United States Patent (19) 11 Patent Number: 4,485,088 Chvapil (45) Date of Patent: Nov. 27, 1984

54 METHOD OF TREATMENT OF FIBROTIC Primary Examiner-Stanley J. Friedman LESIONS BY TOPCAL ADMINISTRATION OF LATHYROGENC DRUGS 57. ABSTRACT The disclosure deals with a method of treating fibrotic 75 Inventor: Milos Chvapil, Tucson, Ariz. lesions related to abnormal collagen polymerization by 73 Assignee: Bio-Products, Inc., Tucson, Ariz. topical administration of lathyrogenic substances, such as 3-aminopropionitrile fumarate, , 21 Appl. No.: 362,241 D-penicillamine, into the site of the injury. The lathyro 22 Filed: Mar. 26, 1982 genic drugs are administered by local injection or onto the skin and percutaneously transported into the site of 51) Int. Cl...... A61K9/70; A61K 31/275 the lesion. Lathyrogens, administered locally as a single 52 U.S. C...... 424/28; 424/304 drug or in combination do not produce any local or 58 Field of Search ...... 424/304, 28 systemic toxic effects and are improving the function of the pathological fibrosis based on the accumulation of 56 References Cited polymerized collagenous protein. PUBLICATIONS Code of Federal Regulations 37 (7-1-82), p. 38. 5 Claims, No Drawings 4,485,088 2 ble and acquiring mechanical strength. This process is METHOD OF TREATMENT OF FEBROTC also called maturation or polymerization of collagen LESIONS BY TOPCAL ADMINISTRATION OF and is the main target of this invention. LATHY ROGENC DRUGS Formation of a scar is the result of a fibroproliferative Y 5 inflammation. Scar is an imperfect method of repairing REFERENCES CITED tissue defects. Several steps could be identified in the E. E. Peacock, Jr. and Walton Van Winkle. Wound course of fibrotic reaction. The injury activates fibro Repair, Sanders Co. Philadelphia, USA, 1976. blasts to produce more collagen and glycosaminogly H. Keiser and A. Sjoerdsma. Clin. Pharmacol. Ther. - cans. Often part of the injury is bleeding by ruptured 8:593, 1967. 10 vessels. Blood is a known factor inducing fibrosis. In E. E. Peacock and J. W. Madden. Surgery 66: 215, ruptured or injured tendons, the presence of blood and 1969. blood proteins, mainly of fibrin, forms bridges between D. J. Prockop. German Pat. No. 2,228,187 (Dec. 14, the tendon and its sheath to form peritendineous adhe 1972). sions which immobilize the joint by impairing the glid In human medicine, there has always been an urgent 15 ing function of the tendon. Once collagen is formed and need for an efficient method of inhibiting abnormal accumulates at the site of the injury, it matures and only accumulation of collagen in the form of fibrotic, cir then do the abnormal functions of the tissue manifest rhotic or excessive scar lesions. Only in the last decade. themselves. have various aspects of collagen metabolism, and colla In the dynamics of the fibroproductive inflammation, gen regulating mechanisms made it possible to apply 20 basic knowledge to more integrated and complex sys various reactions reach their maximum and then decline tems as represented by various models of fibrosis in with time. Activity of lysyl oxidase, the enzyme which animals. The goal of various treatments aiming at the is involved in polymerization of collagen, is highest at inhibition of collagen metabolism was to inhibit selec much later stages after collagen has been accumulated tively the synthesis and deposition of this fibrous pro- 25 in the intercellular space. The maximal activity of lysyl tein specifically in the fibroticlesion, not in intact tis oxidase, coincides with formation of an insoluble colla sues. As will be shown below, so far, not an efficient gen within the injured tissue. Consequently, the optimal method was developed, in spite of the fact that every time of "pharmacological' interference with a certain aspect of collagen metabolism was taken into such a step of the inflammation, is at the time of maximum “therapeutic" consideration. It has been my experience 30 incidence and activity; of this specific process. We that inhibition of maturation (polymerization, stabiliza learned by our experiments that in skin incision wounds, tion of the structure) of collagen has so far been the the inhibition of lysyl-oxidase could be started five days most successful and powerful method of interfering after inflicting the injury, and continued the treatment with the physical properties of scar contractures and for 14-21 days to achieve an effective and permanent fibrotic structures. Apparently, it is not the total volume 35 prevention of collagen maturation. . . . . of collagenous structures within a certain tissue, but The fibrotic, collagenous tissue is in a continuous rather the physical properties of the callogenous matrix increased metabolic turnover. Both synthesis and deg which represent the real danger to the function of the radation of collagen are increased and their relative tissue or organ. When collagen is cross-linked, possibly activities change with time of healing. It could be as by stable covalent cross-links involving the function of 40 sumed that by decreasing the structural stability in the lysyl oxidase, it is less degradable by mammalian colla scar (i.e., decreasing the degree of polymerization of genase and forms a compact rigid scar binding less collagen) by administration of lathyrogenic agent, the water. As will be demonstrated below, formation of a pool of “extractable" collagen is increased. These forms scar, which ultimately shrinks, contracts, forms defor would be more readily available to digestion by collage mities, strictures, represent the real danger for the pa- 45 nase system and by other proteolytic enzymes in the tient. It is the object of this disclosure to present a new later stage. This will result in a reduction of the size and method of topical interference with the formation of volume of the fibrotic mass...... mature scars by a group of drugs, called lathyrogens. Pharmacology of Fibrosis ...... Lathrogens are defined as a group of chemical sub There exist several steps unique for collagen which stances inhibiting by any method the formation of inter-50 could be used as a target for so-called "specific interfer molecular and intramolecular covalent cross-links in ence” with collagen metabolism. These stages are: collagen or elastin structures. 1. Hydroxylation of prolyl and lysyl residues by ap propriate hydroxylases. BACKGROUND INFORMATION 2. Glycosylation of some e-NH2 hydroxylysyl groups In order to justify my reasons for administering cer- 55. and; ...... tain drugs called lathyrogens topically into the site of 3. subsequent secretion of the molecule out of the fibrotic lesion, it is necessary to outline several topics : cell. - related to this new therapeutical method. 4. In the extracellular space, the molecule further' - Dynamics of Scar Formation polymerizes or matures under the effect of lysyl Scar callagen is synthesized by fibroblasts. While still 60 oxidase which forms the basis for development of within the fibroblasts, the collagen polypeptides are stable covalent cross-links. hydroxylated, form a triple helix and undergo glycosy 5. At different stages of collagen synthesis the mole lation to the transport form, known as procollagen. cule is degradable but the "younger" the molecule Procollagen is then secreted from the fibroblasts into is, the faster the degradation by tissue collagenases. the extracellular wound environment, to form tropocol 65 Several sophisticated methods were developed and lagen. Tropocollagen then undergoes intramolecular studied with the aim to reduce collagen deposition or and intermolecular covalent cross-linking to form colla polymerization in the fibrotic lesion (D. J. Prockop gen fibers which are becoming progressively less solu German Pat. No. 2,228,187). In all these situations, the 4,485,088 3 4. medication was administered either perorally or paren densation of aldehydes or formation of Schiff bases as terally, in other words by systemic route, where the shown in Scheme I.

Scheme I Suggested methods of the effect of lathyrogens on the covalent cross-links in the collagen structure.

BAPN

in-chi-Nil, Lysyl oxidase G -- 3-(CH-CHO Penicillamine Hydroxylysine CU C O2 8-Semialdehyde Lysine a-Amino adipic acid

OH -thH >-R- CH2NH- R Hydroxylysine Labile Schiff Stable CH-(CH2)3-CHO base cross-link

--OHC-R- >-R-C-ERCHO Aldol condensate Stable cross-link Since BAPN and D-penicillamine affect two different drug was distributed among all tissue and body fluids. sites in the formation of covalent cross-links, when used All various methods of interference with individual 30 together their effect is additive. The lathyritic proper steps of collagen synthesis have one major deficiency; ties of penicillamine derive from its ability to chelate they work very nicely in isolated, closed systems of aldehydes formed by the action of lysyl oxidase on the cells in tissue cultures and are minimally effective or epsilon amino group of tropocollagen lysine. In addi quite ineffective in vivo in the whole organism. In fact, tion, direct inhibition of of lysyl oxidase by D-penicilla after systemic administration of many of these drugs 35 mine was demonstrated. The overall effectiveness of (proline analogs, chelating agents, colchicine, etc.) their systemically-administered D-penicillamine in decreas toxic effect is close to the therapeutic effect. Thus, there ing the structural stability of collagen has been well is a permanent risk of general toxicity of the drug used. documented. All of the clinical studies using the treat The only exception seems to be the use of a lathyro ment with D-penicillamine have indicated a high inci gen to interfere with collagen polymerization mainly dence of acute hypersensitivity reactions. Striking because of high effectiveness of a typical representative changes in the metal content in various tissues and fluids of lathyrogens, beta-aminopropionitrile (BAPN), to after D-penicillamine treatment were reported. It is inhibit lysyl oxidase at 107M concentrations. Still, the mainly the metabolism of zinc and copper which is fast metabolism of BAPN requires frequent administra affected and which results in various deficiencies and tion of this potent drug (every 6 hours seems to be 45 pathologies. These symptoms are suppressed by sys optimal) which may result in induction of some toxic temic steroids; thus, a combination of D-penicillamine adverse effects in both lab animals as well as in humans. and prednisone should minimize toxic side effects. Topi Maturation (Polymerization of Collagen) cal administration of D-penicillamine by injection route The enzyme forming the basis for the polymerization or by percutaneous absorption similar to BAPN uses of collagen is lysyl oxidase. Lysyl oxidase oxidatively 50 several times lower dose than that used systemically. deaminates specific e-amino groups of peptidyl lysine This avoids the incidence of toxic complications. and hydroxylysine residues contained within the struc Toxicity of Systemically-Administered Lathyrogens tural proteins collagen and elastin. The aldehyde prod The systemic toxicity of BAPN has been the major uct then forms, nonenzymatically, either Schiff base obstacle in using this drug in large scale in human pa adducts with other specific e-amino peptidyl lysine or 55 thology. There is no doubt that at lathyrogenic dosages hydroxylysine residues or forms aldol condensates with of BAPN, the animals stop growing, lose body weight other preformed aldehydric components. These cross and lower their food intake. Also, the animal's behavior linking reactions lend structural integrity to collagenous is changed-they do not clean their fur and are irritated and elastinous connective tissue. if they are touched. All this indicates a general toxicity. In principle, we may interfere directly with the func There is also no doubt that various lathyrogenic drugs, tion of lysyl oxidase by various lathyrogenic agents, mainly BAPN showed to be very effective in the block BAPN and aminoacetonitrile being the most effective. ing the polymerization of collagen in various animal Inhibition of lysyl oxidase by BAPN is irreversible. As models of fibrotic lesion. will be shown below, lysyl oxidase has very high turn Because of success in animal studies, Keiser and Sjo over and its activity in a granuloma tissue after a single 65 erdsma (1967) studied the effect of BAPN in patients systemic moderate dose of BAPN recovers within 6-12 with fibrotic lesions of the skin and other organs called hours. Another possibility is blocking the formed alde scleroderma. In short-term treatment with a dose of 2 hydes, as with D-penicillamine, thus preventing con g/day, no toxic side effects were noted; longer courses 4,485,088 5 6 of treatment were, however, associated with prohibi safe, effective and optimal therapeutic regimen to tive reactions, such as allergic skin rash and hemolytic achieve significant interference with maturation of col anemia. Peacock and Madden (1969) employed BAPN lagenous structure in tissues with an injury. in clinical trials with humans undergoing flexor tendon Peritendineous adhesions as well as stiffness is injured surgery. Although a significant number of the patients or immobilized joints is a major clinical problem. Com exhibited hypersensitivity to the agent, decreased cova plete rehabilitation of a patient may be prolonged or lent bonding of human collagen with significant clinical prevented by such adhesions, scar contractures or joint benefit was demonstrated. stiffness. The lesions are due to the formation and re One way to reduce the systemic toxicity of BAPN modeling of collagen within scar, ligaments, or fascia. It without interfering with its lathyrogenic activity is to O was shown experimentally in rats and dogs that inhibi inhibit the metabolism of this drug. If degradation of tion of collagen cross-linking in and around tenolyzed BAPN is blocked by pargyline, a monoamine oxidase tissue or an immobilized joint using systemically-admin (MAO) inhibitor, prolonged lysyl oxidase inhibition istered BAPN could significantly improve the tendon potentiates wound strength diminution without accom gliding fraction or reduce stiffness. In both species, panying toxicity. Indeed, isoniazid (INH), both a weak 15 however, the systemic administration of the lathyrogen lysyl oxidase and MAO inhibitor potentiate the lathy resulted in undesirable side effects. Still, the beneficial ritic effects of BAPN. The addition of isoniazid or par effects on reducing adhesions or joint stiffness have gyline to an otherwise effective dose of BAPN pro encouraged my efforts to introduce the lathyrogen lo foundly inhibited lysyl oxidase and depressed wound cally at the site of the tendon lesion or over the immobi burst strength. All animals gained weight throughout 20 lized joint by topical administration. the experiment. Percutaneous Resorption In order to overcome the general toxicity of systemi This disclosure proposes to inhibit collagen in spe cally administered BAPN, I studied two methods, both cific fibrosis related lesions either by topical application of which showed to be unsuccessful. In the first method of BAPN or other lathyrogens onto the skin or by di BAPN was bound by amide- and ionic linkage to a 25 rect injection into the lesion. The major limitation of polymer such as polyacrylic acid, with the aim to obtain topical drug administration onto the skin surface is the long lasting sustained release of the drug. It was found, imposing impermeability of the skin, which forms a however, that many carriers for BAPN induced nonac chemical and waterproofing seal. By several studies it ceptable fibrotic tissue reaction when injected in injured was shown that the rate of percutaneous resorption is tissues. The second approach intended to increase the 30 limited mainly by the stratum corneum, formed by 15 percutaneous absorption of BAPN by synthesizing cells thick layer of cornified cells. Substantial knowl BAPN base. Indeed it was demonstrated that BAPN edge has been accumulated and reviewed on the perme base penetrates the skin barrier approximately five times ability of the skin...... ; faster than BAPN fumarate. Unfortunately, the base It was shown that the rate of transport of some sol showed to be unstable under storage, undergoing hy 35 utes through the skin depends on: drolysis forming and toxic . For (a) polar-nonpolar nature of the substance these reasons we discontinued the use of either polym (b) hydration of the skin er-BAPN fumarate complex of BAPN-base. (c) blood supply Based on the evidence of systemic toxicity of BAPN, (d) modification of the stratum corneum by chemi I arrived at the conclusion that the only method of 40 cals. : ; ; ; ; ; ; ; ; ; ; preventing the toxic problems of this clinically impor Accordingly, non-charged molecules, penetrate tant and needed drug is to administer BAPN and other faster. I demonstrated this by showing higher penetra lathyrogens topically. There exist several clinical symp tion of BAPN-base than that of BAPN-fumarate. The toms with collagen pathology, which could be treated instability of the base is, however, the major obstacle in with benefit by topically administered BAPN. These 45 using this drug. Occlusive bandage with the solutes include skin scar contractures, such as after deep burns, enhances penetration considerably. UV-irradiation of peritendineous adhesions after injury to the tendon, the skin, causing erythema, is supporting percutaneous perineural adhesions, stiffness of periarticular tissue of resorption. Detergents, dimethylsulfoxide (DMSO) and immobilized joints and Dupuytren's contracture and several other solvents have been shown to substantially other fibrotic lesions common in human and veterinary 50 increase the process. Beta-aminopropionitrile (BAPN), medicine. - as the term is used herein, includes both free BAPN and Although the pathogenesis of the above disorders BAPN salts, such as BAPN-fumerate, which also func may differ, in everyone it was postulated that the physi tion to inhibit lysyl oxidase. The various solvents and cal properties of the scar tissue were affected by abnor solvent mixtures, including aqueous buffers, and water mal cross-linking of the collagenous component. A 55 containing solvent systems, such as buffer/DMSO, variety of methods was proposed and used to alleviate which are used to carry BAPN for topical administra the problems of abnormal reactivity of polymerized tion are also referred to herein as drug carriers. It will scar tissue (Peacock and Van Winkle 1976). In all the be understood by those skilled in the art that such above manifestations BAPN systemic administration carries may be formulated to contain emulsifiers or the was suggested. The ideal method of topical administra 60 like for use particularly in compounding the BAPN for tion would be the noninvasive percutaneous resorption external topical, i.e., percutaneous administration. The of BAPN painted onto the skin as proposed in this dis drug and its drug carrier are referred to, collectively, as closure. I will show that BAPN is transported through a drug preparation. . . . . the skin and effectively inhibits lysyl oxidase in granu This disclosure will demonstrate that BAPN-fuma loma tissue formed in subcutaneously implanted polyvi 65 rate effectively penetrates the stratum corneum barrier, nyl alcohol sponge. At the same time, no morphological and is excreted into urine, if administered onto the pre or ultrastructural evidence of topical cytotoxicity is treated skin by a method described by this invention. found. These findings assured me that topical BAPN is The most important is, however, that due to this trans 4,485,088 7 8 port, BAPN inhibits collagen maturation (lysyl oxidase phosphate buffer (0.1M, pH 7.2) and for easy detection activity) in the subcutaneously induced granuloma tis 2 uCi of C-6-aminopropionitrile fumarate was painted sue. This makes it possible to use the simplest method of on the shaved skin area 2X2 cm of a rat. In another rat, BAPN administration, i.e., painting the pretreated skin the shaved skin was washed first with 2% Ivory soap, in with BAPN base fluid in combination with occlusive the next group the skin area after painting was covered dressing or to use special delivery system as consisting with occlusive bandage. In another group of rats (Table of hydrophilic pouch containing the solution of the 1) the drug was administered onto the skin the drug in drug described in this disclosure. 60% dimethyl sulfoxide, a solvent known to increase percutaneous absorption. Finally, in the last group OBJECTIVES OF THIS INVENTION O (Table 1), the basic solution was administered into a It is the object of this invention to demonstrate that small container made of a hydrogel polymer. This hy topically administered substances delivered locally to drogel is characterized by containing 80% water. It the site of the fibrotic injury and interfering with colla makes good contact with the skin, allows penetration of gen polymerization and called lathyrogens are not caus the drug through the container wall, hydrates the skin ing any side toxic effects local or systemic. 5 under the occlusive dressing, thus enhancing the percu Another object of this invention is to show that topi taneous transport. TABLE EFFECT OF VEHICLE AND SKN PRETREATMENT ON THE PERCUTANEOUS ABSORPTION OF BAPN-FUMARATE IN THE RAT Drug Recovery in the Urine Group % Recovered in 6 hours 1. BAPN-F W 0.97 in phosphate buffer on intact skin 2. BAPN-F - 2.17 in phosphate buffer skin washed with 2% Ivory soap 3. BAPN-F r 0.56 in phosphate buffer skin washed with 2% Ivory soap, occlusive bandage 4. BAPN-F ... : . 66 in phosphate buffer in 60% DMSO 5. BAPNF Y . . . . 0.10 in phosphate buffer in 60% DMSO . . . skin washed in 2% Ivory soap: 6. BAPN-F 35.00 in phosphate buffer . . . skin washed with 2% Ivory'soap : applied in hydrophilic container, occlusive bandage C-BAPN-fumarate, 2 Ciin 30 mg BAPN-fumarate/ml, 0.1 ml applied/skin area DMSO is dimethylsulfoxide % recovered in 24 hours cally administered lathyrogens are effectively changing the functional characteristics of the fibrotic lesions. 40 Yet another object of this invention is to show that a The results, shown in Table 1 indicate the importance lathyrogen such as 6-aminopropionitrile (BAPN) when of cleaning the skin from contaminating lipids and ke administered topically on the skin adjacent to the under ratinous debris by Ivory soap (Group 2). It also shows lying fibrotic lesion, is penetrating across the skin by a the significant role of occlusive dressing on the magni process called percutaneous absorption and is affecting 45 tude of percutaneous transport (Group 3). The enhance the chemistry and function of the fibrotic injury. ment by DMSO is documented by the results of Groups Yet another object of this invention is to show that 4 and 5. The major accomplishment was the continuous direct injection of lathyrogens into the lesion such as delivery of the drug through a hydrophilic container, injury to the tendon called tendinitis or tendovaginitis is attached to the skin by occlusive dressing formed by effectively improving the clinical picture of the disease. 50 surgical tape Blenderm (3M Company, Minnesota). By Finally, the invention will demonstrate that combina this method (Group 6) the delivery was increased al tion of lathyrogens such as g-aminopropionitrile and most 40 times when compared with Group 1. It is obvi D-penicillamine administered topically into the lesion ous that by this container a sustained release of the by injection or applied onto the intact skin by method of lathyrogen or combination of this drug could be ob percutaneous absorption allowing both drugs to pene 55 tained and that its (their) penetration across the skin trate into the fibrotic lesion is more effective than the barrier is achieved. administration of either substance alone. The invention is further and more specifically ex EXAMPLE 2 plained by the following examples which are given by shows a method of delivering the lathyrogen across the way of illustration and not by way of limitation. 60 skin barrier by sustained release from a bag made of a hydrogel polymer. s EXAMPLE 1 The experimental conditions were similar to those of will demonstrate that a solution of a lathyrogen applied Example 1 for Group 6. A 0.4 ml solution of 1.1M (3- onto the skin penetrates the skin barrier and is detect aminopropionitrile fumarate containing 200 mg/ml and able in the urine. . . . : ...... 65 2 puCi of the same 14C labeled substance was placed into Adult rats, Sprague Dawley strain, were shaved on a hydrophilic bag membrane. This membrane contained the dorsum. A 0.1 ml solution of 0.16M 3-aminopro 80% water. The container bag was placed onto the pionitrile fumarate, containing 30 mg BAPN/ml of shaved skin area of 4 cm2, covered with impermeable 4,485,088 9 10 occlusive dressing represented by Blenderm 'surgical excised and used for the determination of the activity of tape (3M, Minnesota). The rats were placed in meta lysyl oxidase. - bolic cages and at defined time intervals, the urine was The results in Table 4 show significant inhibition of collected and the amount of radioactive drug deter the enzyme activity in the lesion when BAPN was mined. Table 2 shows that during 120 hours of observa painted over the implanted sponge. This documents that tion 22.5% of the drug penetrated across the skin and the drug penetrates across the skin barrier into the site that the release was continuous at the constant rate. of the fibrotic injury and exerts its inhibitory effect on TABLE 2 the enzyme involved in the polymerization of collagen structures. - RECOVERY OF B-AMINOPROPIONITRILE FROM THE 10 URINE AFTER TSADMINISTRATION ON THESKIN OF TABLE 4 RATS THROUGH AHYDROPHILIC BAG CONTAINER EFFECT OF TOPICAL APPLICATION OF g-AMINOPRO Recovery in Urine PIONTRILE FUMARATE ON THE ACTEVITY OF LYSYL Time (hours) (In 26 of the Total Dose) OXIDASE IN THE GRANULOMATISSUE OF RATS 4 0.5 Lysyl Oxidase Activity 8 1.01 15 Group DPM/mg protein 12 2.37 24 5.40 Control (no treatment) -36.27.1 72 9.9 P. < 0.001 96 5.2 BAPN-fumarate, topical 1.8 - 0.3 120 22.5 20 . There were four-rats, in each group. Time indicates the urine sampling period. Recovery is Variability is given by standard deviation. given in percent of the dose administered into the hy Significance of the results tested by Student's t-test. drophilic bag. - EXAMPLE5 EXAMPLE 3 25 will document the therapeutic effectiveness of direct shows that a lethal dose of a lathyrogen administered infusions of g-aminopropionitrile fumarate solution into percutaneously is almost ten times higher than when the dissected peritendinous adhesions in a rooster model administered systemically by intraperitoneal injection. of tendon injury. The determination of lethal dose, killing within a 30 Adult White Leghorn chickens were sedated with certain time after the administration of the tested drug Pentothal, fixed to the operating table and given meta 50% of the exposed animals, is a standard method of carpal local anesthesia with 1% xylocaine. Following evaluation of the toxicity of a drug. ..., surgical prep of the foot, a tourniquet was applied. A total of 40 male CD-1 mice with a body weight of Through a mid-lateral incision the sublimus tendon was 20 g were injected either intraperitoneally with g 35 removed from the long digit of both feet and a 4–0 aminopropionitrile fumarate or the drug was applied in braided stainless steel, wire was passed through the en a solution onto the shaved dorsal aspect of the skin. tire length of the remaining profundus tendon to pro Four dosage levels spaced in geometric progression mote injury to the tendon by "scarification". The inci were evaluated using five animals for each dose. After sion was closed with a continuous nylon suture, 4-0. administration, the animals were observed for a 24-hour Both feet were then incorporated in a plaster of Paris period for incidence of death. The results shown in dressing with the feet in a functional position permitting Table 3 demonstrate that LD50 for intraperitoneally the animals to walk while in the plaster. . . ." administered BAPN-fumarate is at least 10 times higher "Three weeks later the animals were prepared for than when the drug is given percutaneously. surgery as above and the original incision was opened. 45 A complete tenolysis was performed freeing the profun TABLE 3 dus tendon from surrounding fibrotic adhesions. The ACUTE TOXICITY OF B-AMINOPROPIONITRILE FUMA RATE ADMINISTERED INTRAPERTONEALLY OR wound was closed using a continuous 4-0 nylon suture. TOPCALLY ON THESKIN A 1.0 ml silastic tube was prepared with multiple perfo LDSQ (g/kg body weight) rations over 3.0 centimeters of its distal end. This tube 50 was placed along side the digit operated upon and the intraperitoneally percutaneously digit with tube incorporated in a "Saran-Wrap' sheath. BAPN-fumarate 3.0 23.1 The foot was again incorporated in a walking plaster in a functional position with the silastic tube emerging from the proximal end of the cast. A Leur lock was EXAMPLE 4 55 attached to the tube permitting the infusion of 0.3 l of demonstrates the effectiveness of the drug, g-aminopro 1.6M beta-aminopropionitrile fumarate (300 mg/ml) pionitrile fumarate, in the fibrotic lesion in the subcutis every 12 hours for 5 days. Controls received 0.3 ml of after application of onto the intact skin. saline. Two groups of rats, four rats in each group, 250 g After 5 days of BAPN application the animals were body weight, Sprague-Dawley males, were implanted sacrificed, the plaster dressings removed, and the legs subcutaneously with a polyvinylalcohol sponge (Iva amputated. The flexor profundus tendon to the digit lon, Unipoint Labs, New Jersey) in the dorsal region which had undergone tenolysis was isolated at the level through an incision distal from the placement of the of the "ankle' and a traction wire was placed using a sponge. Bunnel suturing technique. The leg was then mounted A solution of 0.1 ml of 6-aminopropionitrile (200 mg 65 in an Instron Tensiometer and measured traction was BAPN/ml) was applied every 12 hours onto the skin applied to the flexor profundus tendon at a rate of 2.5 over the implanted sponge for a total of seven days. cm/min while the flexion of the three digital joints was Then the rats were sacrificed, reactive granuloma tissue measured. Data permitted the calculation of "Work of 4,485,088 11 12 Flexion' which is defined as the tension force required The other method of topical administration of la to flex the digit (toe-tip to the sole) divided by the sum thyrogens consists of the direct injection of sterile solu of the degrees of flexion of the three joints of the digit. tion of 2-5mM 3-aminopropionitrile with a 26 gauge needle into the lesion. Approximately 0.4 ml of the solution is injected at one site, number of injection sites TABLE 5 depend on the type and size of the pathology. In gen TOPCAL BAPN-FUMARATE eral, the injections are placed 2 cm apart, thus infiltrat Application Control (Saline) ing the lesion. The injections are repeated daily for a Work of Flexion (Mean) 13.10 gm/degree 44.8 gm/degree period of 1-6 weeks, depending on the type of the pa Standard Deviation 4.25 9.9 10 thology. In the case D-penicillamine is used, the con t-statistic (paired data) = -3.94 centration of this drug for local injections is 10-30mM. P & 0.01 Same volume per injection site and frequency of the injection is used with D-penicillamine as with 3-amino Table 5 shows that tendons treated with topical . BAPN-fumarate glided more easily; much lower force 5 It is with advantage to use the combination of both was needed to flex the finger. Similar results were ob drugs either for percutaneous application or for local tained by direct injection of BAPN-fumarate solution injections. into the injured tendon. The effective therapeutic dose of D-penicillamine in either application method is always 2-6 times higher DESCRIPTION OF THE INVENTION 20 than that of g-aminopropionitrile dose. The invention deals with a treatment of pathological What is claimed is: situations arising from accumulated collagenous tissue 1. A method of treating a wound to reduce the extent in the form of a scar, fibrotic adhesions or increased of collagen crosslinking at the wound site, comprising formation of stable crosslinks in the collagenous matrix preparing a drug preparation containing beta-amino 25 propionitrile in a drug carrier, and by local administration of lathyrogenic substances, such applying a therapeutically effective amount of the as 6-aminopropionitrile or D-penicillamine either onto drug preparation locally to the wound site. the skin or by local injection of the sterile solution of the 2. The method of claim 1, wherein the drug prepara above drugs, or their combination into the site of the tion is prepared to include beta-aminopropionitrile at a lesion. In the first case, solution of 8-aminopropioni 30 concentration between about 0.2M and 2M, and said trile, containing 100 mg to 300 mg per milliliter of water applying includes placing the preparation in contact is painted at 12 hour intervals onto the soap washed with the skin surface of the wound site. skin. It is possible to apply the drug to the skin over the 3. The method of claim 2, wherein the drug carrier lesion at the same concentrations in the carrier of a gel, includes water or water and DMSO, and said applying ointment, jelly for easier administration. The treatment 35 includes placing the drug preparation on the skin at the should be administered for a period of 2 to 12 weeks, wound site, and covering the site with an occlusive depending on the nature of the fibrotic pathology. It is material to reduce drying at the site. with advantage to wash the skin first with a keratolytic 4. The method of claim 3, wherein the drug prepara agent such as 20% solution of or 20% tion is contained in a porous hydrophilic support which solution of urea to increase the percutaneous transport is placed against the skin surface of the wound site. of the drug or combination of both drugs. It is also with 5. The method of claim 2, wherein said applying advantage to cover the site of treatment with an occlu includes infusing the wound site subcutaneously with sive dressing consisting of impermeable polymer adhe the drug preparation. sive tape. st k 45

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