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US 2017.0049865A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0049865 A1 ROSENBERG et al. (43) Pub. Date: Feb. 23, 2017

(54) PROTEOLYTIC EXTRACT FROM Publication Classification FOR THE TREATMENT OF (51) Int. Cl. CONNECTIVE TISSUE DSORDERS A638/48 (2006.01) A6IR 9/08 (2006.01) (71) Applicant: MEDIWOUND LTD., Yavne (IL) A6IR 9/00 (2006.01) (72) Inventors: Lior ROSENBERG, Omer (IL); Guy A6IR 9/14 (2006.01) RUBIN, Lower Galilee (IL); Eilon A638/17 (2006.01) ASCULAI, Lehavim (IL) A6II 45/06 (2006.01) (52) U.S. Cl. CPC ...... A61K 38/4873 (2013.01); A61K 38/1732 (21) Appl. No.: 15/345,482 (2013.01); A61K 45/06 (2013.01); A61 K 9/0019 (2013.01); A61K 9/14 (2013.01); A61 K (22) Filed: Nov. 7, 2016 9/08 (2013.01); C12Y 304/22031 (2013.01); CI2Y 304/22032 (2013.01) Related U.S. Application Data (57) ABSTRACT (63) Continuation of application No. 14/233,082, filed on The present invention relates to a proteolytic extract Jan. 15, 2014, now Pat. No. 9,511,126, filed as obtained from bromelain for the treatment of connective application No. PCT/IL2012/050261 on Jul. 19, tissue diseases. In particular, the present invention relates to 2012. a pharmaceutical composition comprising a proteolytic (60) Provisional application No. 61/509,612, filed on Jul. extract obtained from bromelain for the treatment of dis 20, 2011. eases such as Dupuytren’s disease and Peyronie's disease.

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PROTEOLYTIC EXTRACT FROM developed and include radiotherapy, ultrasound, injection of BROMELAIN FOR THE TREATMENT OF Vitamin A, vitamin E. Steroids and interferon-Y. CONNECTIVE TISSUE DISORDERS 0007. In vitro studies have demonstrated the ability of collagenase to decrease the tensile modulus and the force CROSS-REFERENCE TO RELATED needed to rupture Dupuytren’s cord tissue, indicating that APPLICATIONS collagenase may be effective in enzymatic fasciotomy. 0001. This application is a continuation of U.S. patent Clinical studies have recently demonstrated that treatment application Ser. No. 14/233,082 filed on Jan. 15, 2014, with Clostridium histolyticum collagenase released DD con which is a 371 filing of International patent application no. tractures and improved the range of motion in affected PCT/IL2012/050261 filed on Jul.19, 2012, which claims the joints. An 8-year follow-up of collagenase injection in benefit of U.S. provisional patent application No. 61/509, patients with DD showed that the MCPJ contracture was less 612 filed on Jul. 20, 2011, the entire contents of each of severe following the recurrence of the disease, when com which are incorporated herein by reference thereto. pared to the initial contracture before applying the collage nase treatment. It has also been shown that Type-III colla FIELD OF THE INVENTION gen, which is usually absent from normal adult palmar fascia, is abundant in the tissue of patients with DD. 0002 The present invention relates to a proteolytic 0008 Peyronie's disease is a connective tissue disorder extract obtained from bromelain for the treatment of con involving the growth of fibrous plaques rich in collagen in nective tissue diseases. In particular, the present invention the soft tissue of the penis affecting up to 10% of men. relates to a pharmaceutical composition comprising a pro Specifically, the fibrous plaques are formed in the tunica teolytic extract obtained from bromelain for the treatment of albuginea, the thick sheath of tissue Surrounding the corpora diseases Such as Dupuytren’s disease and Peyronie's dis cavernosa, cause abnormal curvature which is often associ CaSC. ated with pain. 0009 Surgery is the only approach to treating Peyronie's BACKGROUND OF THE INVENTION disease which appears to have predictably repeatable effi 0003 Collagen is the main component of the connective cacy. Surgery is usually only indicated in long-term cases tissue and it is mostly found in fibrous tissues such as where the disease is stabilized and the deformity prevents tendon, ligament and skin. Numerous diseases and condi intercourse and/or causes extreme pain. However, compli tions are associated with excess collagen deposition, the cations can develop from Surgery, including a permanent most common are Dupuytren’s disease and Peronei's dis shortening of the penis. CaSC. 0010 Non-surgical approaches to Peyronies disease 0004 Dupuytren’s disease (DD) is a connective tissue treatment are also available, although they are all largely disorder of abnormal collagen production and deposition in ineffective. Attempts to dissolve the plaques by direct intra the hand that is commonly characterized by contracture of lesional injections have been tried. Of the injection meth metacarpophalangeal joints (MCPJs) and proximal interpha odologies, those involving clostridial collagenase appear to langeal joints (PIPJs) in the ring and little fingers. Fibroblast exhibit the most consistent efficacy, though still quite limited proliferation and differentiation into myofibroblasts with in effect and duration. In addition, radiation therapy and excess collagen deposition at the level of the palmar fascia laser technology have been tried. cause nodule and fibrotic cord formation in the palm and/or 0011 U.S. Pat. Nos. 5,589,171, 6,086,872 and Reissued digits. The fibrotic cords or nodules can be of varying U.S. Pat. No. RE39,941 disclose methods of treating an thicknesses, from 1 millimeter in diameter for the fibrotic individual suffering from Dupuytren’s disease which meth cords to nearly 10 millimeters in diameter for the fibrotic ods comprise applying collagenase to a fibrous affected nodules. As the disease progresses, cords begin to contract, palmar fascia. causing finger flexion-deformities (flexion contractures) 0012 U.S. Pat. No. 6,022,539 discloses methods of treat which interfere and decrease hand function. ing an individual suffering from Peyronie's disease which 0005. The prevalence of DD increases with age and methods comprise injecting collagenase into a fibrous Pey males are more often affected. Genetic Susceptibility, Smok ronie's plaque in the penis of the individual. ing, alcohol, diabetes mellitus, epilepsy and repetitive (0013 U.S. Pat. No. 6,353,028 discloses topical medica manual work are thought to be common risk factors for DD. ment which comprises calcium channel blocker agents and The severity and progress of DD can be classified by the carrier agents facilitating transdermal delivery of the cal affected degree of digital flexion-contracture. cium channel blocker for the treatment of connective tissue 0006 Surgical fasciectomy is currently the most widely disorders: Peyronie's disease, Dupuytren’s disease and Led available treatment for DD which provides positive, though derhose Fibrosis. temporary outcomes for most patients. However, Surgical (0014 U.S. Patent Application Publication No. 2008/ fasciectomy usually involves common Surgical complica 0206228 discloses a medicament containing tions (e.g., infection, hematoma, tissue loss) as well as or derivatives thereof in association with collagenase for the specific complications such as digital nerve damage, loss of treatment of various kinds of wounds, , pressure Sores, fingers, skin flap loss, wound healing problems and postop vascular ulcers, and diabetic foot ulcers as well as for the erative stiffness. In addition, fasciectomy involves a long treatment of hypertrophic and keloid scars. Treatment of recovery and does not offer a definitive cure as DD has an Dupuytren’s disease is explicitly disclosed. extremely high recurrence rate. Minimally invasive proce 0015 International Patent Application Publication No. dures using needles or thin blades have been tried; such WO 2004/037183 discloses methods and compositions for procedures though cause less complications, increase the treatment of conditions involving fibrosis, among which recurrence rate. Non Surgical interventions have also been Peyronie's disease and Dupuytren’s disease are disclosed. US 2017/00498.65 A1 Feb. 23, 2017

The compositions comprise a phosphodiesterase (PDE)-4 0025. According to one aspect, the present invention inhibitor, a PDE-5 inhibitor or a compound that elevates provides a method of treating a connective tissue disease cGMP, to list some. comprising administering to a subject in need of Such 0016 Use of cell cycle inhibitors, including anti-micro treatment a pharmaceutical composition comprising a thera tubule agents, antimetabolites, alkylating agents, Vinca alka peutically effective amount of a proteolytic extract obtained loids, PDE inhibitors, matrix metalloproteinase including from bromelain and a pharmaceutically acceptable carrier, , for treating a contracture such as Dupuytren’s wherein the proteolytic extract comprises at least one cys contracture or Peyronies contracture is disclosed in Inter teine selected from the group consisting of stem national Patent Application Publication No. WO 2005/ bromelain and , and wherein the connective tissue O749 13. disease is associated with excess collagen deposition. 0017 Nowhere in the background art is it disclosed or 0026. According to additional embodiments, the connec Suggested that proteolytic from plant Sources are tive tissue disease is selected from the group consisting of useful for treating connective tissue disorders involving Dupuytren’s disease, Peyronie's disease, frozen shoulder, excess collagen deposition. and Ledderhose disease. According to a certain embodiment, 00.18 Extracts derived from the stem of the the connective tissue disease is Dupuytren’s disease. plant (Ananas comosus) have been found to selectively According to another embodiment, the connective tissue remove devitalized tissue. Such extracts, also named bro disease is Peroneis disease. melain, contain various proteolytic and hydrolytic enzymes. 0027. According to one embodiment, the proteolytic 0019 International Patent Application Publication No. extract comprises and ananain. According to WO 2006/054309 to the applicant of the present invention another embodiment, the proteolytic extract further com discloses a debriding composition obtained from bromelain prises a protease precursor. According to a further comprising most of the proteolytic enzymes present in embodiment, the proteolytic extract further comprises a bromelain, the proteolytic enzymes having an average fragment. According to an exemplary molecular weight of 23 kDa. WO 2006/054309 further embodiment, the proteolytic extract comprises stem brome discloses uses of said debriding composition for debriding lain, ananain, and a cysteine protease precursor. non-viable tissues. 0028. According to further embodiments, the pharmaceu 0020. There remains an unmet need for improved non tical composition further comprising an agent selected from invasive methods for treating connective tissue diseases the group consisting of an anesthetic agent, antibacterial involving excess collagen deposition. agent and an anti-inflammatory agent. 0029. According to still further embodiments, the anes SUMMARY OF THE INVENTION thetic agent is selected from the group consisting of ame 0021. The present invention provides a proteolytic thocaine (tetracaine), lignocaine (lidocaine), Xylocaine, extract obtained from bromelain for the treatment of con bupivacaine, prilocaine, ropivacaine, benzocaine, mepivo nective tissue diseases. Particularly, the present invention caine, cocaine and combinations thereof. Each possibility is provides a proteolytic extract obtained from bromelain for a separate embodiment of the invention. the treatment of connective tissue diseases which are asso 0030. According to additional embodiments, the antibac ciated with excess of collagen deposition, including terial agent is selected from the group consisting of aman Dupuytren’s disease and Peyronie's disease. fadine hydrochloride, amanfadine Sulfate, amikacin, amika 0022. It is now disclosed for the first time that a pro cin Sulfate, amoglycosides, amoxicillin, amplicillin, teolytic extract obtained from bromelain comprising one or amsamycins, bacitracin, beta-lactams, candicidin, capreo more of the cysteine present in bromelain, e.g., mycin, carbenicillin, cephalexin, cephaloridine, cephalothin, stem bromelain or ananain, is capable of degrading native, cefazolin, cephapirin, cephradine, cephaloglycin, chi non-denatured collagen. Unexpectedly, injection of the pro lomphenicols, chlorhexidine, chloshexidine gluconate, chlo teolytic extract into a Dupuytren’s cord resulted in rupture of rhexidine hydrochloride, chloroxine, chlorquiraldol, chlo the cord while maintaining the normal healthy connective rtetracycline, chlortetracycline hydrochloride, ciprofloxacin, tissue intact. circulin, clindamycin, clindamycin hydrochloride, clotrima 0023 The present invention further discloses that the Zole, cloxacillin, demeclocycline, dicloSXacillin, diiodohy efficacy of the proteolytic extract to rupture or dissolve droxyquin, doxycycline, ethambutol, ethambutol hydrochlo Dupuytren’s cords is similar to or even higher than that of ride, erythromycin, erythromycin estolate, erhmycin collagenase. However, while collagenase may cause damage Stearate, farnesol, floxacillin, gentamicin, gentamicin Sul to non-diseased ligaments or tendons due to its affinity to the fate, gramicidin, giseofulvin, haloprogin, haloquinol, various types of collagen, the proteolytic extract of the hexachlorophene, iminocylcline, iodochlorhydroxyquin, present invention shows specificity to the diseased cords. kanamycin, kanamycin Sulfate, lincomycin, lineomycin, Thus, the proteolytic extract of the present invention pro lineomycin hydrochloride, macrollides, meclocycline, meth vides an improved and safe medication for connective tissue acycline, methacycline hydrochloride, methenine, meth diseases which involve excess collagen deposition, particu enamine hippurate, methenamine mandelate, methicillin, larly for the treatment of Dupuytren’s disease and Peronei's metonidazole, miconazole, miconazole hydrochloride, disease. minocycline, minocycline hydrochloride, mupirocin, nafcil 0024. Due to the fact that high concentrations of the lin, neomycin, neomycin Sulfate, netimicin, netilmicin Sul proteolytic extract can be prepared in Small Volumes, such fate, nitrofuraZone, norfloxacin, nyStatin, octopirox, olean small volumes can be injected into the diseased fibrous cords domycin, orcephalosporins, oxacillin, oxyteacline, or plaques, thus avoiding extravasation and damage to oxytetracycline hydrochloride, parachlorometa Xylenol, Surrounding tissues, simplifying the clinical procedure and paromomycin, paromomycin Sulfate, penicillins, penicillin hence increasing patient's compliance. G. penicillin V, pentamidine, pentamidine hydrochloride, US 2017/00498.65 A1 Feb. 23, 2017 phenethicillin, polymyxins, quinolones, streptomycin Sul 0040 FIG. 4 is a graph showing the gelatinase activity of fate, tetracycline, tobramycin, tolnaftate, triclosan, trifam the proteolytic extract. Increasing concentrations of two pin, rifamycin, rollitetracycline, silver salts, spectinomycin, batches of the proteolytic extract (designated J-01-19 and spiramycin, struptomycin, Sulfonamide, tetracyclines, tetra J-14-45) were incubated for 20 minutes in the presence of cycline, tobramycin, tobramycin Sulfate, triclocarbon, tri fluorescently labeled gelatin and thereafter fluorescence was closan, trimethoprim-sulfamethoxazole, tylosin, Vancomy measured. cin, and yrothricin. Each possibility is a separate 0041 FIGS. 5A-C are photographs showing surgical embodiment of the invention. excision-removal of the Dupuytren’s cord from a patient. 0031. According to further embodiments, the anti-in FIG. 5A is a photograph showing the abnormal contracture flammatory agent is selected from the group consisting of of the ring finger in a patient with Dupuytren’s disease. FIG. non-steroidal anti-inflammatory agents and steroidal anti 5B is a photograph showing the Surgical removal of the inflammatory agents. pathological cord. FIG. 5C is a photograph showing the cord 0032. According to yet further embodiments, the phar after removal from the palmar bed. maceutical composition further comprises a component 0042 FIG. 6 is a photograph showing the dissection of selected from the group consisting of a stabilizing agent, an the Dupuytren’s cord to two. anti-oxidant, a preservative, a buffering agent, a chelating 0043 FIG. 7 is a photograph showing the anchoring of agent, and a tonicity agent. the cord. 0033 According to still further embodiments, the phar 0044 FIG. 8 is a photograph showing the injecting step maceutical composition is formulated in a form selected of a solution into the Dupuytren’s cord. from the group consisting of a solid formulation, a semi 0045 FIG. 9 is a photograph showing the tensile stretch Solid formulation, a liquid formulation and a foam formu ing machine. lation. According to a certain embodiment, the Solid formu 0046 FIGS. 10A-B are photographs showing the cord lation is a powder. According to another embodiment, the before and after tensile force application. liquid formulation is an injectable solution of a pH of about 0047 FIG. 10A shows the cord before tensile force 6 to about 7. application. FIG. 10B shows the cord after tensile force 0034. According to an exemplary embodiment, the phar application and before cord rupture. maceutical composition is administered by injection into the 0048 FIG. 11 shows the effect of saline on Dupuytren’s diseased fibrous tissue. The pharmaceutical composition can cord elongation as a function of tensile strength application. be injected as a single dose or in aliquots at two or more Dupuytren’s cord was injected with saline and incubated in locations in the diseased fibrous tissue. saline for 24 hours. Thereafter, the cord was subjected to 0035. According to another aspect, the present invention tensile stress, and cord elongation and rupture was evalu provides a pharmaceutical composition comprising a pro ated. teolytic extract for use in the treatment of a connective tissue 0049 FIG. 12 shows the effect of the proteolytic extract disease, wherein the proteolytic extract comprises at least on Dupuytren’s cord elongation as a function of tensile one cysteine protease selected from the group consisting of strength application. Dupuytren’s cord was injected with the stem bromelain and ananain, and wherein the connective proteolytic extract and incubated in the presence of the tissue disease is associated with excess collagen deposition. proteolytic extract for 24 hours. Thereafter, the cord was 0036. These and other embodiments of the present inven Subjected to tensile stress, and cord elongation and rupture tion will be better understood in relation to the figures, was evaluated. description, examples, and claims that follow. 0050 FIG. 13 shows the effect of a single injection of the proteolytic extract on Dupuytren’s cord elongation as a BRIEF DESCRIPTION OF THE DRAWINGS function of tensile strength application. Dupuytren’s cords 0037 FIG. 1 is a graph showing the collagenolytic activ were injected with the proteolytic extract and incubated in ity of two batches of the proteolytic extract. Increasing saline for 24 hours. Thereafter, the cords were subjected to concentrations of the proteolytic extracts (designated MD2 tensile stress, and cord elongation and rupture were evalu H-05-27 and MD5 H-10-46) were incubated for 20 minutes ated. in the presence of fluorescently labeled collagen type IV. At the end of the incubation fluorescence was measured. DETAILED DESCRIPTION OF THE Results are presented in relative fluorescence units (RFU). INVENTION 0038 FIG. 2 is a graph showing the collagenolytic activ 0051. The present invention provides methods of treating ity of the proteolytic extract on collagen type I and type IV. connective tissue diseases involving excess collagen depo The proteolytic extract was incubated in the presence of sition comprising administering to a Subject in need of Such fluorescently labeled collagen type I or collagen type IV for treatment a proteolytic extract obtained from bromelain. various time periods. At the end of the incubation fluores 0.052 A debriding composition obtained from bromelain cence was measured. Results are presented in relative fluo (also termed Debrase(R) was first disclosed in WO 2006/ rescence units (RFU). 054309 to the applicant of the present invention, the content 0039 FIG. 3 is a graph showing the collagenolytic activ of which is incorporated by reference as if fully set forth ity of the proteolytic extract as compared to that of colla herein. The debriding composition disclosed in WO 2006/ genase. Clostridium histoliticum collagenase was incubated 054309 comprises cysteine proteases such as stem brome with either fluorescently labeled collagen type IV or fluo lain and ananain. WO 2006/054309 further discloses that the rescently labeled gelatin and fluorescence was measured at debriding composition debrided burned skin, i.e., devital the end of 20 minutes of incubation. The proteolytic extract ized tissue, more efficiently than bromelain. However, the was incubated with fluorescently labeled collagen type IV debriding composition was found to be inactive in debriding for 20 minutes and thereafter fluorescence was measured. healthy or vital skin or dermis (see, for example, Singer et US 2017/00498.65 A1 Feb. 23, 2017

al., 2010, J. Care Res. 31: 304-309). The debriding 0.063 (f) adjusting the suspension of (e) to a pH from composition was therefore shown to be active against devi about 2.5 to about 4: talized tissues, not against viable tissues. 0.064 (g) incubating the suspension of (f) at 3°C.-10° 0053. Unexpectedly, the present invention discloses that C.; a proteolytic extract obtained from bromelain exerted coll 0065 (h) centrifuging the suspension of (g) to yield an genolytic activity in vitro and was capable of dissolving the ammonium Sulfate precipitate; palmar fibrotic cords obtained from subjects suffering from 0.066 (i) dissolving the ammonium sulfate precipitate Dupuytren’s disease. As the proteolytic extract of the pres in an acidic solution optionally comprising an anti ent invention does not degrade healthy connective tissue, the oxidant having a pH in the range from about 2.4 to present invention thus provides safe and efficient enzymatic about 4: medicament for dissolving fibrous tissue rich in collagen, specifically in Subjects Suffering from Dupuytren’s disease 0067 () filtering the solution of (i) through a 10 kDa or Peronei's disease. ultra-filter; and 0054 The terms “proteolytic extract obtained from bro 0068 (k) lyophilizing the retained solution of (). melain” and “proteolytic extract are used interchangeably 0069. According to some embodiments, suspending bro throughout the specification and claims and refer to an melain can be performed in any acidic Solution having a pH enzymatic preparation partially purified from bromelain. between about 2.4 to 4. Examples of acidic solutions or 0055. The term “bromelain” refers to any of a number of buffers that can be used according to the present invention presently commercially available bromelain powder prepa include, but are not limited to, acetic acid in water, acetate rations. Examples of manufacturers of bromelain include, buffer and acetate buffer containing 1% thioglycolic acid, but are not limited to, Sigma and Challenge Bioproducts Co. pH 2.4-4. According to certain exemplary embodiments, the Ltd., Taiwan. Bromelain is prepared from the stem of acidic solution is selected from the buffers and solutions pineapple plant. A typical procedure to obtain bromelain is disclosed in U.S. Pat. Nos. 5,830,739 and 4,197,291, the as follows: the juice from the stem of pineapple plant is first content of which is incorporated by reference as if fully set adjusted to a pH of about 3 or 4 with phosphoric acid, and forth herein. sodium hydride or sodium sulfhydride is added to protect 0070 The acidic solution can optionally comprise an against Sulfhydryl oxidation. The inert material is precipi anti-oxidant. Examples of anti-oxidants include, but are not tated at about 30% acetone and, after filtration, the clarified limited to, ascorbic acid, dihydroquinon, butylated hydroxy fluid is precipitated with 70% acetone. This precipitate is toluene and dithiothreitol. The anti-oxidant can be added at collected by centrifugation and either redissolved in water a concentration of about 0.5% to about 2%, preferably at containing sodium hydride or Sodium Sulfhydride which has 19/6. been acidified with phosphoric acid and reprecipitated, or 0071. The acidic solution can further comprise a wetting dried in a vacuum oven directly. If the material is reprecipi agent. Examples of wetting agents include, but are not tated, 70% acetone is utilized. The dried material from either limited to, n-octanol. process is suitable as a starting material to obtain the 0072 The pH of the acidic solution, which optionally debriding composition of the present invention. comprises an anti-oxidant, can be in the range from about 0056. The proteolytic extract of the present invention can 2.4 to about 4. According to a certain preferred embodiment, comprise one or more of the cysteine proteases present in the pH of the acidic solution, which optionally comprises an bromelain. According to an exemplary embodiment, the anti-oxidant, ranges from about 2.4 to about 2.6. proteolytic extract (also termed Debrase(R) or NexobridR) 0073. According to additional embodiments, a filter aid is comprises the cysteine proteases stem bromelain (EC 3.4. added to the Suspension of (a). According to one embodi 22.32) and ananain (EC 3.4.22.31). The proteolytic extract ment, the filter aid comprises silica. Preferably, the filter aid can further comprise one or more of the cysteine protease is natural diatomite that is calcined so that faster flow rates precursors of bromelain such as, for example, ananain (EC are achieved. 3.4.22.31) precursor, (EC 3.4.22.33) precur 0074 Precipitating the desired proteins is performed by sor, and stem bromelain (EC 3.4.22.31) precursor. The adding to the filtered solution of step (d) ammonium sulfate proteolytic extract can further comprise cysteine protease salt. Ammonium sulfate salt can be added to yield Saturation fragments (see, for example, WO 2006/054309), a jacalin of the ammonium sulfate at a range of between about 40% like lectin (see, for example, Raval et al., Glycobiology, to about 50%. Preferably, ammonium sulfate salt can be 2004, 14(12): 1247-1263), and/or bromelain inhibitors. added to yield 40% saturation of ammonium sulfate. 0057 The proteolytic extract can be prepared by a 0075. The suspension of step (f) is then incubated at a method comprising the following steps: temperature between 3°C. to 10° C. Preferably, the suspen 0.058 (a) suspending bromelain with an acidic solution sion of step (f) is incubated for at least 10 hours at tem optionally comprising an anti-oxidant, the acidic solu peratures between 3° C. to 10° C. More preferably, the tion having a pH in the range from about 2.4 to about suspension of step (f) is incubated for 12-24 hours at 4°C. 4. 0076. At the end of the incubation, the suspension of step 0059 (b) adjusting the suspension of (a) to a pH in the (g) is centrifuged to precipitate the desired proteins, i.e., the range from about 2.4 to about 4. proteolytic enzymes. The precipitate is then dissolved in 0060 (c) adding a filter aid to the suspension of (b): acidic Solution optionally comprising an anti-oxidant. 0061 (d) filtering the suspension of (c) to remove According to an exemplary embodiment, the Suspension is insoluble components; incubated for at least 10 hours at 4° C. 0062 (e) adding to the filtered solution of (d) ammo 0077. The solution of step (i) is subjected to a step of nium sulfate salt to yield Saturation of ammonium filtering to retain proteolytic enzymes having molecular sulfate in the range from about 40% to about 50%: weights in excess of about 10 kDa. According to a preferred US 2017/00498.65 A1 Feb. 23, 2017

embodiment, the solution of step (i) is filtered through a 0094. It is to be understood that the proteolytic extract of membrane filter having a molecular weight cut off of about the invention is useful for treating individuals having other 10 kDa. diseases associated with excess collagen deposition. Other 0078. The proteolytic extract can be lyophilized after fibrous tissue malformations and abnormalities which filtration, can be washed with distilled water and then involve collagen deposition include Peyronie's disease, Led lyophilized or can be filtered and then lyophilized. Accord derhose Fibrosis, and fibrosis of joint-capsules, tendons and ing to a currently preferred embodiment, the proteolytic ligaments sheaths. Thus, the proteolytic extract can also be extract is filtered through a filter membrane having a pore useful for treating frozen shoulder (adhesive capsulitis). size of at least about 0.5 um to obtain a sterile solution, These connective tissue diseases involving excess collagen which is then lyophilized and stored. Preferably, the pro deposition or fibrous tissue malformations are not associated teolytic extract is stored as a lyophilized powder as its with wounds or burns. stability is prolonged in the absence of moisture. Prior to (0095. As used herein, the terms “treating” or “treatment” use, the proteolytic extract is dissolved in a solution so as to refer to amelioration or elimination of at least one or more obtain a solution with a pH of about 6 to about 7. of the symptoms associated with a connective tissue disease. 0079 According to an exemplary embodiment, the pro For example, symptoms associated with Dupuytren’s dis teolytic extract can be prepared by the method comprising ease include joint contracture, decrease range of motion of the following steps: the joints, to list some. Symptoms of Peyronie's disease 0080 (a) suspending bromelain with 0.3 Macetic acid include, for example, pain, abnormal curvature, and erectile comprising 1% ascorbic acid and n-octanol having a dysfunction. pH from about 2.4 to about 2.6; (0096. The term “therapeutically effective amount” of the I0081 (b) adjusting the suspension of (a) to a pH in the proteolytic extract is that amount of the proteolytic extract range from about 2.5 to about 3.5; which is sufficient to provide a beneficial effect to the subject I0082 (c) adding a filter aid comprising silica to the to which the composition is administered. Suspension of (b): (0097. The term “about” when refers to a pH of a solution I0083 (d) filtering the suspension of (c) through a filter or suspension is meant to indicate that 0.5 pH units above or press to remove insoluble components; below the indicated pH are within the scope of the present I0084 (e) adding to the filtered solution of (d) ammo invention. nium sulfate salt (285 g/L) to yield 40% saturation of 0098. The pharmaceutical composition of the present ammonium sulfate; invention comprises the proteolytic extract and a pharma I0085 (f) adjusting the suspension of (e) to a pH from ceutically acceptable carrier. about 2.5 to about 3.5; 0099. The term “pharmaceutically acceptable” means I0086 (g) incubating the suspension of (f) for approxi approved by a regulatory agency of the Federal or a state mately 12-24 hours at 4°C.; government or listed in the U.S. Pharmacopeia or other I0087 (h) centrifuging the suspension of (g) to yield an generally recognized pharmacopeia for use in animals, and ammonium Sulfate precipitate; more particularly in humans. I0088 (i) dissolving the ammonium sulfate precipitate 0100. The term “carrier” refers to a diluent, excipient, or in 0.3 M acetic acid comprising 1% ascorbic acid vehicle with which the proteolytic extract is administered. having a pH from about 2.4 to about 2.6; Such pharmaceutical carriers can be sterile liquids, Such as I0089 () filtering the solution of (i) through a 10 kDa water and oils, including those of petroleum, animal, Veg ultra-filter; etable or synthetic origin, such as peanut oil, Soybean oil, 0090 (k) filtering the retained solution of () to yield a mineral oil, sesame oil and the like, polyethylene glycols, sterile solution; and glycerine, propylene glycol or other synthetic solvents. 0091 (1) lyophilizing the filtered solution of (k). Saline solutions, aqueous NaCl/CaCl2 solution, aqueous 0092. The term “Dupuytren's disease” and “DD” are dextrose, glycerol Solutions and albumin Solutions can be interchangeably used herein and refer to a disease where the employed as liquid carriers, particularly for injectable solu fingers cannot fully extend and are usually flexed towards tions. Water can also be used as a carrier when the pharma the palm of the hand. Specifically, Dupuytren’s disease ceutical composition is administered intravenously; begins with the formation of fibromatous nodules in the 0101 The pharmaceutical composition can further com palmar fascia, usually in the ulnar side. The nodules progress prise Stabilizing agents such as lactose, dextrose, Sucrose, and form a fibrous band or cord lying from the palm to the Sorbitol, mannitol, starch, gum acacia, calcium phosphate, fingers. Eventually this leads to permanent finger flexion alginates, tragacanth, calcium silicate, polyvinylpyrrolidone contractures. The ring finger is most commonly affected, and cellulose. The composition can additionally include followed by the little finger. lubricating agents, such as, magnesium Stearate and mineral 0093. The terms “Dupuytren’s cord” and “the diseased oil; wetting agents; emulsifying and Suspending agents; cord are used herein interchangeably and refer to the bands preservatives such as Thimerosal, benzyl alcohol, parabens, of fascial fibers that run longitudinally bellow the palmar methyl- or propylhydroxybenzoates; anti-oxidants such as skin. These bands lead to contractures of the overlay skin ascorbic acid, dihydroquinon, butylated hydroxytoluene and and distal digits that are attached to the bands and eventually dithiothreitol; and buffering agents such as monobasic progresses to permanent flexion-contracture of the affected Sodium phosphate, dibasic sodium phosphate, sodium ben digits. Typically, the Dupuytren’s cord comprises high num Zoate, potassium benzoate, sodium citrate, Sodium acetate, bers of fibroblasts, increased deposition of extracellular and Sodium tartrate; chelating agents such as ethylenedi matrix (ECM) proteins, particularly collagen, and myofi aminetetraacetic acid; and agents for the adjustment of broblasts. tonicity Such as sodium chloride or dextrose. US 2017/00498.65 A1 Feb. 23, 2017

0102 The pharmaceutical composition can further com Zone, azapropaZone, and trimethaZone. Extracts of these prise an anesthetic agent. non-steroidal anti-inflammatory agents may also be employed. 0103) Anesthetic agents include, but are not limited to, 0108) Non-limiting examples of steroidal anti-inflamma amethocaine (tetracaine), lignocaine (lidocaine), Xylocaine, tory drugs include, corticosteroids such as hydrocortisone, bupivacaine, prilocaine, ropivacaine, benzocaine, mepivo hydroxyl-triamcinolone, alpha-methyl dexamethasone, dex caine, cocaine and combinations thereof. amethasone-phosphate, beclomethasone dipropionates, clo 0104. The pharmaceutical composition can further com betasol Valerate, desonide, desoxymethasone, desoxycorti prise an antibacterial agent. costerone acetate, dexamethasone, dichlorisone, diflorasone 0105 Antibacterial agents include, but are not limited to, diacetate, diflucortolone Valerate, fluadrenolone, fluclo amanfadine hydrochloride, amanfadine Sulfate, amikacin, rolone acetonide, fludrocortisone, flumethasone pivalate, amikacin Sulfate, amoglycosides, amoxicillin, amplicillin, fluosinolone acetonide, fluocinonide, flucortine butylesters, amsamycins, bacitracin, beta-lactams, candici din, capreo fluocortolone, fluprednidene (fluprednylidene) acetate, flu mycin, carbenicillin, cephalexin, cephaloridine, cephalothin, randrenolone, halcinonide, hydrocortisone acetate, hydro cefazolin, cephapirin, cephradine, cephaloglycin, chi cortisone butyrate, methylprednisolone, triamcinolone lomphenicols, chlorhexidine, chloshexidine gluconate, chlo acetonide, cortisone, cortodoxone, flucetonide, fludrocor rhexidine hydrochloride, chloroxine, chlorquiraldol, chlo isone, difluorosone diacetate, fluradrenolone, fludrocorti rtetracycline, chlortetracycline hydrochloride, ciprofloxacin, Sone, difluroSone diacetate, fluradrenolone acetonide, circulin, clindamycin, clindamycin hydrochloride, clotrima medrysone, amcinafel, amcinafide, betamethasone and the Zole, cloxacillin, demeclocycline, dicloSXacillin, diiodohy balance of its esters, chloroprednisone, chlorprednisone droxyquin, doxycycline, ethambutol, ethambutol hydrochlo acetate, clocortelone, clescinolone, dichlorisone, diflurpred ride, erythromycin, erythromycin estolate, erhmycin nate, flucloronide, flunisolide, fluoromethalone, fluperolone, Stearate, farnesol, floxacillin, gentamicin, gentamicin Sul fluprednisolone, hydrocortisone Valerate, hydrocortisone fate, gramicidin, giseofulvin, haloprogin, haloquinol, cyclopentylpropionate, hydrocortamate, meprednisone, hexachlorophene, iminocylcline, iodochlorhydroxyquin, paramethasone, prednisolone, prednisone, beclomethasone kanamycin, kanamycin Sulfate, lincomycin, lineomycin, dipropionate, triamcinolone, and extracts thereof. lineomycin hydrochloride, macrollides, meclocycline, meth 0109 The pharmaceutical composition can be formulated acycline, methacycline hydrochloride, methenine, meth as a dry or lyophilized formulation, semi Solid formulation, enamine hippurate, methenamine mandelate, methicillin, liquid formulation or a foam formulation. Thus, the phar metonidazole, miconazole, miconazole hydrochloride, maceutical composition can be formulated in the form of a minocycline, minocycline hydrochloride, mupirocin, nafcil powder, Solution, Suspension, emulsion, gel, spray, or a lin, neomycin, neomycin Sulfate, netimicin, netilmicin Sul patch. fate, nitrofuraZone, norfloxacin, nyStatin, octopirox, olean 0110. The pharmaceutical composition can be adminis domycin, orcephalosporins, oxacillin, oxyteacline, tered into the affected site topically, Subcutaneously, intra oxytetracycline hydrochloride, parachlorometa Xylenol, cutaneously, or intramuscularly. paromomycin, paromomycin Sulfate, penicillins, penicillin 0111. According to a certain embodiment, the pharma G. penicillin V, pentamidine, pentamidine hydrochloride, ceutical composition is administered by injection. According phenethicillin, polymyxins, quinolones, streptomycin Sul to an exemplary embodiment, the pharmaceutical composi fate, tetracycline, tobramycin, tolnaftate, triclosan, trifam tion is injected directly into the diseased fibrous nodules or pin, rifamycin, rollitetracycline, silver salts, spectinomycin, cord or into the fibrous plaque. Alternatively, the pharma spiramycin, struptomycin, Sulfonamide, tetracyclines, tetra ceutical composition is implanted into a Surgical incision. cycline, tobramycin, tobramycin Sulfate, triclocarbon, tri Sterile injectable preparations may be formulated as aqueous closan, trimethoprim-sulfamethoxazole, tylosin, Vancomy Solutions or oleaginous Suspensions as known in the art. cin, and yrothricin. 0112 For topical use on the skin the pharmaceutical composition can be formulated in the form of an ointment, 0106. According to yet another embodiment, the phar cream, lotion, paste, spray, or aerosol. Examples of Suitable maceutical composition can further comprise an anti-inflam vehicles include, but are not limited to, petrolatum, aqua matory agent. phor, neobase, propylene glycol, glycerin and the like. 0107 The anti-inflammatory agent can be non-steroidal, Combinations of two or more of these vehicles can also be steroidal, or a combination thereof. Non limiting examples used. of non-steroidal anti-inflammatory agents include, oxicams, 0113. The pharmaceutical composition may be formu Such as piroxicam, isoxicam, tenoxicam, Sudoxicam, Salicy lated as controlled or sustained release formulations allow lates, such as aspirin, disalcid, benorylate, trilisate, Safapryn, ing for extended release of the active components over a Solprin, diflunisal, and fendosal; acetic acid derivatives. Such predetermined time period. In a certain embodiment, the as diclofenac, fenclofenac, indomethacin, Sulindac, tolme pharmaceutical composition is administered in combination tin, isoxepac, furofenac, tiopinac, Zidometacin, acematacin, with a biodegradable, biocompatible polymeric implant, fentiazac, Zomepirac, clindanac, oxepinac, felbinac, and which releases the proteolytic extract over a controlled ketorolac, fenamates, such as mefenamic, meclofenamic, period of time at a selected site. Examples of polymeric flufenamic, niflumic, and tolfenamic acids; propionic acid materials include polyanhydrides, polyorthoesters, polygly derivatives, such as ibuprofen, naproxen, benoxaprofen, colic acid, polylactic acid, polyethylene vinyl acetate, copo flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, lymers and blends thereof (See, Medical applications of pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, controlled release, Langer and Wise (eds.), 1974, CRC Pres. tioxaprofen, Suprofen, alminoprofen, and tiaprofenic; pyra Boca Raton, Fla.). Alternatively, the pharmaceutical com Zoles, such as phenylbutaZone, oxyphenbutaZone, fepra position is applied topically as a gel. Examples of polymeric US 2017/00498.65 A1 Feb. 23, 2017

materials that can be used are polysaccharides, particularly I0122) To each well, a Debrase reaction buffer (0.15 M cellulose derivatives such as, for example, hydroxypropyl Tris-HCl and 10 mM EDTA, pH 7.6) was added in order to cellulose, carboxymethyl cellulose, and hydroxyethyl cellu obtain a final volume of 100 uL. Ten uI of 0.5ug/ul of DQ lose, chitin, chitosan, and alginates. The gel formulation collagen type IVTM solution were then added to the wells. would allow for extended release of the active components Thereafter, different volumes (10-80 LL) of freshly prepared over a predetermined period of time. Debrase at a concentration of 0.225-1 ng/u were added to 0114. The pharmaceutical composition can be formulated the wells in Debrase buffer to achieve concentrations of as foam. Gas propellants are used to generate and administer 1.5-20 ng/well. Debrase buffer was used as a negative a foamable composition as foam. Examples of Suitable gas control. Reaction plate was incubated at room temperature propellants include Volatile hydrocarbons such as butane, for 20 minutes. To stop the reaction, 20 uL of stop reaction propane, isobutane or mixtures thereof, and fluorocarbon solution (0.324 mM iodoacetic acid in Debrase buffer) were gases. The composition may be acqueous, oil-in-water emul added. Fluorescence intensity was measured by a fluores sion or water-in-oil emulsion, further comprising a stabiliz cence micro-plate reader (Analyst AD, L.JL) equipped with ing agent. The stabilizing agent increases the Viscosity of the standard fluorescein filters. Background fluorescence from composition, can contribute to the composition stability, wells incubated in the absence of was subtracted. and/or slows foam collapse rate. Examples of stabilizing I0123 FIG. 1 shows the collagenolytic activity of two agents include, but are not limited to, naturally-occurring batches of Debrase. As shown in the figure, the two batches polymeric materials (e.g., alginate, albumin, carrageenan, of Debrase exerted similar collagenolytic activity indicating Xanthan gum, starch), semi-synthetic polymeric materials that the experimental procedure for obtaining the proteolytic Such as cellulose ethers (e.g. hydroxyethyl cellulose, methyl extract of the present invention yields consistent enzyme cellulose, carboxymethyl cellulose, hydroxy propylmethyl preparations. cellulose), and synthetic polymeric materials (e.g., polyvinyl 0.124. Next, the ability of the proteolytic extract to alcohol, carboxyvinyl polymers, and polyvinylpyrrolidone). degrade collagen types I and IV was determined. For that 0115 Techniques for formulation and administration of end, DQ collagen type IVTM and DQ collagen type ITM drugs can be found in “Remington’s Pharmaceutical Sci labeled with fluorescein were used as substrates. The assay ences.” Mack Publishing Co., Easton, Pa., latest edition, was performed as described herein above and continued for which is incorporated herein by reference. the time periods as indicated in FIG. 2. The reaction was 0116. The pharmaceutical composition can be adminis stopped by the addition of 20 uL stop reaction solution tered as a single dose, or in aliquots at two or more locations (0.324 mM iodoacetic acid in Debrase buffer) and the in the affected fibrous tissue. The amount of the proteolytic fluorescence intensity was measured as described herein extract to be administered is an effective amount which above. softens and/or ruptures the plaque. An effective amount of the proteolytic extract can range from about 0.2 mg/day to 0.125 Collagenase purified from Clostridium histolyti about 40 mg/day. In a certain embodiment, the pharmaceu cum served as a positive control with predefined activity tical composition is administered in two or more aliquots, (one unit was defined as the amount of enzyme required to each comprising about 0.5-1.5 mg. optionally in 0.2-0.5 ml liberate 1 micromole of E-leucine equivalents from collagen of Solution or Suspension. in 5 hours at 37° C. pH 7.5). 0117. In certain embodiments, the organ into which the 0.126 For the assay with Clostridium histolyticum colla pharmaceutical composition comprising the proteolytic genase, a reaction buffer for collagenase (0.05 M Tris-HCl, extract is administered is immobilized for several hours, 0.15 M NaCl, 5 mM CaCl2, 0.2 mM sodium azide, pH 7.6) e.g., 2 to 12 hours. was added to obtain a final volume of 100 uL per each well. 0118. Each possibility disclosed throughout the specifi Then, different volumes (10-80 uL) of Clostridium collage cation is a separate embodiment of the invention. nase, 0.4-1 mu/uIL, in Clostridium collagenase buffer where 0119 The following examples are presented to provide a added to reference wells to reach concentrations ranging more complete understanding of the invention. The specific from 5 to 80 mU/well. To stop the Clostridium collagenase techniques, conditions, materials, proportions and reported reaction—20 uL of 2 mg/ml 1,10-phenanthroline in colla data set forth to illustrate the principles of the invention are genase buffer was added. exemplary and should not be construed as limiting the scope I0127. Data from Clostridium collagenase were used as a of the invention. reference value per munit. Data from the proteolytic extract (also named Debrase) samples were divided by the reference EXAMPLE 1. value to determine mu/ng Debrase. I0128 FIG. 2 shows that the proteolytic extract degraded The Collagenolytic Activity of the Proteolytic collagen type I and IV with a specific activity of 1.58 and Extract 1.27 mu/ng, respectively. 0120. The proteolytic extract was obtained from brome I0129. Next, the activity of the proteolytic extract was lain as described in WO 2006/0543.09. compared to Clostridium histolyticum collagenase activity 0121 The ability of two batches of Debrase to degrade against collagen. The proteolytic activity of collagenase collagen type IV was first determined. The assay was based against gelatin was also measured using the EnzChek.R. on EnzChek R. Gelatinase/collagenase Assay Kit (Invitro Gelatinase/collagenase Assay Kit (Invitrogen) and DQ gen) which contained DQ collagen type IVTM labeled with gelatinTM (Invitrogen) as a substrate. fluorescein as a substrate. This substrate is known to be I0130 FIG. 3 shows that the proteolytic extract exerted efficiently digested by collagenases to yield highly fluores collagenolytic activity against collagen type IV, which activ cent peptides. The increase in fluorescence is proportional to ity was higher than that obtained by the commercially the proteolytic activity. available collagenase against collagen. US 2017/00498.65 A1 Feb. 23, 2017

0131 FIG. 4 shows that the proteolytic extract exerted was further demonstrated at higher doses of the proteolytic gelatinase activity. As shown in the figure, gelatinase activ extract, i.e., up to 150 mg/ml. ity of the proteolytic extract was linear at a concentration 0.138. In order to evaluate the effect of a single injection range of 2-8 ng/well. of the proteolytic extract on the stretching of Dupuytren’s cord, the cords were injected with the proteolytic extract at EXAMPLE 2 three different sites along the cord and then incubated in saline in the absence of the proteolytic extract for 24 hours The Proteolytic Extract Facilitates Rupture of at 37° C. Dupuytren’s Cord I0139 FIG. 13 shows that injections of the proteolytic 0132 Dupuytren’s cords were obtained from patients extract given at three sites of the Dupuytren’s cords were undergoing fasciectomy (FIGS. 5A, 5B and 5C). Consent capable of reducing the tensile strength of the cords by a forms were signed by all Subjects prior to Surgery and the factor of 4-5 (see FIG. 13, test 1a and 2 showing cord rupture study was approved by a Helsinki committee. The experi at a stress of 18 N and 15 N, respectively 13). One cord (FIG. ment examined the capability of the proteolytic extract to 13, test 1b) underwent rupture at a stress of 0.4 N. These perform fasiectomy of the cord. results demonstrate the efficiency of the proteolytic extract to dissolve Dupuytren’s cords and clearly imply that the Tissue Preparation proteolytic extract of the present invention is a highly effective enzymatic medication for Dupuytren’s disease as 0133. The cords obtained from the patients were divided well as for other connective tissue diseases involving excess to two or three pieces, depending on their length (FIG. 6). collagen deposition. The cords were connected by the Krackov technique to a 0140. It will be appreciated by persons skilled in the art mechanical testing device via a prolene 1 Suture (Ethicon, that the present invention is not limited by what has been Somerville, N.J.) (FIG. 7). One of the two cords was injected particularly shown and described herein above. Rather the with the proteolytic extract (0.3-0.5 ml of the proteolytic scope of the invention is defined by the claims that follow. extract according to the cord size) and the second, control 1. A method of treating a connective tissue disease com cord, was injected with saline (FIG. 8). The cords of the prising injecting to a subject in need of Such treatment a proteolytic extract group were immersed in the proteolytic pharmaceutical composition comprising a therapeutically extract Solution and the cords of the control group were effective amount of a proteolytic extract obtained from immersed in saline. Both groups were incubated at 37 for bromelain and a pharmaceutically acceptable carrier, 24 hours. wherein the proteolytic extract comprises at least one cys Mechanical Testing teine protease selected from the group consisting of stem bromelain EC 3.4.22.32, and ananain EC 3.4.22.31, said 0134. After 24 hours of incubation, all cords were con proteolytic extract further comprises at least one cysteine nected to a mechanical tensile stress testing device (Zwick protease precursor, wherein the connective tissue disease is 1445 testing system, Zwick Co., Germany). Each cord was associated with excess collagen deposition, and wherein the Subjected to an increasing load until the cord or the con step of injecting is repeated two or more times. necting Suture was ruptured. The device measured the 2. The method according to claim 1, wherein the connec applied tensile force until rupture. tive tissue disease is selected from the group consisting of Dupuytren’s disease, Peyronie's disease, frozen shoulder, Histological Analysis and Ledderhose disease. 0135 A sample of each specimen was obtained for his 3. The method according to claim 1, wherein the connec tological analysis and for determination of the disease stage. tive tissue disease is Dupuytren’s disease. 4. The method according to claim 1, wherein the connec Statistical Analysis tive tissue disease is Peyronies disease. 5. The method according to claim 1, wherein the pro 0136. The efficiency of the control group compared to the teolytic extract comprises stem bromelain EC 3.4.22.32, study group was tested using Fisher exact test. ananain EC 3.4.22.31, and at least one cysteine protease precursor. Results 6. The method according to claim 1, wherein the phar 0.137 All the cords treated with the proteolytic extract maceutical composition further comprises an agent selected (n=10) were ruptured following stretching (FIGS. 10A and from the group consisting of an anesthetic agent, an anti 10B). Some of the cords treated with the proteolytic extract bacterial agent, and an anti-inflammatory agent. were almost completely ruptured, practically dissolved prior 7. The method according to claim 7, wherein the anes to the mechanical test. All control cords (n=9) did not thetic agent is selected from the group consisting of ame rupture following the tensile force application. As depicted thocaine (tetracaine), lignocaine (lidocaine), Xylocaine, in FIG. 11, all the control cords exhibited a similar stress bupivacaine, prilocaine, ropivacaine, benzocaine, mepivo elongation pattern represented by a limited elongation with caine, cocaine and combinations thereof. increasing stress of the cord until ruptured when a very high 8. The method according to claim 7, wherein the antibac load applied. In contrast, all the cords treated with the terial agent is selected from the group consisting of aman proteolytic extract exhibited loss of the cords tensile fadine hydrochloride, amanfadine Sulfate, amikacin, amika strength, culminating in rupture of the cord at a very low cin Sulfate, amoglycosides, amoxicillin, amplicillin, stress (FIG. 12). The results demonstrated that low doses, amsamycins, bacitracin, beta-lactams, candicidin, capreo i.e., 0.8 mg/ml, of the proteolytic extract were capable of mycin, carbenicillin, cephalexin, cephaloridine, cephalothin, rupturing the Dupuytren’s diseases cords and that this effect cefazolin, cephapirin, cephradine, cephaloglycin, chi US 2017/00498.65 A1 Feb. 23, 2017 lomphenicols, chlorhexidine, chloshexidine gluconate, chlo 10. The method of claim 1, wherein the pharmaceutical rhexidine hydrochloride, chloroxine, chlorquiraldol, chlo composition further comprises a component selected from rtetracycline, chlortetracycline hydrochloride, ciprofloxacin, the group consisting of a stabilizing agent, an anti-oxidant, a preservative, a buffering agent, a chelating agent, and a circulin, clindamycin, clindamycin hydrochloride, clotrima tonicity agent. Zole, cloxacillin, demeclocycline, dicloSXacillin, diiodohy 11. The method according to claim 1, wherein the phar droxyquin, doxycycline, ethambutol, ethambutol hydrochlo maceutical composition is formulated in a form selected ride, erythromycin, erythromycin estolate, erhmycin from the group consisting of a solid formulation, a semi Stearate, farnesol, floxacillin, gentamicin, gentamicin Sul Solid formulation, a liquid formulation, and a foam formu fate, gramicidin, giseofulvin, haloprogin, haloquinol, lation. hexachlorophene, iminocyldine, iodochlorhydroxyquin, 12. The method according to claim 11, wherein the solid kanamycin, kanamycin Sulfate, lincomycin, lineomycin, formulation is a powder. lineomycin hydrochloride, macrollides, meclocycline, meth 13. The method according to claim 11, wherein the liquid acycline, methacycline hydrochloride, methenine, meth formulation has a pH of about 6 to about 7. enamine hippurate, methenamine mandelate, methicillin, 14. The method according to claim 1, wherein the phar metonidazole, miconazole, miconazole hydrochloride, maceutical composition is injected into the diseased fibrous minocycline, minocycline hydrochloride, mupirocin, nafcil tissue. lin, neomycin, neomycin Sulfate, netimicin, netilmicin Sul 15. The method according to claim 5, wherein the pro fate, nitrofuraZone, norfloxacin, nyStatin, octopirox, olean teolytic extract further comprises at least one cycteine domycin, orcephalosporins, oxacillin, oxyteacline, protease fragment. oxytetracycline hydrochloride, parachlorometa Xylenol, 16. The method according to claim 15, wherein the paromomycin, paromomycin Sulfate, penicillins, penicillin proteolytic extract comprises stem bromelain EC 3.4.22.32, G. penicillin V, pentamidine, pentamidine hydrochloride, ananain EC 3.4.22.31, at least one cysteine protease precur phenethicillin, polymyxins, quinolones, streptomycin Sul Sor, at least one cysteine protease fragment, and a lectin. fate, tetracycline, tobramycin, tolnaftate, triclosan, trifam 17. The method according to claim 1, wherein the con pin, rifamycin, rollitetracycline, silver salts, spectinomycin, nective tissue disease associated with excess collagen depo spiramycin, struptomycin, Sulfonamide, tetracyclines, tetra sition is selected from the group consisting of fibrous tissue cycline, tobramycin, tobramycin Sulfate, triclocarbon, tri malformations and fibrous tissue abnormalities. closan, trimethoprim-sulfamethoxazole, tylosin, Vancomy 18. The method according to claim 17, wherein the fibrous cin, and yrothricin. tissue malformations and fibrous tissue abnormalities are 9. The method according to claim 7, wherein the anti selected from the group consisting of fibrosis of joint inflammatory agent is selected from the group consisting of capsules, fibrosis of tendons, fibrosis of ligament sheaths, non-steroidal anti-inflammatory agents, and steroidal anti Scars, hypertrophic scars, and keloid scars. inflammatory agents. k k k k k