# Patent

## Title
Soy protein-based thermoplastic composition for preparing molded articles

## Abstract

The present invention comprises a method of alleviating restenosis, post-PTCA or other coronary arterial intervention, and more particularly, to the use of ancrod to prevent restenosis in the coronary arteries.

## Background
1. Field of the Invention

The present invention relates to methods of alleviating restenosis post-percutaneous transluminal coronary angioplasty (PTCA) or other coronary arterial intervention, and more particularly, to the use of ancrod to prevent restenosis in the coronary arteries.

2. Background of the Prior Art

Restenosis following PTCA is a significant limitation to the application of this otherwise useful revascularization technique. Restenosis occurs with varying frequency, and is generally accepted to be 25%-45%, depending on a number of factors including the site of the lesion, residual gradient post PTCA, sex (male), smoking and/or diabetes, skill of operator, method and timing of followup, and balloon size.

The problem typically manifests within 12 weeks to six months post procedure, and presents as recurrent angina in many cases, although "silent" restenosis occurs as well where ischemia recurs without overt clinical manifestation. This problem will become increasingly severe, as estimates that 400,000 or more procedures will be performed annually by the year 1993.

Clinical trials with other interventional methods such as atherectomy and intracoronary laser ablation suffer from the same restenosis problem, probably with similar frequencies of occurrence. Attempts to prevent restenosis with systemic drug therapy such as anti-platelet agents, anticoagulants, corticosteroids, and calcium channel blockers have uniformly failed to reduce its occurrence.

The histologic appearance of the recurrent lesion in restenosis is distinctly different from the atheromatous process of the original lesion. The atherosclerotic process evolves over many decades, beginning as flat, fatty streaks consisting of lipid-laden smooth muscle cells. Over time, these lesions evolve in complexity, becoming grossly fibrous plaques with ulceration, calcification, thrombosis and/or hemorrhage into the vessel wall. Microscopically, the atherosclerotic lesion has histological features of disruption and replication of the internal elastic lamina, spindle and smooth cell proliferation, interstitial fibrosis, intracellular and interstitial lipid accumulation, fibrin deposition, calcification, hemorrhage, thrombosis, capillary proliferation and macrophage infiltration. This process may be a response to injury. Most atherosclerotic lesions are endothelialized continuously with adjacent vascular endothelium.

The histopathology of restenosis after PTCA has been recently elucidated with the advent of percutaneous atherectomy procedures whereby such lesions are removed intact. In contrast to the atheromatous lesion, restenotic segments consist of a proliferation of cells and interstitial material resembling normal connective tissue with smooth muscle involvement. The lesion of restenosis thus differs distinctly from the original atherosclerotic disease both in time course of development and histologic appearance. It is likely that restenosis represents a zealous natural healing process of damaged coronary arterial walls with neointimal tissue impinging significantly on the vessel lumen.

If an agent or process could be found whereby restenosis could be significantly reduced or eliminated, a major advance in clinical care would be achieved. Savings of much morbidity, time, emotional investment, and economic cost could be realized by patients with coronary artery disease undergoing PTCA or other percutaneous coronary revascularization, in addition to improving patient safety.

The present invention has met the above-described needs by providing methods for reducing restenosis by the use of an effective amount of ancrod, a snake venom protein used as a defibrinogenating agent.

It is an object of the present invention to provide a method of reducing restenosis in coronary arteries.

It is another object of the present invention to provide a method of treating patients undergoing PTCA or other percutaneous coronary revascularization.

These and other objects of the present invention will be more fully understood from the following description of the invention.

The present invention provides a method of reducing restenosis in coronary arteries by using an effective amount of ancrod. Ancrod is commercially available under the trademark ARVIN. In addition, the present invention provides a method of treating patients undergoing PTCA or other percutaneous coronary revascularization by an effective amount of ancrod.

Ancrod is a snake venom protein used as a defibrinogenating agent. Ancrod does not alter coagulation factors other than fibrinogen, nor does it induce serious bleeding.

While not wishing to be bound by any particular theory, it is believed that the proposed mechanism is that an early, space-occupying lesion after injury is composed chiefly of a fibrin/platelet/red blood cell thrombus. Because ancrod is a potent defibrinogenating/fibrinolytic agent, it is believed that ancrod-mediated reduction of lesion size early in the development of restenosis will result in a small final lesion.

Ancrod may be absorbed intramuscularly, subcutaneously, rectally and intraperitoneally. Ancrod may be administered parenterally, intravenously, subcutaneously, intramuscularly, rectally or orally by methods well know in the art.

The particular dosage regimen and schedule of ancrod administration is dependent on the aim of therapy. In any event, it is important to administer ancrod relatively slowly in order to prevent excessive coagulation.

For example, when thrombus formation is to be prevented, the intravenous route is preferred. Ancrod may be administered intravenously, for example, in a dosage unit of about 1 to 3 units (a unit is defined as the specific biological activity contained in 0.307 mg of the International Standard for ancrod) per kg of body weight, diluted with isotonic sodium chloride solution. Depending upon the fibrinogen level at the end of the initial infusion period, a similar second infusion in 50 mL of isotonic saline may be administered in order to achieve the desired fibrinogen level. In this example, the desired fibrinogen level to be achieved is about 100 mg/mL. Thereafter, maintenance doses may be administered after determining the patient's plasma fibrinogen concentration. In general, about 0.05 to about 1.5 u/kg of body weight diluted in 50 mL isotonic saline may be administered once or twice a day.

Ancrod may be administered subcutaneously in a dosage of about 0.3 to 4 units per kg of body weight. In this example, the desired fibrinogen level to be achieved is about 100 mg/dL. As with the intravenous route of administration, the number of doses and the intervals administered is dependent upon the patient's plasma fibrinogen level and response.

The use of ancrod for treatment of the above-mentioned condition may extend from 1 to 2 weeks for intravenous and 3 to 4 weeks for subcutaneous administration. However, depending upon patient response, a longer period of administration may be used.

The following examples serve to illustrate the invention and should in no way be construed as limiting the scope thereof.

An accurate model of this proliferative restenosis and obstructive neointima was developed in the coronary arteries of domestic crossbred swine, Sus scrofa. This model was developed in the Mayo Clinic Cardiac Laboratory in order that pharmacologic approaches to restenosis might be first tried in animals.

The advantage to using this model is that it is an accurate model of human restenosis, it utilizes the coronary arteries of swine (with known similarity to humans), and direct histopathologic correlation with therapy is readily assessed. Furthermore, results can be available within a short period of time. Schwartz et al., Circulation, vol 82(6), pps. 2190-2200 (1990).

The model utilizes severe coronary artery injury by a metallic coil implant with an oversized PTCA balloon. More specifically, metallic coils of either stainless steel or tantalum were implanted into the media of pig coronary arteries using oversized balloons and high inflation pressures. (0.125 mm stainless steel or tantalum wire coils implanted percutaneously in pig coronary arteries with oversized PTCA balloons inflated to 14 atm).

In all pigs studied at necropsy a substantial proliferative response was observed, and the histological appearance of the proliferative tissue was identical to that in human specimens obtained via atherectomy in post PTCA restenosis cases.

The study showed that ancrod can successfully inhibit the restenotic response to injury in a porcine model of human restenosis. Specifically, quantitative measurements of luminal stenosis and proliferative response in treated and untreated groups of pigs were used to determine inhibition of restenosis.

The lesions were seen at time intervals of 11 days minimum to 42 days. Typically, 28 days were required to generate these lesions.

In some animals, some proliferative tissue was noted, but not to the same severe degree of tight luminal stenosis. Histology typically showed less vessel damage in these specimens, without rupture of the internal elastic lamina. The results indicate that coil oversizing and high balloon implant pressures (up to 14 atm) should be used. Severe stenotic lesions are reliably generated.

Concern for the efficacy and practicality of ancrod in the pig motivated a preliminary study of the ability of ancrod to defibrinogenate this animal. Two animals were used, both of which received a 70u intravenous administration over 10 minutes. Measures of clotting (thrombin time, activated clotting time, APTT) are shown in the following table. ACT         Thrombin  Protime  APTT  Fibrinogen (see)       Time (sec) (sec)    (sec) (mg %) ______________________________________ Pig #728 Pre   149         40      11     26    420 30 min          >600      ***    ***   184 1 hour 112       >600      26     33     0 2 hr  192       --        --     --    -- 4 hr  >1500     >600      --     --     0 24 hr             17      11     --    361 Pig #733 Pre    58         32      10     25.5  586 30 min 90       >600      ***    ***   397 1 hr  129       >600        11.5 62    204 4 hr  156       >600      18     35     0 24 hr  92         21      12     61    319 ______________________________________

The data in this table clearly show the defibrinogenation effects in the two pigs which receiving the ancrod. The *** symbols indicate that immediate clot formed upon addition of calcium to the plasma. The dash (--) above indicates not measured.

After these studies, each animal appeared to suffer no ill effects from the relatively rapid infusion. These results demonstrate that ancrod is effective in safely defibrinogenating the pig.

Cutdown was performed on the ventral neck, with isolation of the right external carotid artery and internal jugular vein. The internal jugular vein was cannulated and an indwelling catheter (polyethylene 0.060") placed in the superior vena cava. This catheter was attached to an implantable infusion pump which has been filled with ancrod 700 units. An arterial hemostatic sheath was placed in the carotid artery for access to the coronary tree.

The method of metallic coil implantation to generate neointimal hyperplasia and proliferation in young domestic crossbred pigs is a procedure which can now be accomplished with comparative ease and efficiency. The pig model has been chosen since their large and small vessel coronary anatomy is comparable to humans, their coagulation system resembles the human system, and because the neointimal hyperplasia resulting from coil implantation is identical to human tissue found post PTCA in restenosis.

For this study there were 10 pigs in each group, ancrod treated and untreated Assuming an accidental death rate of 20% 4 extra pigs were requested (24 total).

After insuring adequate hemostasis (cautery and silver nitrate) from cutdown, a custom-curved PTCA guide catheter was advanced to the aortic root and the left main coronary artery engaged under fluoroscopic guidance. Metal coils were implanted in at least 2 predetermined coronary arteries of the animal, so that at least 20 lesions in each group were available for study.

Animals were randomized to either treated or untreated groups.

All animals received 1) a single oral dose of sustained released verapamil (commercially available from Knoll Pharmaceutical Company, Whippany, N.J. as Isoptin.RTM.SR) 120 mg one day prior to coil implant; and 2) oral aspirin 650 mg 1 day prior to coil implant and continuing for the 10 day period of ancrod infusion after implant.

The verapamil was used to reduce coronary artery spasm during implant. Aspirin has been shown to reduce platelet aggregation acutely, and to increase survival rate without affecting the degree of neointimal proliferation.

The treatment group received ancrod intravenously 70 units (about 2 units/Kg) over 1 hour during the cutdown procedure. As noted, the continuous infusion osmotic pump contained ancrod, 700 units infused continuously at about 70 units/day into the superior vena cava. Fibrinogen levels were monitored to assess efficacy and adequate dose of ancrod. For the untreated group, the same procedure was followed except the pump contained normal saline only.

The parameters below were measured daily for 3 days, then every 3 days thereafter for 10 days.

All animals were sacrificed at 28 +/- days for histopathologic examination and quantification. The animals were sacrificed using a commercial intravenous euthanasia solution ("Sleepaway") as approved by the Mayo clinic section of Veterinary Medicine. The heart was removed and placed on a fixation pump under pressure. Formalin 10% was the fixation medium.

The left atrial appendage was removed for access to the proximal left coronary artery system. The coronary artery segments with coils were dissected free of the epicardium. The segment containing the coil was isolated allowing enough tissue to contain uninvolved vessel at either end.

These arterial segments, each roughly 2.5 cm in length, were sectioned grossly with a scalpel by transverse cuts 3 mm apart. The cut wire coil was removed via forceps after gross photography of each 3 mm section.

The 3 mm sections with coils removed was imbedded and stained with hematoxylin and eosin, and elastin stains. These sections were observed with quantitative low and high power light microscopy where a movable calibrated reticle was utilized for making length measurements in the plane of microscopic view. Each section was be photographed with both high and low power visualization. This allowed for observation of the normal vessel, progressing into proliferative vessel where the coil was located, and back to the normal vessel beyond the point where the coil was located.

Quantitative measurement of the degree of proliferation was assessed by three methods: 1) maximum, minimum, and mean thickness of neointimal tissue; 2)maximum percent luminal stenosis; and 3) medial thickness. Measurements were done via calibrated microscope reticle.

Neointimal proliferative area was calculated by assuming the native and residual lumina to be prolate ellipses. A major and minor axis was measured, and area calculated by the expression:

Mean neointimal thickness was calculated by measuring proliferative thickness at each wire site and dividing by the number of wires present in that section.

Quantitative maximum percent area stenosis was calculated according to the following expression:

Maximum medial thickness was also measured.

All three parameters of neointimal area, maximum percent stenosis, and mean neointimal smooth muscle cell density were compared between the treated group and the control group.

Pathologic lesions in 5 of 5 implanted animals (at 11 days to 63 days) included a severe proliferative response of medial smooth muscle cells with significant luminal compromise (mean area stenosis 70%+/-24% SD). Disruption of the internal elastic lamina was present in all vessels showing the proliferative response, as was mild chronic inflammation surrounding the wire itself. One animal died spontaneously at 11 days from a tight, proliferative left anterior descending coronary artery (LAD) stenosis. In all animals, histopathologic features of the proliferative neointimal tissue were identical to those of human restenotic tissue post PTCA obtained in 38 patients by atherectomy.

Whereas particular embodiments of the invention have been described above for purposes of illustration, it will be appreciated by those skilled in the art that numerous variations of the details may be made without departing from the invention as described in the appended claims.

## Claims

1. A method of reducing restenosis in coronary arteries of a patient following percutaneous coronary arterial intervention comprising administering to said patient, post-coronary arterial intervention, a restenosis reducing effective amount of ancrod.

2. The method of claim 1 wherein ancrod is administered parenterally, intravenously, subcutaneously, intramuscularly, rectally or orally.

3. The method of claim 2 wherein ancrod is administered intravenously.

4. A method of treating percutaneous transluminal coronary angioplasty (PTCA) patients to reduce restenosis in coronary arteries comprising administering to patients in need of said treatment post-PTCA a restenosis reducing effective amount of ancrod.

5. The method of claim 4 wherein ancrod is administered parenterally, intravenously, subcutaneously, intramuscularly, rectally or orally.

6. The method of claim 5 wherein ancrod is administered intravenously.

7. A method of treating patients undergoing percutaneous coronary revascularization to reduce restenosis comprising administering to patients in need of said treatment a restenosis-reducing effective amount of ancrod.

8. A method as in claim 7, wherein ancrod is administered parenterally, intravenously, subcutaneously, intramuscularly, rectally or orally.

9. A method as in claim 1, 4 or 7, wherein ancrod is administered intravenously in a dosage of about 1 to 3 units per kg of body weight.

10. A method as in claim 9, wherein ancrod is administered to achieve a fibrinogen level of about 100 mg/mL.

11. A method as in claim 1, 4 or 7, ancrod is administered subcutaneously in a dosage of about 0.3 to 4 units per kg of body weight.

12. A method as in claim 11, wherein ancrod is administered to obtain fibrinogen level of about 100 mg/dL.