illustration and description, but are not intended to limit the scope of the invention in any way. Preferred embodiments of some aspects of the invention are shown in the drawings, in which:

FIG. 1 shows the pathological features (12 week duration) of a chronically occluded femoral artery in rabbits. M is media, a is adventitia, a, B is Movat, 10 × original; c ═ Movat, 20 ×; d ═ hematoxylin (hematoxylin) and eosin 20 ×.

A denotes the lumen (L) occluded by fibrotic intimal lesions.

B denotes the occluded lumen which also contains small vessel passages.

C represents the enlargement of the small vessel channel (indicated by the arrow).

D denotes fibrosis and cellular components of the occluded lumen (L).

FIG. 2 illustrates one embodiment of a method of treating chronically occluded tubes and lumens according to the present invention.

A indicates chronic total arterial occlusion.

B shows the use of a linear angioplasty balloon catheter with two different guidewires (ChoicePT)^TMAnd Wizdom^TM) By failure of the occlusion.

C denotes an inflated angioplasty balloon catheter adjacent to the total occlusion. The guidewire has been removed from the port of the angioplasty balloon catheter and collagenase is infused into the small space between the inflated angioplasty balloon and the occluded arterial segment (to prevent proximal outflow).

D represents the diffusion of collagenase solution along the arterial occlusion and degraded portions of the segment of the occlusion.

E indicates that the wire successfully passed through the artery 72 hours after collagenase placement. The occluded arterial segment has been partially degraded by collagenase, allowing the guidewire to pass through the true lumen of the artery into the unoccluded artery distal to the arterial occlusion.

Figure 3 shows the results of a pilot wire pass-through attempted angiography in a rabbit femoral chronic total occlusion model. A-C: successful attempts at 72 hours after collagenase infusion. D-F: unsuccessful attempts at 72 hours after ineffective control infusion. BI ═ comparison in bladder.

A indicates that in the collagenase treated artery there was a significant occlusion between the two arrows in the angiogram before the lead-through attempt was made.

B indicates that the guidewire (indicated by the arrow) has successfully passed the occlusion.

C indicates no anatomical evidence after the lead was passed.

D indicates that in the arteries treated with no effective contrast agent, there was a significant occlusion between the two arrows in the angiogram before the guidewire pass attempt was made.

E indicates that the guidewire (arrow) cannot be advanced through the occlusion.

F indicates that contrast extravasation of the lead anatomy (D) was evident in unsuccessful attempts to pass a total occlusion.

4 lead passage 72 hours after collagenase infusion. Movat10 × native, P ═ intimal plaque (which occludes the lumen), W ═ the location through which the guidewire passes, M ═ media, Ad ═ adventitia.

FIG. 4A shows successful lead passage (region filled with red blood cells) in collagenase treated arteries (450 μ g). Evidence of some plaque degradation is evident. The inner elastic layer (arrows) and the middle layer remain intact.

Figure 4B shows an artery treated with ineffective contrast with unsuccessful guidewire transit. Extensive and occluded intimal plaques (P) are present.

Fig. 5 the lead was passed 72 hours after collagenase infusion. Movat10 × native, P ═ intimal plaque (which occludes the lumen), W ═ the location through which the guidewire passes, M ═ media, Ad ═ adventitia.

A indicates successful lead passage (area filled with open space and red blood cells) in collagenase treated arteries (450 μ g). Evidence of some plaque degradation is evident. The inner elastic layer (arrows) and the middle layer remain intact.

B indicates an ineffective control treated artery with unsuccessful lead passage. There are extensive and occluded intimal plaques (P) with microvasculature. A portion of the middle layer (between 3 and 5 points) is degraded and atrophied.

FIG. 6 shows statistically significant differences in successful wire passage (p < 0.03) in collagenase treatment compared to ineffective control treatment 72 hours after treatment. The treatments were random and the operator was not aware of the treatment assignments.

FIG. 7 shows Western blot analysis of interstitial collagenase (MMP-1) in collagenase and placebo treated arteries 24 hours after treatment. Chronic arterial occlusion with either treatment showed a band at ≈ 93kD, confirming the presence of interstitial collagenase (MMP-1). This band was significantly enhanced in collagenase treated arteries (lanes 1 and 2) compared to non-control treated arteries (lanes 3 and 4), indicating that interstitial collagenase was increased in collagenase treated arteries. Lane 5 shows the MMP-1 protein present in the collagenase preparation and served as a positive control.

FIG. 8 shows the gelatinase profiles of collagenase treated artery (lane 1) and placebo treated artery (lane 2) 24 hours after treatment in a chronic occluded artery. There was an increase of 92-kD gelatinase (MMP-9) only in collagenase treated arteries, and no MMP-9 activity was found in arteries treated with ineffective control. There are lytic bands at 92 and 82kD, which reflect the zymogen and activated form of MMP-9 in collagenase treated arteries. Collagenase and ineffective control treated arteries also had evidence of 72-kD gelatinase (MMP-2).

FIG. 9 shows Western blot analysis of collagen degradation fragments (carboxy-terminal of collagen fragments) after 24 hours of treatment with collagenase (lanes 1 and 2) or ineffective control (lanes 3 and 4) in chronically occluded arteries. In collagenase treated arteries, there was a significant increase in collagen fragments.

Figure 10 shows the effect of collagenase and ineffective control treatment at 24 hours without attempting to pass the lead. Movat10 × native, P ═ intimal plaque (which occludes the lumen), M ═ media, Ad ═ adventitia.

A represents collagenase treated arteries with extensive plaque degradation within the previously occluded lumen (L). The inner elastic layer (arrows) and the middle layer remain intact.

B represents the non-control treated artery with extensive and occluded intimal plaque (P) where the microvessels were present. After occlusion, there is also considerable extensive breakdown of the inner elastic layer (indicated by arrows) and atrophy of the middle layer during chronic changes. Arteries treated with ineffective control agents have the same pathological characteristics as the arteries described above in the chronic total occlusion model.

Detailed Description

In accordance with the present invention, methods are described for significantly improving the outcome of chronic occlusion treatment. The method of topically delivering a therapeutically effective amount of a proteolytic enzyme containing formulation is effective to alter the matrix content in the occlusive plaque in the following manner: significantly facilitating the passage of the guide wire and substantially increasing the success rate of the treatment without causing adverse effects of these enzymes on the occluded artery and adjacent uninccluded arterial segments, said formulation having matrix degrading enzymes belonging to the following families: matrix metalloproteinases, serine elastase, trypsin, neutral protease, chymotrypsin, aspartase, cysteinase and clostripain.

Pathological features of chronic total occlusion in human coronary arteries

The main factor in the formation of atherosclerotic plaques in CTO is fibrocalcification³⁹Consisting essentially of smooth muscle cells, extracellular matrix and calcium, and variable amounts of intracellular and extracellular lipids⁴⁰. Inflammatory cells are usually present³⁹. Collagen is the major structural component of the extracellular matrix and accounts for up to 50% of the dry weight^41，42Types I and III (and minor amounts of IV, V and VI) predominate in the fibrous matrix of atherosclerotic plaques. In CTO, which lasts less than 1 year, proteoglycans are also commonly present in the inner membrane. Thrombosis can be of varying degrees of influence depending on the severity of the atherosclerotic plaque forming factors and can result in the formation of one or more layers of a clot. Over time, the thrombus becomes organized and converted to collagen-rich fibrous tissue (known as fibrointimal hyperplasia), which ultimately becomes a factor in atherosclerotic plaque formation⁴⁰. The most recently developed fibrointimal hyperplasia is the most likely structure that an angioplasty guidewire must pass through to pass through a complete occlusion. Previously organized collagenous fibrous tissues are barriers to the successful passage of current angioplasty techniques. The presence of newly formed fibrous tissue within the lumen is a target for collagenase treatment according to the invention. Intimal plaque neovascular passages are also common (> 75%) in CTO, regardless of the duration of occlusion³⁹. Several new passages are formed via occlusion (intra-arterial artery), and/or the vasa vasorum (i.e., the juxtaskeleton tissue) dilates to supply vascular supply and possibly active agents such as collagenase via the occluded segment. However, these small channels are insufficient to provide adequate distal coronary perfusionPreventing symptoms.

The present invention relates to methods of treating chronically occluded animal tubes and lumens. The term "animal tubes and lumens" refers to humans and other animals in which the methods of the present invention have medical and veterinary applications, and further, the methods of the present invention are applicable to occluded tubes and lumens containing collagen-rich tissue, such as root canals, fallopian tubes, bile ducts, sinuses, ureters and urethra, arteries, veins, and vein grafts for arterial catheters. The method of the invention is primarily for coronary artery occlusion, but may also be used for non-coronary arteries occluded, such as the iliac, popliteal, femoral, carotid, or subclavian veins. Routine adaptation of the method is included herein to be applicable to occluded body ducts and lumens, including veins, vein grafts, root canals, fallopian tubes, bile ducts, sinuses, ureters, and urethra.

Animal model

The present invention provides a previously untried method of treating chronically occluded arteries that are not amenable to treatment with angioplasty because the guidewire cannot pass through the site of injury. There has not previously been an easily available experimental model of chronic occlusion rich in homogenated collagen. The unique properties of chronic occlusions that make them difficult to pass through include high collagen content and an occlusion length that limits direct contact of the occlusion portion with the treatment. To evaluate the effect of collagenase, an in vivo animal model of chronic total occlusion was developed according to the invention (figure 1). The animal model may be established using any typical laboratory animal including, but not limited to, rabbits, pigs, dogs, sheep, rats, and non-human primates. Dosage and timing may need to be adjusted according to species and body size changes. For illustration, the animal model of the invention is described below with male New Zealand white rabbits weighing 3.0-3.5 kg.

The first step of the method is to isolate an arterial segment of an animal artery (such as the femoral artery shown in figure 1) and stop the flow of blood through the isolated arterial segment of the animal artery with an occlusive ligature. In a preferred embodiment of the invention, this step is accomplished by: male rabbits were anesthetized with isoflurane and incisions were made on both sides of the inguinal ligament. Ligatures spaced at least about 5mm apart are then placed to isolate the femoral artery segment. In a preferred embodiment, ligatures spaced at least about 15mm apart are placed. The ligature not only separates out, but also actually occludes the arterial segment.

The next step in the method is the local injection of thrombin into the arterial segment to form an acute thrombotic occlusion. This step is carried out by mixing 100 IU of bovine thrombin solution (Thrombostat) using a 27 gauge needle^TMParke-Davis) into the isolated arterial segment. After waiting at least 20 minutes, the suture is released to determine if an occlusion has formed. This may be accomplished by loosening the ligature (typically a suture) to determine if there is still a forward blood flow. If there is still a forward blood flow, another thrombin injection or injections are performed using the same technique until an acute occlusion occurs. Typically, the ligature is applied for 60 minutes before removal.

A waiting period follows during which acute thrombotic occlusion translates into chronic fibrotic occlusion. The waiting period is a period of about 10 weeks to 25 weeks. To determine the appropriate waiting period, arterial patency was assessed by angiography (using the left carotid artery) at a mean duration of 16(± 4) weeks.

It is believed that the method of the present invention, which has been developed into an in vivo animal model of chronic occlusion, can be adapted to other body ducts and cavities containing fibrotic collagen-rich tissue, such as root canals, fallopian tubes, bile ducts, sinuses, ureters and the urethra, veins and vein grafts. Such a method would include the following steps: isolating a segment of the selected animal tube, wherein liquid flow through the tube is stopped with an occlusion ligature spaced at least about 5mm apart, and then a sclerosing agent (e.g., tetracycline or other active agent suitable for the selected lumen or tube) is locally injected into the segment to form an acute occlusion; wait, allow acute occlusion to transform into chronic fibrotic occlusion.

Pathological features of chronic total occlusion model

In the first 2 rabbits (at weeks 10 and 15) that exhibited persistent femoral artery occlusion, the arteries were removed and pathologically examined to confirm the histological characteristics of the developed chronic occlusion. In chronic fibrotic occlusions (fig. 1), there was little to no significant fibrin residue. In addition to mature fibrous tissue, there are many small intraluminal blood vessels and sometimes extracellular lipid deposits, pigment-laden macrophages and lymphocytes. There is no evidence of vascular calcification and/or inflammation in the media. The occluded segment has also undergone a substantial inward change as compared to the adjacent arterial segment. A common feature in chronic occlusion models is the breakdown of the inner elastic layer at several locations and the appearance of fibrous tissue. All these changes resemble a chronic total occlusion of the coronary arteries in humans.

The model reflects the characteristics of a variety of therapeutic approaches. The mean degree of occlusion is approximately 28mm (14mm-56mm), which is much longer than most clinical coronary occlusion that would take a percutaneous coronary intervention. Moreover, the occluded lumen and the overall vessel size are very small due to the inward change.

Method for treating chronically occluded animal tubes and lumens

In the present specification and claims reference is made to "proteolytic enzyme containing preparations". In the present invention, the proteolytic enzyme is selected from the group consisting of matrix metalloproteases, serine elastases, trypsin, neutrase, chymotrypsin, aspartase, cysteinase and clostripain. Matrix Metalloproteinases (MMPs) are a class of zinc-containing enzymes that degrade extracellular matrix (ECM) components, including fibronectin, collagen, elastin, proteoglycans, and laminin (laminin). These ECM components are important components of occlusive atherosclerotic plaques. MMPs play an important role in normal embryogenesis, inflammation, wound healing and tumor invasion^45，46. These enzymes are broadly coveredClassification into 3 categories: collagenase, gelatinase, and stromelysin. Collagenase is the primary mediator of the extracellular pathway of interstitial collagen degradation⁴⁷Cleavage occurs at specific sites in the collagen molecule, making the collagen susceptible to breakdown by other neutral proteases in the extracellular space (e.g., gelatinases). The proteolytic enzyme containing formulation preferably comprises a matrix metalloproteinase selected from the group consisting of: collagenase, collagenase type 1A, gelatinase, and stromelysin. Most preferably, the proteolytic enzyme containing formulation comprises collagenase alone or in combination with other enzymes. It should be understood that reference to the use of "collagenase preparations" in this specification is intended to illustrate preferred embodiments of the invention, and is not intended to be limiting.

A method of treating chronically occluded animal tubes and lumens, such as coronary arteries, that cannot be passed through by a conventional angioplasty guidewire (0.014 "or 0.018" diameter) (fig. 2A) includes the following steps: a therapeutically effective amount of a proteolytic enzyme containing formulation is administered adjacent to an occlusive atherosclerotic plaque, subjected to a pre-angioplasty waiting period prior to passage of the angioplasty guidewire through the occlusive plaque, and then the angioplasty guidewire is passed through the occlusive plaque.

The step of administering a therapeutically effective amount of a proteolytic enzyme containing formulation is performed as follows. After complete occlusion of the coronary artery was confirmed by angiography, a linear angioplasty balloon catheter over a guidewire was introduced into the occluded coronary artery using fluoroscopic guidance. If the occlusion cannot be passed by a conventional 0.014 "or 0.018" coronary angioplasty guidewire (FIG. 2B), the guidewire is removed. The angioplasty balloon is inflated at a low pressure of about 1-5 atmospheres to prevent the collagenase formulation from flowing out during administration. It is preferred to inflate the angioplasty balloon to a pressure of about 4 atmospheres (fig. 2C). The collagenase containing formulation is slowly infused into the small space between the inflated balloon and the occlusion. As shown in fig. 2C, the collagenase containing formulation was infused directly through the wire port of the angioplasty balloon catheter. Infusion is performed at a pressure of about 0.5 atm to 3.5 atm. Infusion is preferably performed at low pressures of about 1-2 atmospheres. The formulation may also be infused directly into the vicinity of the occlusion itself via an infusion needle or catheter.

After infusion, the collagenase-containing formulation was held in place by an inflated angioplasty balloon for a waiting period of about 10-100 minutes of formulation exposure. The waiting period is preferably from about 50 to about 80 minutes. According to a preferred embodiment, the formulation exposure waiting period is about 60 minutes, after which the angioplasty balloon is deflated and then removed (fig. 2D).

It has been found that a therapeutically effective amount of a proteolytic enzyme containing formulation comprises collagenase type about 50-2000 μ gIA.

A therapeutically effective amount of the proteolytic enzyme containing formulation is administered and the angioplasty balloon is deflated and then removed, requiring about 1-108 hours of pre-angioplasty waiting period. It has been found that a waiting period of about 12 hours to about 86 hours is preferred, with best results being obtained after a waiting period of about 72 hours. This waiting period is required to spread the enzyme-containing formulation along the length of the occlusive segment and to sufficiently degrade the collagen and "soften" the occlusive plaque.

If the arterial wall (medial) collagen is easily destroyed by the treatment as plaque collagen in the occluded lumen, the collagenase dose will be ineffective due to being too low, or too high to prevent excessive destruction and weakening of the arterial wall. However, the newly formed collagen within the occlusive plaque is most susceptible to matrix metalloproteinases. Collagen in the middle layer of normal arteries is formed early in the development of the vessel wall and is extensively cross-linked at a very slow turnover rate. In contrast, intimal plaque development, where occlusive thrombopoiesis exists, is a very dynamic process that contains more recently synthesized collagen, has variable cross-linking, and is susceptible to degradation by MMPs such as collagenase. The newly organized thrombus is the portion of the lesion most likely to be crossed by an angioplasty guidewire to pass through a complete occlusion. Thus, this recently formed looser fibrous tissue within the lumen is a primary goal of the treatment method of the present invention. At 72 hours, the patient returns to the catheterization laboratory and the operator again attempts to pass a conventional angioplasty guidewire prior to performing angioplasty (FIG. 2E).

A number of in vitro and in vivo tests have been performed to evaluate the feasibility and efficacy of treatment. The test was performed with collagenase type IA (Sigma), a commercially available bacterial collagenase preparation obtained by fermentation of Clostridium histolyticum. The enzyme preparation is generally used for isolating cells from tissues for cell culture. The preparation also contains small amount of clostripain, neutral protease and trypsin-like active substance. Collagenase type IA (Sigma) is a bacterial collagenase preparation obtained by fermentation of clostridium histolyticum. The dose range was determined based on the results of in vitro assays that evaluated the effect of collagenase preparations on arterial wall structure over a range of doses and incubation times. Human coronary arteries containing stenotic atherosclerotic plaques were obtained at autopsy. The arterial segment was cut into 3mm cross-sectional sections and fixed on an agar gel in culture wells. This enables the collagenase to be selectively delivered directly into the lumen with a small pipette. Thus, only the occlusive plaque is in direct contact with the collagenase preparation, similar to the method of intraluminal delivery of collagenase. After a culture period of 4-18 hours, the results show that 100-500. mu.g/ml collagenase type IA causes a well-defined in vitro degradation of occlusive plaques, although some damage is caused in the deeper layers of the vessel wall in the higher dose range.

After confirming the continued presence of the occlusion, a linear angioplasty balloon catheter (3.0 mm diameter) was placed into the iliac artery adjacent to the occluded femoral artery via a 5F sheath in the left carotid artery and under fluoroscopic guidance. Attempts were made to leave a conventional 0.014 "coronary angioplasty guidewire [ Wizdom^TM(Cordis) and Choice PT^TM，(Boston Scientific)]And (4) passing. If the operator is unable to pass the wire, the injury can be carried into the trial. An angioplasty balloon catheter is placed at the occlusion and the length of the occlusion is determined using the known balloon length (20 mm between markers) as a calibration device. The balloon was inflated to 4 atmospheres to prevent proximal efflux of enzyme solution. The lead was then removed and the tap used to administer a composition containing collagenase type IA (n-33 arteries)Total dose 100-. The enzyme preparation was slowly delivered at 1-2 atmospheres.

The angioplasty balloon is kept inflated for a maximum of 60 minutes. The first attempts through chronic occlusion (n-10) within 1 hour of complete collagenase administration were unsuccessful. All other attempts were performed 72 hours after collagenase (n-23) or no control (n-24) administration. At 72 hours, when one of the first 2 collagenase-treated arteries successfully passed the occlusion, the remaining arteries (n ═ 45) were randomly assigned to the placebo or collagenase-treated group, which was unknown to the operator. Attempts at 72 hours through occlusion were performed after assessment of arterial circulation via right carotid artery truncation and placement of an angioplasty balloon catheter as described above. Repeated injections of contrast agent to evaluate angioplasty guidewires (Wizdom)^TMAnd Choice PT^TM) The distance traveled, and the assurance that the wire remains within the true lumen. The wire passing attempts were continued until the wire passed through the damaged portion, a large incision was made, or until multiple attempts with different wires failed to achieve any success. With angiography, successful lesion passage is determined by the free movement of the lead tip across the occlusion segment in the distal vascular bed. After the guidewire is passed, no angioplasty is performed, thus leaving the arterial structure intact for analysis. At the end of the procedure, rabbits were sacrificed, femoral arteries excised, and histological analysis (Movat and H) performed&E) In that respect At least 3 cross-sectional slices were determined for each occluded segment.

Statistics of

Fisher's exact test was used to assess the difference between the success rates of the leads through occlusion. P values < 0.05 were considered statistically significant.

Length of occlusion

There was no significant difference in occlusion length between the collagen-treated artery (29.5+/-8.6mm) and the ineffective control-treated artery (27.9+/-8.7 mm).

Success rate through chronic occlusion at 72 hours as demonstrated by angiography:

there was a significant increase in the success rate of lead passage (p < 0.03) in the collagen-treated artery (14/23, 61%) compared to the non-control treated artery (7/24, 29%), respectively, fig. 3 and 5.

TABLE 1

Treatment of	Incubation time	Success example/attempt example	% success
				Collagenase	1 hour	0/10	0％
Collagenase	72 hours	14/23	61％
				Ineffective contrast agent	72 hours	7/24	29％

By the pathological features of the post-trial artery:

when angiography indicated successful guidewire passage, histological analysis confirmed the presence of a blood-filled vascular channel in the area where the guidewire passed through the occluded intimal plaque (fig. 4A and 5A). There was also some evidence of plaque breakdown. For failed lead passes without angiographically indicated incisions, the pathological features were identical to the chronic total occlusion model with thick fibrotic plaques, neovascular plaques, some inflammatory cell infiltration and frequent rupture of the internal elastic layer and the appearance of fibrous tissue (fig. 4B and 5B). In these cases, there is no evidence of any wire damage. For failed guidewire passes due to angiographically indicated incisions, there is a clear guidewire passage outside the intimal plaque in the media and occasionally in the adventitia or periadventitial region. There was no detectable difference in the degree of vessel wall damage (e.g., rupture of the inner elastic layer or medial wall) in collagenase-treated versus ineffective control-treated arteries.

24 hour test for collagenase effect on chronic total occlusion

To determine that the collagenase preparation actually affected the structure and extracellular matrix proteins in the occluded arterial plaque, an additional 6 arteries were removed 24 hours after administration (3 for collagenase [450 μ g ], 3 for no-effect controls). In order to evaluate collagenase action without confounding effects of the lead, no attempt was made to pass the lead through these occlusions. MP-1 protein, collagen degradation products and gelatinase activity present in the arteries were also evaluated.

Western blot analysis of interstitial collagenase (MMP-1)

The frozen arteries were crushed in liquid nitrogen and extracted in cold extraction buffer (codylicic 10mM, NaCl 150mM, ZnCl)₂ 20mM，NaN₃1.5mM and SDS 1% w/v). To determine collagen degradation products, extracts containing 50. mu.g of protein were separated on a 4-20% triglycine gel under reducing conditions and electrotransferred onto nitrocellulose membranes (Bio-Rad). Using COL 2^3//4C short polyclonal Rabbit IgG (HDM Diagnostics)&Imaging Inc, Toronto) as a primary antibody at a dilution of 1: 1000 and anti-rabbit IgG-HRP (Santa Cruz Biotechnology) as a secondary antibody. To detect interstitial collagenase (MMP-1) protein, an extract containing 50 μ g of protein was isolated under non-reducing conditions and electroblotted onto nitrocellulose membranes. anti-MMP-1 monoclonal antibody (Calbiochem) was used at a dilution of 1: 100 as the primary antibody and anti-mouse IgG-HRP (Santa Cruz Biotechnology) as the secondary antibody. To reveal the stimulating antibodies, a chemiluminescent detection system (ECL Plus, Amersham) was used, followed by autoradiography.

Western blot analysis of interstitial collagenase (MMP-1) in collagenase and placebo treated arteries showed the presence of a band at 93kD, confirming the presence of interstitial collagenase (MMP-1) (FIG. 7). This band was significantly enhanced in collagenase treated arteries compared to non-effective control treated arteries, indicating that interstitial collagenase (MMP-1) was increased in collagenase treated arteries 24 hours after treatment.

Gelatin zymogram

Gelatin zymography as described in the prior art⁵². There was an increase of 92-kD gelatinase (MMP-9) only in collagenase treated arteries, and no significant activity was found in arteries not treated with control (figure)8). There are lytic bands at 92 and 82kD, which reflect the zymogen and activated form of MMP-9 in collagenase treated arteries. Collagenase and ineffective control treated arteries also had evidence of 72-kD gelatinase (MMP-2).

Collagen degradation products

Degraded collagen was evaluated by Western blot analysis under reducing conditions using a polyclonal antibody against human type II collagen (col 23/4C, 1/1000 dilution, Diagnostic Imaging). Collagen degradation products were identified in both failed control and collagenase treated arteries, with a significant increase in collagenase treated arteries (figure 9).

Pathological features of chronically occluded arteries treated with collagenase or ineffective control

Of the 3 arteries treated with collagenase, two arteries had extensive degradation of the plaque of the occlusion evident, while no such degradation was present in any of the arteries treated with the failed control (fig. 10). Arteries treated with ineffective control agents have the same pathological characteristics as the arteries described above in the chronic total occlusion model.

Experimental models have been developed for studying the composition and amount of chronic arterial occlusion and collagenase-containing agents that facilitate the passage of the guidewire. A chronic arterial occlusion model was developed in rabbit femoral arteries by the following method: a temporary occlusion ligation was applied, thrombin was injected, and then a waiting period of 16 weeks was averaged to allow the acute thrombotic occlusion to progress to a chronic fibrotic occlusion similar to a chronic human arterial occlusion. Local delivery of a formulation containing 450 μ g collagenase over a 60 minute period via a wire port in the form of a balloon of a linear angioplasty balloon while inflating the balloon, resulted in collagen degradation, increased MMP-1 and MMP-9 activity, and demonstrable plaque component degradation at 24 hours compared to arteries treated with ineffective control agents. Such local delivery of collagenase may improve the success rate of lead passage 72 hours but not 1 hour after collagenase administration. Therefore, a 24-72 hour waiting period is required for collagenase to degrade the plaque before passing the wire. These effects of collagenase on occlusive plaques can be achieved without damaging the outer layers of the vessel wall (media and adventitia) and without forming an aneurysm.

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Claims (5)

1. Use of collagenase in the manufacture of a formulation for the degradation of collagen in an occlusive atherosclerotic plaque in a chronic occlusive artery or lumen.

2. The use of claim 1, wherein the formulation is an infusion formulation for administration and retention of a therapeutic dose of collagenase adjacent to atherosclerotic plaques during waiting periods for exposure of the formulation.

3. The use of claim 1, wherein the formulation is an infusion formulation for administration and retention of a therapeutic dose of collagenase within atherosclerotic plaques during waiting for exposure of the formulation.

4. The use of claim 2 or 3, wherein the infusion formulation is formulated for administration of a collagenase type 1A dose in the range of about 50 μ g to about 2000 μ g.

5. The use of claim 2 or 3, wherein the infusion formulation is formulated for administration of a collagenase dose of about 150 μ g to about 500 μ g.