Title of Invention	"A METHOD OF PREPARING ON A COMMERCIAL SCALE A PURIFIED PREPARATION OF COD TRYPSIN ISOENZYMES"
Abstract	Fish derived serine proteinases including trypsins and chymotrypsin derived from cod such as Atlantic cod is used for treating and/or preventing a variety of diseases and disorders such as inflammatory diseases, infectious diseases caused by viruses, bacteria and fungal species and diseases where a receptor binding mechanism is involved in the pathogenesis. Pharmaceutical and cosmetic compositions comprising the proteinases are described.

Title of Invention

"A METHOD OF PREPARING ON A COMMERCIAL SCALE A PURIFIED PREPARATION OF COD TRYPSIN ISOENZYMES"

Abstract

Fish derived serine proteinases including trypsins and chymotrypsin derived from cod such as Atlantic cod is used for treating and/or preventing a variety of diseases and disorders such as inflammatory diseases, infectious diseases caused by viruses, bacteria and fungal species and diseases where a receptor binding mechanism is involved in the pathogenesis. Pharmaceutical and cosmetic compositions comprising the proteinases are described.

Full Text	I FISH SERINE PROTEINASES AND THEIR PHARMACEUTICAL AND COSMETIC USE FIELD OF INVENTION The present invention relates in its broadest aspect to the field of treatment and prevention of diseases and to the cosmetic field, in particular the pharmaceutical and cosmetic use of serine proteinases derived from fish. Specifically there are provided pharmaceutically or cosmetically active compositions comprising as an active component, trypsin and/or chymotrypsin isolated from Atlantic cod, and serine proteinases related to such enzymes. TECHNICAL BACKGROUND AND PRIOR ART U.S. Patent Nos. 4,801,451 and 4,963,491 disclose a mixture of exo- and endopeptidases isolated from Antarctic krill (Euphasia superba) and the use of this mixture as a cleaning solution. U.S. Patent No. 4,801,451 discloses the use of such enzymes to remove foreign matter and dead tissue from wounds. International patent application WO 85/04809 discloses the use of krill enzymes as a digestion-promoting agent. EP-A1-0170115 discloses the use of krill enzymes to dissolve blood clots. However, all of these references disclose impure or poorly characterized materials. A purified peptidase or mixture of purified peptidases is desirable to provide a pharmaceutically useful product. Furthermore, no disclosure is found wherein a purified peptidase or mixture of purified peptidases is useful for treating e.g. arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid arthritis, septic arthritis, phlebitis, eczema. rash, psoriasis or infectious diseases. International patent application WO 96/24371 discloses the use of a krill-derived multifunctional proteolytic enzyme and a family of crustacean and fish derived proteolytic enzymes having substantial structural similarity to the multifunctional enzyme derived from Antarctic krill. This application also relates to methods of purifying the multifunctional enzyme and to pharmaceutical, cosmetic and other uses of the multifunctional enzyme.. Substantial structural similarity to the multifunctional enzyme derived from Antarctic krill is defined in that applcatior, as at least 70% homology with the killi derived multifunctional hydrolase. The cod derived trypsin and chymotrypsiri as disclosed herein have less than 70% homology with the krill derived multifunctional enzyme. Additionally, the cod derived trypsins a~-d ohymotrypsins are not multifunctior,al enzymes. Their major known activity is that of trypsin proteases and chymotrypsin proteaseu respectively. A major objective of the present invention is to provide the use of the cod derived trypsins and chymbtrypsins ir pharmaceutical compositoni or medicaments for local and topical application, to treat internal diseases and disorders and cosmetic use of such enzymes. The topical use for treating local, internal disorders, without the enzyme having to penetrate through open wounds or mucous tissue, was found to be surprisingly effective as described In the accompanying examples. This i; contrary to prior art teachings of related use of other enzymes where, e.g., the enzyme k~ill muitifunctional hydrotase is injected for treatment of various local pain. Another objective of the present invention is the use of the cod derived trypsins and chymctiypsns for use in pharmaceutical compositions or mecicaments for the transport of other proteineous or nori-proteineous active compounds t-irou~h the skin for local an: topcal application to treat internal diseases and disorders. It has been found that such serine proteinases are pharmaceutically highly active against a variety of diseases. SUMMARY OF THE INVENTION Accordingly, the present invention pertains in a first aspact to a fish serine proteinase for use as a r-~edicament. In this context, particularly useful proteinases include trypsins and chymotrypsins derived from cod such as the Atlantic: cod. In a further aspect the invention relates to the use of a fsh serine protein ase in the manufacturing of a medicament for treating and/zr ~rev~nting a disease in a human or an animal. Diseases and disorders which may be treati~d or prevented include pain, acute inflammaton, chronic inflammation. arthrit~s, inflame d jcints, oursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic artnrttis, fibromyalgia, systemic lupus erytnematosus, phlebitis, tendinitis, rash, psoliasis, acne, eczema, facial seborrheic eczema, eczema of the hands, face or neck, foreskn ln~ectlons, athlet&s foot, fistulae infections, infected topical ulcers, navel infections ir newborns, wrinkles, scars, kelloids, boils, warts and allergic itch, hemorrhoids, wounds, wound infections, wounds from burns, a fungal infection and an immunological disorder including an autoimmune disease. In still further aspects, the invention provides the use of a fish serine proteinase in the manufacturing of a medicament for removing dead or peeling skin from otherwise healthy skin and the use of a fish serine proteinase in the manufacturing of a medicament for treating or preventing a disease in whose pathogenesis a receptor-mediated binding is involved. In yet further aspects, the invention relates to pharmaceutical and cosmetic compositions comprising a fish serine proteinase and to a method of treating and/or preventing a disease in a human or an animal, the method comprising administering to said human or animal a pharmaceutically effective amount of a fish serine proteinase. In accordance with the invention there are also provided a method of preparing on a commercial scale a purified preparation of cod trypsin isoenzymes, the method comprising the steps of (i) preparing an aqueous extract of cod viscera, (ii) subjecting the aqueous extract one or more chromatography steps including an affinity chromatography step using a p-aminobenzamidine affinity ligand, and (iii) desorbing and eluting the trypsin bound to the p-aminobenzamidine affinity ligand and a purified preparation on a commercial scale of cod trypsin. DETAILED DISCLOSURE .OF THE INVENTION The present invention provides the use of cod derived trypsins or other related trypsins or serine proteinases such as chymotrypsins derived from animals, in particular aquatic animals such as fish including cod, in a variety of medical and cosmetic contexts. As used herein, the term "enzyme' is used to indicate an active enzyme if not otherwise specified. Also, as used herein the term 'trypsin or other related peptidases" indicates peptidases of the trypsin type (EC 3.4.21 .4) and all peptidases that have 90% or more sequence homology with the Atlantic cod trypsins (Asgeirsson et al., Eur. J. Bioch em. 180:85-94, 1989; Gudmundsdottiret al., Eur. J. Biochem. 217:1091-1097, 1993). As used herein, the term 'chymotrypsin or other related peptidases" means all peptidases of the chymotrypsin type (EC 3.4.21 .1) and all peptidases that have 90% or more sequence homology with the Atlantic cod chymotrypsins A and B (4sgeirsson and Bjamason, Comp. Biochem. Physiol. 998: 327-335-9~, 1991; Gudmundsdottir et al., Biochim etSlophy.s. Acta 1219:211-214, 1994). In particular, the enzymes according to tne invention are useful for treating pain, inflammation, acute or chronic inflammation, arthritis, inflamec joints, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemic lupus eythematosus, phlebiti~, eczema, rash, psoriasis, acne, wounds and candidiasis. In useful embodiments, cod trypsin or cod chymotrypsin or a mixture of these enzymes are administered locally arid topically at the affected site in a pharmaceutical composition comprising the purified peptidase or mixture of purified peptidases and a pharmaceutically acceptable diluerit or carrier, in particular but not limited to a hydrogel. In addition, the enzynes are useful for treating viral infections such as herpes outbreaks, fungal, bacterial or parasitic infections, including colitis, ulcers, hemorrhoids corneal scarring, dental plaque and immune disorders including autoimmune d:sease. Enzymes that are substantially structurally similar to the cod-delved trypsin and chymnotrypsin enzymes have the same utility as the cod enzymes In particular, these trypsin and chymotrypain enzymes are useful for treating arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid ar hrit's, juvenile rheumatoid arthritis. septic arthritis and phlebitis. In such treatments, the irypsin or chymotrypsin or a mixture of both enzymes are administered locally and topically az the site of arthritis or irilammation in a pharmaceutical compc'sition comprising the trypsins or the chymotrypsins or both enzymes and a ~harmaceutically acceptable diluent or carrier, in particular bur not limited to hydrogel. n addition, the trypsin end chymotrypsin enzymes according to the invention are useft.:l for treating viral infections such as herpes outbreaks. fungal, bacterial or parasitic Infections, including colitis, ulcers, hemorrhoids, corneal scarring, dental plaque, acne, wounds, candidiasis a~d immune disorders including a~jtoimmune disease. In particular, the invention relates to t~e medical, pharmaceutical and cosmetic uses of trypsins derived from Atl~intic cod or other animals. There are three major isoenzymes of trypsin in Atlantic cod That have been purified and characterized. They have been termed Trypsin 1,11 and Ill (Asgeirsson et al., Eur. J. Biochem. 180:85-94, 1989). The cod trypsins have the amino terminal sequence l-V-G-G-Y-Q/E-C-E/T-K/R-H-S-Q-A-H-QV-S-L-N-S while mammalian trypsins such as bovine trypsin have the amino terminal sequence l-VG-G-Y-T-C-G-A-N-T-V-P-Y-Q-V-S-L-N-S. All three isoforms of cod trypsin have a similar molecular mass of about 24 kDa. The invention also relates to the medical, pharmaceutical and cosmetic uses of chymotrypsins derived from Atlantic cod or other animals. There are two major isoenzymes of chymotrypsin in Atlantic cod that have been purified and characterized. They have been designated Chymotrypsin A and B (Asgeirsson and Bjarnason, Comp. Biochem. Physiol. 99B:327-335-94, 1991). The cod chymotrypsins have the dual amino terminal sequences of one of its active forms C-G-R/S-P-A-l-S/Q-P-V/Q-IiV-T-G-Y (A chain) and J-V-N-G-E-E-A-V-P-H-SFF-W-S/PIY-W-Q-V-S-LQ-D/Q (B chain) whereas mammalian chymotrypsins such as bovine chymotrypsin A have the amino terminal sequences C-G-V-P-A-l-Q-P-V-L-S-G-L (A chain) and l-V-N-G-E-E-A-V-P-G-S-W-P-W-QV-S-L-Q-D (B chain). Both isoforms of cod chymotrypsin have a similar molecular mass of about 26 kDa. Cod trypsins and chymotrypsins are isolated and purified from cod viscera except liver, spleen and roe. The inventors have found the methods described below useful for a commercial scale production of cod trypsins and chymotrypsins. The enzymes are extracted from the whole or minced viscera by aqueous extraction with gentle stirring at any ratio of viscera to water, preferably a ratio .(w/w) of from 1:1 to 1:10 of offal to water. After separation of the crude extract solution and residual offal, the extract is clarified by sedimentation or filtration. The clarified solution is concentrated preferably by ultrafiltration and/or optionally ion exchange and then preferably microfiltered to achieve a true solution of low bioburden suitable for packed column chromatography that may include several steps. Subsequently, cod trypsin is purified by affinity chromatography preferably by amino benzamidine affinity chromatography and chymotrypsin by hydrophobic interaction chromatography preferably by Phenyl Sepharose chromatography. As used herein, commercial scale production of cod trypsins and chymotrypsins refers to methods that are reliable and robust and allow the production of batches on the order of 1 to 10 g or more, such as 10 to 100 g or more. The preferred method of application of the purified enzymes or mixture of purified enzymes is in a preparation of hydrogel and water containing 0 to 85% (vol/vol) of a polyvalent alcohol (polyol) such as glyceroL A suitable concentration of trypsin activity is 0.1 to 10,000 enryme units of activity for CBZ-C~ly-Pro-Arg-pNA (carbobenzoxy Gly-ProArg-para nitroanalide) per 100 milliliters of the rinal hydrogel preparation arid the appropriate concentration of chyrnotrypsin ac~ivlty is 0.1 to 10,000 enzyme units of activity for Succinyl-Ala-Ala-Pro-Phe-pNA per 100 milliliters of the final hydrogel preparation. The invention also orovides a pharmaceutical compcsition comprising the cod trypsins and related peptidases and a pharmaceutically acceptable diluent or carrier. The Invention further provides (a) methods relating t~ certain conditions using effective amounts of the purified enzymes described above, (o) compositions or substances for use in such methods, (c) pharmaceutical compositions containing effective amounts of enzymes for use in such methods, and (d) uses of the eizyme or enzyme composition for manufacturing a medicament for use in such methods. The methods are for: treating or prophylactically preventing an indication ~electec~ from the group consisting of pain, inflammation, acute or chronic inflammation, arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatcdd arthritis, septic arthritis, fibromyalgia, systemic lupus erythematosus and pnl~bitis where preferably the amount is treating or preventing effective amount; treating or prophylactically preventing derinatologca~ cc iditioris such as e.g. acne, rash, psoriasis or eczema, including facial seborrheic eczema or eczema of the hands, face, scalp or neck, hemorrhoids and the like, where prefe~abry the amount of the cod trypsins and related peptidases administered is a dermatolo~ical condition treating or preventing effective amoun.t treating or prophylactically prevent~ng wound nfectYi and debriding wounds (by applying to the wound a microbial infection-pr3ventin~ effect..e a!T1~unt of the enzyme or by enhancing the healing of wounds by admini~:ering ~ micrc'bs inhibiting effective amount of the enzyme), when treated the wound can Le substamial~,' free of necrotic tissue; removing dead or peeling skin from otherwise healthy skin to improve the skin's appearance, where preferably the amount of the enzyme administered is a dead skin removing effective amount; treating or prophylactically preventing cystic fibrosis, cancer, e.g. by administering a tumor treating effective amount or a tumor metastasis preventing or inhibiting amount of enzyme, atherosclerosis, asthma, septic shock, toxic shock syndrome, tissue adhesions such as tendon-sheath, abdominal post-surgical or joint adhesions, reperfusion injury, malaria, immune disorder such as an autoimmune disease, apoptosis, colitis and enteritis such as Crohn's disease, where preferably the amount of the cod trypsins and related peptidases administered are effective for treating or preventing; treating or prophylactically preventing a microbial infection, e.g. a viral infection such as a herpes virus infection (e.g. HSV-1, HSV-2, herpes zoster or genital herpes infection), HIV, hepatitis, influenza, coronavirus, cytomegalovirus, rhinovirus or papilloma virus infection; an infection causing a gastrointestinal disease such as ulcer or diarrhoea; a fungal infection such as systemic, skin, oral, vaginal or esophageal fungal infection, including e.g. yeast infection, including a fungal nail infection and candida infections; microbial infections of the eye, preferably treated with ocular administrations; bacterial infections including infection by Staphylococcus spp., Streptococcus spp., Kiebsiella spp., Pseudomonas spp., Neisseria gonorrheae, Haemophllus spp., Chiamydia spp., syphilis and E. co/i infections and bacterial infections causing chancroid; opportunistic microbial infections in immunocompromised patients where preferably the administered amount of the cod trypsins is a microbial infection-treating or -preventing effective amount or has inhibitory activity against cell-cell or cell-virus adhesion; removing dental plaque, where preferably the amount of the enzyme administered is a dental plaque removing effective amount; and lysing blood clots, where preferably the amount of the enzyme is a clot lysing effective amount. The method comprises administering a composition comprising the cod trypsins and/or chymotrypsins, or related peptidases as described above. The composition of the invention can also be used to remove dead or divergent cells. The invention provides topical cosmetic and medical compositions comprising the purified cod trypsin, chymcti-ypsin or other re~ated peptidase~ described above; and gel, cream or suppository composition. The invention also provides (a) methods for treating or prophylactically preventing a cell-cell or cell-virus adhesion related syndrome, compriiiing administering an anti-adhesion effective amount of the cod trypsins or chyrr'otrypsins or related peptidases effective to remove or inactivate a cellular or viral acceptor or receptor adhesion component that is involved In the cell-cell or cell-vIrus adhesion, (b) corn pc'sitions or substances for use In such methods, (c) pharmaceutical compositions containing effective amounts of enzyme for use in such methods, and (d) uses of me enzyme composition for manufacturing a medicament for use in such methods. Generally, the syndrome involves inflammation, arthritis, inflamed joints, bursitis, osteoarthi-itis, rneurnatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, phlebitis, eczema, rash, psoriasis, acne, wounds, candidiasis and immune disorders including autoimmure diseases, i;hock, tumor metastasis, transplantation rejection reactions or microbial infeclions. Preferably, (a) the syndrome is selected from the group consisting of r~~icrobal infection, immune disorder, cystic fibrosis, atherosclerosis, cancer, asthma, seFtic shock, toxic shock syndrome, conjunctivitis, reperfusion injury and pain, and (b~ cell surface re~eptor, associated with the cell-cell or cell-virus adhesion syndrome. semected from followirg, which cod trypsin has been shown to cleave, consisting of CD4, O')8, CD54 (ICAM-I), CD31, CDS2L, CD1O2 (ICAM-2), CDI la/CD1S, is removed or hactivated by the administered enzyme. Preferably, a microolal infection is treated ~r prevented and th~ microbial infection is herpes, HIV, hepatitis or papilloma infee:ron, an infection causing colitis or diarrhoea; a Candida infection, such as an oral, vaginal or esophageal Caridida infection; a cold or influenza infection; a StapP~yioco~cus, Streptococcus, KiebsieJa, Pseudomonas, Haemophilus or E. coli infection; a primary or secondary infection of leprosy; or an infection causing conjunctivitis or tuberculosis. l~ one embodiment, the inventor, provides a method of inhibiting or prophylactically preventing the transmission of a pathogenic microbe: by administering the cod trypsin or chymotrypsin enzymes. preferably, the coc tr~psin 2ind/or chymotrypsin enzyme is applied to the portion of the body that comprises the primary transmission entryway for me mIcrobe in question. In one preferred embodiment, a spray, ointment or wash is applied to a body orifice involved in sexual activity, for instance, to prevent HIV or hepatitis transmission. In another preferred embodiment, the cod trypsin or related peptidases is applied to the upper airways, for example. via an aerosol, to inhibit or prevent the transmission of a common cold virus, such as a rhinovirus or a coronavirus. In one aspect, the method of extra-corporeally treating a tissue, body fluid or composition of cells to remove cell adhesion components reduces the immune rejection of tissue, body fluid or composition of cells that is transplanted from one individual to another. In another aspect, such treatments remove or inactivate the cell adhesion components found in the treated tissue, body fluid or composition of cells involved in a microbial infection. In treating or prophylactically preventing septic shock or toxic shock syndrome by administering the cod trypsins or related peptidases, appropriate routes of administration include systemic administration. For vaginal infections associated with shock, vaginal flushes, creams, gels or suppositories may be used as a method of administration. In treating or prophylactically preventing pain, inflammation, acute or chronic inflammation, arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemic lupus erythematosus, phlebitis by administering the cod trypsins or related peptidases, appropriate routes of administration would include without limitation creams, gels or suppositories, in particular but not limited to hydrogels containing glycerol or other polyols. In treating or prophylactically preventing rash, psoriasis, acne, eczema, including facial seborrheic eczema or eczema of the hands, face, scalp or neck, foreskin infections, athlete's foot, fistulae infections, infected topical ulcers, navel infections in newborns, wrinkles, scars and kelloids, boils, warts and allergic itch, hemorrhoids and the like, wounds, wound infections, wounds from burns, removing dead or peeling skin from otherwise healthy skin to improve the skin's appearance, a fungal infection such as systemic, skin, oral, vaginal or esophageal fungal, including for example, yeast infection, including a fungal nail infection and Candida infections and immune disorders including autoimmune diseases by administering the cod trypsins, chymotrypsins or peptidases related to these enzymes, appropriate routes of administration would include creams, gels or suppositories, in particular but not limited to hydrogels containing glycerol or other polyols. Accordingly, it is one objective of the present invention to provide a composition for the treatment of pain, inflammation, acute c- chronic inflammation, arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemIc lupus erythematosus, phlebitis, rash, psoriasis, acne, eczema, including facial sebortheic eczema oreozeira of the ~ands, face, scalp or neck,foreskin infections, athlete's foot, fistulae infections, nfected topical ulcers, navel infections in newborns, wrinkIes,~ scars and kelloids, boi!s, warts and allergic itch, hemorrhoids and the like, wounds, wound infections, wounds frcrn bums, removing dead or peeling skin from otherwise heal:hy skin to improve the skin a appearance, a fungal infection such as systemic, skin, oral, vaginal or esophageal fungal infE'ctic'n including e.g. yeast infection, including a fungal nail infection and Canc Ida spp. infections, and immune disorders including autoimrnune diseases. Another object is to provide a method for the preparation of a composition to be used for the treatment of said diseases. A third object is to provide a method for tie application of said coriposition for t~e treatment of said diseases, including dose levels for external arid topical applications. The invention hence is based on compositions comprising enzymes where previously n~ enzymes have been used or a better enzyme wnere enzymes have been used. The above objectives are achieved by using a composition containing an effective amount of the cod trypsins and/or chymotryr~sins which is capable of relieving the pain, inflammation, arthritis, swelling, edema, psoriasis ecz3me, dermatitis, rash and/or other symptoms of the diseases mentior'ed in the introductcry part. The cod enzymes can be obtained at a irigh yield and in a relatively simple man ier from cod viscera. It has been found that the cod enzymes can oe purified on a commercial scale in a reistively straightforward manner wIth rea~onabie yields. T~ie pL.rified cod trypsin contains three major isozymes of the enzyme and cod chymotrypsin ~on:ains two major isozymes. One or all isozymes can be used for the purpose of the invEmtion, It has now been established that the cod trypsin and c'iymotrypsin enzymes of the invention effectively remove or inactivate certain cell-s irface adhesion molecules, bacterial enter~toxins, cytokines, inflammatory mediators and matrix metallop roteinases (MMPs) such as CD4, CD8, C~54 (lZAM-i), CDeI, CDe2L, CDIO2 (teAM-2), CD1la/CD18, TNF-alfa, IL-i, and MMP-9, without affecting the cell viability. This adhesion site cleavage or inactivaton phenomenon of cytckines, Inflammatory mediators and matrix metalloproteinases is believed to provide at least a partial explanation for the effectiveness of the cod trypsins and chymotrypsins against many, though probably not all, of the indications against which the cod trypsins and chymotrypsins are effective. Thus TNF-alpha is implicated in both arthritis, psoriasis, cancer and inflammation (See Cytokines, Eds. Mire-Slui and Thrope, Academic press, 1998, p 350). The cod trypsins and chymotrypsins appear to be more effective than other conventional proteases in many respects. When a panel of 13 of the most active proteases in this respect were compared for activity towards 16 of the aforementioned certain cell-surface adhesion molecules, bacterial enterotoxins, cytokines, inflammatory mediators and MMPs, as well as their sensitivity to serum inhibition, the Atlantic cod trypsin and chymotrypsin were found to be far the most active. The proteases were scored 2 points for high to very high activity, 1 point for low to moderate activity and 0 point for very low to no activity (reverse scores for serum inhibitory activity for the proteases). On the basis of this scoring scheme the Atlantic cod trypsin scored 30 points and the Atlantic cod chymotrypsin scored 29 points, whereas the known krill multifunctional protease scored 16 points. The bovine trypsin and chymotrypsin proteases were found lacking in this respect. Furthermore, the cod trypsins and chymotrypsins are 2 to 20-fold more active than their bovine counterparts when conventional small chromogenic substrates are used to compare their activities (Asgeirsson et al., Eur. J. Biochem. 180:85-94, 1989; Asgeirsson and Bjarnason., Comp. Biochem. Physiol. 99B:327-335-94, 1991). Cod viscera are useful sources for the cod trypsin and chymotrypsin enzymes of the invention. For instance, the enzymes can be extracted on a commercial scale from frozen cod viscera as described above. The clarified and concentrated extract solution can be optionally fractionated by commercial scale ion exchange chromatography, preferably a first cation exchange chromatography step followed by an anion exchange chromatography step to bind and thereby remove related serine proteases such as elastase and serine collagenases. In a subsequent and final step, cod trypsin is purified by commercial scale affinity chromatography preferably using p-aminobenzamidine affinity column chromatography and chymotrypsin by hydrophobic interaction column chromatography preferably using Phenyl Sepharose. The cod trypsin used in the invention can be desorbed from the affinity matrix by applying conditions that will destabilize the interaction between the enzyme and the affinity ligand. Such conditions include high salt followed by low pH, preferably in 30% or more glycerol. The column eluent is allowed to flow Into a neutralizing buffer to stabilize the cod trypsin after the acid elution step (Asgeirsson at al., Ear. J. ffoohem. 180:85-94, 1989). The cod chymotrypsin used in the invention can be released from the Phenyl Sepharose column by applying conditions that will destabilize the interaction between the enzyme and the hydrophobic interaction Igand. Such conditions induce high glycerol content in water or buffer, preferably 50% or more glyc&ol in water or buffer (Asgeirsson and Bjarnason, Comp; Biochem. Physio/, 99B:327-335-94, 1991). By these methods the cod trypsin and chymotrypsin enzymes with a purity in excess of about 90% can be isolated. The cod trypsin fraction thus obtained contains 3 major forms or isozyn,es of cod trypsin as manifested by SDS electrophoresis which yields one band, isoelectric focusing showing three bands and chromatofocusing chromatography which gives three peaks associated with the three ~rypsin isoforms (Fig. 1). At least four i~ozymes of trypsin IV are additionally included in the trypsin fraction if anior. exchange chromatography is not employed. These may be separated as subfracticns with subsequent mono-Q or DEAE anion exchange chromatography. The serine proteases from otner sources than Atlantic cod can be compared to the isolated Atlantic cod trypsin enzymes for molecular mass, isoeleztric point, amino acid sequence. temperatu-~ or pH stability, temperature or pH maximum, proteclytic specificity and kinetic parameters, or for other properties of the Atlantic cod trypein enzymes exemplified in the examples (Asgeirason et al., Eu. J. Bicchem. 180:85-94, 1989). The activity of cod trypsin can be measured using tyrosirie arginine methyl este- (TAME) as substrate. Th. isolated cod trypsin from the affinity column purification (90 —130% purity) will prefera~ly have specific activity of at least 140 U/mg at 250C and pN 8.1, as compared to 60 U/ma for the krill muitifunctional ~ydrolase. Also, tryptic activity can be measured usIng CK-Gly-Pro-ArQ-p-nltrcanllide (Zbz-GPR-pNA) a~ substra:e yielding a specific acivity preferably at least about 100 U/mg. Furthermore when Bz-Arg-pNA is used as suostrete catalytic efficiency constants (Kat/Km) of 52, 20 ~,d 7 s1mM1 are obtained for cod t.ypsin 1,11 and Ill respectively as compared to 3 s1m!.K1 for bovine trypsin (Asgeirsson et al., Eur. J. Biochem. 180:85-94. 1989). Similar differences are seen for the Qd chymotrypsins and bovine chymotrypsin (Asgeirsson and Bjarnason, Cornp. Biochem. Pnys!oI. 99B:327-335, 1991). The molecular mass of the cod trypsins is about 24 kDa, whereas their isoelectric points are 6.6, 6.2 and 5.5 for trypsin 1,11 and Ill respectively, and even lower for trypsin IV isozymes. The amino acid sequences of the isozymes of cod trypsin can be expressed with the following sequence which contains point variability due to the multiple isoforms: l~V-G-G-Y-E-C-T-KR-H-S-Q-A-H-Q-V-S-L-N-S-G-Y-H-Y/F-C-G-G-S L-l-N-KIE-D/Q-W-VV-S-A-A-H-C-Y-K-S-V-L- R-V-R-L-G-E-H-H-l-R-V-N-E-G-T-E-Q-Y/F-l -S-S-S-S-V-l/X-R-HP -N-Y-S-S-Y-N -l -N/D-N-D-l-M -L-l-K-L-T-KIE-P-A-T-L-N-Q-Y-V-H -A-V-A-L-P-T-E-C-A-A T-M-A-N/S-Y, wherein X is any amino acid or no amino acid, and cod trypsin I has amino acid residue K in position 9, whereas cod trypsins II and Ill contain R in this position (Gudmundsdottir at al. 1993 and unpublished data). Generally, the cod trypsin isoenzymes will be sufficiently stable so that at least about 500/c of the TAME activity is retained after incubation at 5 0C for 18 hours at pH 7.0 in a buffer solution containing 10mM calcium chloride. However, only about 10% of the TAME activity is retained after incubation at 5 0C for 18 hours at pH 2.0 (Figure 2). The pH optimum of the cod trypsins foc TAME as~ substrate is preferably between about 7.0 and 8.7 more preferably about 8.0 (Figure 3). Using TAME as the substrate, the Km at about pH 8.1 and 25 0C in the presence of 10 mM calcium chloride is preferably about 0.029 mM, 0.021 mM and 0.049 mM for cod trypsins 1,11 and Ill respectively. Preferably, cod trypsin has a temperature maximum of activity for TAME as substrate between 50 and 60 0C (Figure 4). The cod trypsin and chymotrypsin enzymes of the invention are administered topically, orally, rectally, vaginally, by instillation (for instance into the urinary tract or into fistulas), by the pulmonary route e.g. by use of an aerosol, by application of drops to the eye, or systemically, such as parenterally, including, for example, intramuscularly, subcutaneously, intraperitoneally, intraarterially or intravenously. The cod enzymes are administered in solution or combined with a pharmaceutically-acceptable carrier or excipient according to standard pharmaceut'cal practice. For the oral mode of administratIon, the cod enzymes are used in the form of tablets, capsules, lozenges, chewing gum, troches, powders, syrups, elixirs, alucous solutions and suspensions, and the like. ~or parenteral administration, ster~e ~ of the cod enzymes are usually prepared, and the pH values of the solutions are sui~ably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. For ocular administratIon, ointments or dropable liquids may be delivered by ocular delivery systems known to the sit such as applicators or eye droppers. For pulmonary administration, diluents and/or carriers will be selected to be appropriate to allow the formation of an aerosol. Fo!- topical admlnl'~trations, the cod trypsin enzymes are typically administered in a hydrogel, containing 0 to 35% glycerol, such as about 20% to 30% glycerol and possibly up to 85 % glycerol. For topical treatments, a suitable dose of the cod typsin enzymes per application ranges from about 0.01 ~±g/crri2 to about 1 mg/cm2, preferably from about 0.1 j4cm2 to about 0.01 mg/cm2 (using e.g. about 0.01 mg/mi enzyme gel). For systematic treatments, dosages will generally be selected to maintan a serum level of the cod trypsin enzymes between about 0.1 mg/100 ml and abzut 100 mg!100m1, preferably between about 0.5 mg/laO ml and about 2.0 mgflOO ml. In an alternative measure of preferred systematic administration amounts, preferably from about 0.1 mg/kg to about 10 mg/kg, more preferably about 1 mg/kg, will be used. For vaginal and urinary tract treatments, suitable flushing/instillation solutions of the cod trypsin enzymes will generally have concentrations from about 1 ~±g/ml to about 15 mg/mI, preferably fro,, about 100 jig/mI to about 3 mgfml. For all treatments, the enzyme compo'~itior. will generally be applied from about 1 to about 10 times per day, preferably from abcut 2 to about 5 timt~s per day. These values, of course, will vary with a number of factors including the type and severity of the disease, and the age, weight and medical condition of the pat~ent, as ~viI~ be recognized by those of ordinay skill in the medical arts. It is believed that sLlbstiintia[y higher doses can be used without substantial adverse effect. For wound healing, the cod trypsin enzymes are preleraoly applied more often than simply at the time at which the wound is first dressec. Prefe'~ably, the cod trypsin is applied at least about every time the wounc~ dressing is ~Thanged. The cod trypsin enzymes can also be applied at least about every other day, more preferably, every day, or a 'ew times per day. For dermatological situation5 such as eczema, psoriasis and the like, the cod trypsin and/or chymotrypsin enzyme hydrogel is preferably applied every day, more preferably twice per day. For acute or chronic inflammation, arthritis, inflamed joints, bursitis, osteoarthritis, and the like, the cod trypsin enzymes are preferably applied every day more preferably twice every day. As illustrated in many of the clinical examples below, the cod enzyme hydrogel of the invention is effective to treat or prevent inflammatory conditions. Typically, inflammations are reduced to acceptable levels within 2 or 3 days from initiation of treatment. The examples also illustrate that the enzyme is effective to alleviate pain. Pain relief is often reported within 20 minutes to 2 hours of the start of treatment. Pain relief was not accompanied by loss of feeling in the treated tissue. More complete pain relief, such that the patient only suffered mild pain or a feeling of tenderness, was often experienced within 1 day of the start of treatment. Generally, the cod enzymes according to the invention will be administered in an effective amount. An effective amount is an amount effective to either (1) reduce the symptoms of the disease sought to be treated, (2) induce a pharmacological change relevant to treating the disease sought to be treated, (3) inhibit or prevent infection or re-infection by an infective agent, or (4) prevent the occurrence of a non-infectious disease (for instance a disease treatable by blocking a cell adhesion phenomenon). For wound treatment, in one aspect, an effective amount includes an amount which, if regularly applied, prevents the occurrence of infection. In another aspect, for wound healing, an effective amount includes an amount effective to reduce the average time it takes for a wound to heal. Numerous methods for determining percent homology of proteins are known in the art. One preferred method is to use version 6.0 of the GAP computer program for making sequence comparisons. The program is available from the University of Wisconsin Genetics Computer Group and utilizes the alignment method of Needleman and Wunsch, J. Mol. Biol. 48, 443 (1970) as revised by Smith and Waterman, Adv. Appi. Math., 2, 482 (1981). Another available method uses the FASTA computer program. The invention will now be further illustrated in the following non-limiting examples and the drawings wherein Fig. 1. shows a chromatofocusing chromatograph of the Atlantic cod trypsin fraction off the aminobenzamidine affinity c~l~mn purification stEp. The fraction eluted from the para ~rriirtuWrii.~riiidiii~ ~ulumn was ge~ filtered on a Sepl,adex G-25 column equilibrated in 25 n,M monoethanolan,ine/acetic acid pH 9.4 containin~l 10mM calcium chloride and subsequently applied to a column of ~BE-94 ion exchange material (Pharmacia) equilibrated in the same buffer. The column was then eluted with Polybuffer 94 (adjusted to pH 5.5 with acetic acid) contai,ing 10 mM calcium chloride. Activity for the substrate TAME (filled circles), absorbance at 28' nm (open circles) and pH (open diamonds) was monitored. The irst eluted fraction was termed trypsin I, the second fraction trypsin II and the third fraction trypsin Ill; Fig. 2. illustrates the effect of pH on :~e stability of Atlantic cod trypsin purified by para aminobenzamidine as measured b~; residual TAME activity after incubation at various pH values and two different time per~ds The Atlantic ccd trypsin samples were Incubated at 5 0C for 30 minutes (open circi~s) and 18 hours (closed aquares) at varying pH values. Residual activity for the substrate TAME was subsequently measured ~t pH 8.1 and 25 Fig. 3 shows the pH dependence of the activity c~Atl antic cod trypsin purified by paraaminobenzamid~ne as measured by using the TAME substrate. The buffers used at a final concemration 0.1 M were acetate (pH 3.0-5.9), hepe~~/HCl (pi-l 6.0-8.0) and glycinate (pH 8.3-9.0), all containing 10 mM calcium chloride; and Fig. 4 shows the temperature dependence of the activity of Atlantic cod trypsin I compared to the activity of bovine trypsin measured by u3ing the TAME substrate. The enzymes were added to a preheated therrnostated cuveti:e and after a short equilibration period the average rate of hydrolysis was determined during the following 3 minutes. EXAMPLE 1 Preparation o' a mixture of oroteases from cod About 100 kg of frozen cod viscera were thawed and adoed to a four-fold volume of cold potable water in an extraction tank and the pH adjustt~d to pH 8 to 9 with a sodium hydroxide solution. The mixture was stirred for about 2-6 hours at 0 to 50C. After a brief period of crude sedimentation (about 30 minutes) the aqueous extract was run off the remaining insoluble viscera with a pump and collected in a sedimentation tank. The aqueous extract was allowed to stand in the cooled sedimentation tank to sediment for about 24 to 60 hours. The supernatant was decanted from the supernatant tank to a holding tank using a pump. The supernatant was concentrated 10 to 20-fold by ultrafiltration and diafiltered to an acceptable level of ionic strength with conductivity below about 3 mS/cm. About 10-15 liters of ultrafiltered and diafiltered protein concentrate was obtained. EXAMPLE 2 Purification of cod try~sin from concentrated cod viscera extract About 10 liters of ultrafiltratered and diafiltered concentrate as obtained in. Example 1 was applied to a continuous connected series of about 1 liter packed chromatography columns, the first containing a CM fast flow cation exchange resin (Pharmacia, Sweden), the second one a DEAE fast flow anion exchange resin (Pharmacia, Sweden) and the third one a p-aminobenzamidine affinity ligand coupled to a sepharose resin (Pharmacia, Sweden). The columns were pre-equilibrated with about 10 column volumes of 25 mM Tris buffer of pH 7.8, containing 2.5 mM calcium chloride (buffer A). The concentrate was pumped onto the columns at a flow rate of about 100~ml per minute. When the application of the concentrated solution onto the columns was completed, residual material was washed off the continuous column system with about 8 liters of buffer A. After this wash was completed, the p-aminobenzamidine affinity column was disconnected from the other columns and washed with about 5 column volumes of a high salt solution of 25 mM Tris buffer pH 7.5 containing 0.5 M NaCI and 2.5 mM calcium chloride. The cod trypsins were then desorbed from the affinity ligand and eluted off the column with an acid solution of 25 mM acetic acid pH 3.2 containing 10 mM calcium chloride and 30% glycerol. The cod trypsin fraction was collected into a neutralizing buffer of 200 mM Tris pH 8.5 containing 30% glycerol. The purified cod trypsin preparation was homogeneous by SOS PAGE electrophoresis and FPLC Mono Q chromatography and showed the three trypsin isozyme bands on isoelectric focusing. The enzyme preparation had a specific activIty of about 100 U/mg using to Cbz-GPR-pNA as substrate as previously descr:bed. The purified preparation was filter sterilized through a 0.22 micron filter and stored frozen at about -20CC. EXAMPLE 3 Altemative commercial scale ourification of cod trvosin from concentrated cod viscera extract About 10 liters of ultrafiltratered and diafiltered zoncentrate as obtained In Example I was applied to a p-aminobenzamidine affinity ligand coupled Ic a sepharose resin (Pharmacia, Swede-i). The column was pre-equilibrated witt- about IC' column volumes of 25 mM Tris buffer of pH 7.8, containing 2.5mM calcium chloride bufer A). The concentrate was pumped onto the column at a flow rate of about 100 r~l psr minute. When the application of the co-icentrated solution onto the column was completed, residual material was washed off the column with about 8 liters of buf~r A. The affinity column was subsequently washed with about 5 column volumes of a high salt solution of 25 mM Tris buffer pH 7.5 containing 0.5 M NaCI and 2.5 mM calcium chloride. The cod trypsins were then desorbed from the affinity ligand and eluted off the column with an acid solutiort of 25 mM acetic acid pH 3.2 con tain!ng 10 mM calcium chloride and 30% glycerol. The cod trypsin fraction was collected into a neutralizing buffer of 200 mM Tris pH 8.5 containing 30% glycerol, and contained a~ orcxlmately 5 to 6 g of trypsin. The purified cod trypsin preparation was homogeneous by SDS PAGE electrophoresis and showed the th-ee trypsin isozyn~e banos on isoelectric focusIng. A minor fraction ( XAMPLE 4 Preparation of a hvdroQel preparation of cod trvpsins The purified cod trypsin preparation of Example 2 was mixed with hydrocolloid gel comprising an aqueous gel containing 0.8% w/v Carbomer 940, 30% glycerol and 0.08% paraoxybenzoate. The cod trypsin preparation was mixed in a 1:1 ratio with the hydrogel to give a final concentration of 1 enzyme unit per mg (U/mg) of the final gel-enzyme mixture (the enzyme hydrogel ointment), the enzyme unit being determined using CbzGPR-pNA as substrate as previously described. Thus, the resulting enzyme hydrogel ointment contained about 0.01 mg/mI, or 1 U/mI of the cod trypsin enzymes, 0.4% Carbomer 940, 20% glycerol and 0.04% paraoxybenzoate. In the following, this enzyme hydrogel ointment is referred to as Penzyme 100. EXAMPLE 5 Treating osteoarthritis with Penzyme 1 00 Seventeen individuals with osteoarthtitis, some with acute or very severe symptoms, were treated with the enzyme hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied once to twice per day, always once in the evening before retiring to bed and in most cases also in the morning. About 5 milliliters of the enzyme gel was applied on each affected area, such as each knee or hip or both hands. Larger quantities may be applied in severe cases. The gel was left to dry for about 10 to 30 minutes, depending on the amount applied, time available and the needs and requirements of the patient. This drying can be accelerated by the use of an air blowing device such as a hair dryer. All these patients experienced relief from symptoms within one week, most of them within 2 to 4 days. Patient No. 71 was unable to walk stairs before treatment but has had no problems doing so after treatment. These results are summarized in the following Table 5.1: (Table Removed) A few of the patients (Nos. 19 and 21) have been able to discominue treatment and thus appear to have enjoyed permanent or long term reliet from the disease, but most of them keep the symptoms under control witn cons:ant or int3rrnittent use of Penzyme 100. EXAMPLE S Treatment of oatients sufferino from tendinitis Eight individuals with tendinitis, some with very seve e symptoms causing problems with sleep at night, were treated with the enzyme hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied topically on the affected area once to twice per day, about 5 to 10 milliliters on each affected area, .ii~pending on the size of the area. The gel was left to dry for about 10 to 30 minutes, depending on the amount applied, time available and the needs and requirements of ~he patit~nt. All these patients experienced relief from symptoms with in two weeks, mos'~ of therm within 2 to 4 days but others took longer, especially those with tendinitis of the elbow (tennis elbow or golfer's elbow). These results are summarized in the following Table 6.1 All of these patients have enjoyed permanent relief from the condition and have discontinued treatment. EXAMPLE 7 Treatment of fibromyalpia Two patients suffering from severe fibromyalgia were treated with the enzyme hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied topically on the affected area twice per day, about S to 10 milliliters on each affected area, depending on size of the area. The gel was left to dry for about 10 to 30 minutes, depending on the amount applied, time available and the needs and requirements of the patient. Both patients (No. 78, F-SO and No. 92, F-53) experienced relief from symptoms within three weeks. Both use the enzyme gel treatment when pain reappears. EXAMPLE 8 Treatment of rheumatoid arthritis Five patients with rheumatoid arthritis, some with vey severe symptoms, were treated with the enzyme hydrogel ointment (Penzyme 1 ~0) of Example 4. The enzyme gel was applied topically on the affected area usually at east twice per day, about 3 to 5 milliliters on each affected area, depending on the size of the area. The gel was left to dr5' for about 15 minutes, depending on the thickness of the gel applied. This drying period can be shortened by the use of an air-blowing device such ~ts a hair dryer. All five patients experienced relief from symptoms within 5 days. ThE~se results are summarized in the following Table 8.1: Patient No. 61 could not get out of bed in tne momin~ and dress without assistance before she began the treatment, but was fully capabl'3 of doing so on her own after treatment. Patient No 94 ~ad pain in the chest when breathing due to a complIcation of pleural effusion. She was relieved from these symptoms after 3 days of treatment with Penzyme 100. All of these patients keep the symptoms under control with constant or intermittent use of the enzyme gel treatment. EXAMPLE 9 Treatment of phlebitis Five patients with various forms of phlebitis, such as thrombophlebitis, phlebothrombosis, chronic postphlebitic syndrome and varicose veins, in all cases in the calves, some with very severe symptoms causing pain with difficulty of sleep, were treated with the enzyme hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied topically on the calf at least once per day. i.e. in the evening and in some cases also in the morning, about 5 milliliters on each calf. The gel is left to dry for about 15 minutes. All five patients experienced relief from symptoms within a few weeks. These results are summarized in the following table 9.1: Patient No. 2 was diagnosed with chronic postphlebtic syndrome. Patient No. 68 does not sleep properly at night due to pain if she does not apply the gel before bedtime. All of these patients keep the symptoms under control with constant or intermittent use of the enzyme gel treatment. EXAMPLE 10 Treatment of psoriasis Six patients with psoriasis, some chronic cases with very severe symptoms, were treated with the enzyme hydrogel of Example 4, but in the majority of the cases of psoriasis a hydrogel composition designated Penzyme 200 with double the cod trypsin concentration or 2 U/mI was used, since Penzyme 100 (lU/mI cod trypsin) yielded rather slow and in some cases uncertain results. Patients claimed greal: improvements in treatment with the gel containing the higher cod trypsin content (Penzyrne 2D0) as compared with the gel containing the lower trypsin content (Penzyme 100). A combination of cod trypsin and cod chymotrypsin, with a concentration of 1 U/mI of each enzyme, designated Penzyme 200-C, was also tested, and gave similar posit~e results as Penzyme 200. The enzyme gel was applied once to twice per day, always once In the evening before bedtime, after which the enzyme gel was left on the akir overnight and in some cases also in the morning after bathing and before dressitig. The gel i~ applied as a thin layer or film and takes about 5 mInutes to dry. All six patients experienced relief from symptoms within two weeks. The treatment was in some cases supplemented with conventional self-help methods such as sun bathing or UV light treatment. The results are summarised in the following Table 10.1: Patient No. 3 has suffered from chronic and very severe psoriasis since the age of 5. She has tried all available treatments with limited results. The treatment with Penzyme 200 has yielded the only long-term relief from symptoms for her. She claims that relief from symptoms is noticeable after about Z. days and total relief is accomplished within 2 to 3 weeks of treatment with one application per day before bedtime. Patient No. 69, also with severe chronic psoriasis, has experienced the permanent disappearance of some spots. All of these patients keep tne symptoms under control with constant or intermittent enzyme gel treatment. EXAMPLE 11 Treatment of acne and boils Two individuals (patient Nos. 25, F-16 and no. 63, M-17) suffering from acne and one suffering from boils (No. 64, M-22) were treated with the cod trypsin hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied topically on the affected area (face) once per day, about 5 milliliters each time. The gel is left to dry for about 15 minutes after which it is left on the skin. All three individuals experienced total relief from symptoms within two weeks, and only used the enzyme gel treatment occasionally when symptoms started to reappear. EXAMPLE 12 Treatment of eczema. dermatitis and other skin conditions Eighteen patients suffering from eczema, dermatitis and various other skin conditions, were treated with the cod trypsin hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied once per day (occasionally twice), usually in the evening before retiring to bed. The gel was applied as a thin layer or film. Drying occurred after about 5-10 minutes. A higher amount may be applied in severe cases. This drying can be accelerated by the use of an air-blowing device such as a hair dryer. All these patients experienced relief from symptoms within one week, most of them in 2 to 4 days. The results are summarised in the following Table 12.1: Some of the patients (Nos. 40,41,48,66,50,20,46, 87, 97, 100) have been able to discontinue treatment and thus appear to have enjoyed permanent or long term relief from the disease, but others keep the symptoms under control with constant or intermittent ~se of Penzyme 100. EXAMPLE 13 Wound healing Two individuals (patient Nos. 44, M-4 anc 85, M-9) with bums were treated with the cod trypsin hydrogel ointment (Penzyme 100) of Example 4. Two o:her patients (No. 62, F-47 and 96, M-70) were treated for post-operationa wounds that would not heal. Tne enzyme gel was applied topically on the wounds at least twice per day and left on the affected area. All four individuals experienced good and complication free healing of their wounds within one to three weeks. EXAMPLE 14 Treatments with cod chvmotrvpsin For each of examples 5-13, the cod trypsin enzyme hydrogel is substituted with a cod chymotrypsin enzyme hydrogel or a hydrogel containing a mixture of cod trypsin and cod ch y m ot ryp sin. CLAIMS 1. A fish serine proteinase for use as a medicament. 2. A proteinase according to claim 1 that is selected from the group consisting of a trypsin, a chymotrypsin and any mixture hereof. 3. A.proteinase according to claim 1 or 2 that is not multifunctional. 4. A proteinase according to any of claims 1-3 that is derived from Atlantic cod. 5. A proteinase according to daim 1 that is a trypsin derived from Atlantic cod. 6. A proteinase according to claim 5 that is selected from the group consisting of trypsin I, trypsin II. trypsin III, trypsin IV a, trypsin IV b, trypsin IV c, and trypsin IV d. 7. A proteinase according to claim 4 that is selected from the group consisting of chymotrypsin A and chymotrypsin B. 8. A proteinase according to claim 1 that is a trypsin having at least 90% amino acid sequence homology with any of trypsin I, trypsin II, tiypsin 111, and trypsin IV derived from Atlantic cod. 9. A proteinasa according to claim 1 that is a chymotrypsin having at least 90% amino acid sequence homology with any of chymotrypsin A and chymotrypsin B isolated from Atlantic cod. 10. A proteinase according to claim 1 for use as a medicament for treating and/or preventing a disease in a human or an animal, said disease is selected from the group consisting of pain, acute inflammation, chronic inflammation, arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemic lupus erythematosus, phiebitis, tendinitis, rash, psoriasis, acne, eczema, facial Beborrheic eczema, eczema of the hscxmds, face or neck, foreskin infections, athlete's foot, fistuiae infections, infected topical ulcers, navel infections in newborns, wrinkles, scars, kelloids, boils, warts and allergic itch, hemorrhoids, wounds, wound infections, wounds from burns, a fungal infection and an immunological disorder including an autoimmune disease. 11. A proteinase according to claim 1 for use as a medicament for removing dead or peeling skin from otherwise healthy skin. 12. Use of a fish serine proteinase in the manufacturing of a medicament for treating and/or preventing a disease in a human or an animal, said disease is selected from the group consisting of pain, acute inflammation, chronic inflammation, arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemic lupus erythematosus, phlebitis, tendinitis, rash, psoriasis, acne, eczema, facial seborrheic eczema, eczema of the hands, face or neck, foreskin infections, athlete's foot, fistulae infections, infected topical ulcers, navel infections in newborns, wrinkles, scars, kelloids, boils, warts and allergic itch, hemorrhoids, wounds, wound infections, wounds from burns, a fungal infection and an immunological disorder including an autoimmune disease. 13. Use of a fish serine proteinase in the manufacturing of a medicament for removing dead or peeling skin from otherwise healthy skin. 14. Use of a fish serine proteinase in the manufacturing of a medicament for treating or preventing a disease in whose pathogenesis a receptor-mediated binding is involved. 15. Use according to claim 14 where the disease is caused by a pathogenic organism selected from the group consisting of a virus, a bacterium, a fungus, a parasite and a protozoan. 16. Use according to any of claims 12-14 where the fish serine proteinase is a proteinase according to any of claims 2-9. 17. A pharmaceutical composition comprising a fish serine proteinase. 18. A composition according to claim 17 wherein the fish serine proteinase is a proteinase according to any of claims 2-11 19. A composition according to claim 17 or 18 that is a composition for topical use. 20. A composition according to claim 19 that is a hydrogel. 21. A composition accordJng to claim 20 further comprising a polyvalent alcohol including gly cerol. 22. A composition according to claim 17 comprising a further pharmaceutical!/ active compound. 23. A cosmetic composition comprising a fish serine proteinase. 24. A composition according to claim 23 wherein the fish serine proteinase is a proteinase according to any of claims 2-11 25. A composition according to claim 23 that is a hydrogel. 25. A composition according to claim 23 further comprising a polyvalent alcohol including glycerol. 27. A composition according to claim 23 comprising £ further cosmetically active compound. 28. A method of treating and/or preventing a disease in a human or an animal, the method comprising administering to said human or animal a pharnaceutically effective amount of a fish serine proteinase. 29. A method according to claim 28 wherein the proteinase is a proteinase according to any of claims 2-11. 30. A method according to claim 28 wherein the proteinase is administered topically. 31. A method according to claim 30 wherein the prote nase is administered gastroenteraliy, 32. A method of preparing on a commercial scale a purified preparation of cod trypsin isoenzymes comprising trypsin I, II, III and IV isozymes, the method comprising the steps of (i) preparing an aqueous extract of cod viscera, (ii) subjecting the aqueous extract to one or more chromatography steps including an affinity chromatography step using a p-aminobenzamidine affinity ligand, and (iii) desorbing and eluting the trypsin bound to the p-aminobenzamidine affinity iigand. 33. A method of preparing a purified preparation of cod trypsin isoenzymes, according to claim 32, wherein the method further comprises one or more steps using a cation exchange resin, and one or more steps using an anion exchange resin prior to the affinity chromatography step. 34. A method according to claim 32 or 33 wherein the purification is continued until the preparation contains a specific activity of at least 50 U/mg protein using Cbz-GPR-pNA as substrate. 35. A method according to claim 34 wherein the specific activity is 100 U/mg or more. 36. A method according to claim 32 comprising as a further step that the purified preparation is sterile filtered using a 0.22 fim filter. 37. A purified preparation on a commercial scale of cod trypsin. 38. A purified preparation of cod trypsin according to claim 37 comprising, when it is subjected to SDS-PAGE electrophoresis and FPLC Mono Q chromatography as the only protein bands three to seven bands of trypsin isoenzymes. 39. A preparation according to claim 37 or claim 38 containing a specific activity of at least 50 U/mg protein using Cbz-GPR-pNA as substrate. 40. A preparation according to claim 38 where the saecific activity is 100 U/mg or more. 41. A fish serine proteinase for use as a medicament substantially as herein described with reference to the accompanying drawings. 42. Use of a fish serine proteinase in the manufacturing of a medicament for treating and / or preventing a disease in a human or an animal substantially as herein described with reference to the accompanying drawings. 43. A pharmaceutical composition substantially as herein described with reference to the accompanying drawings. 44. A method of treating and / or preventing a disease in a human or an animal substantially as herein described with reference to the accompanying drawings. 45. A method of preparing on a commercial scale a purified preparation of cod trypsin isoenzymes comprising trypsin I, II, III and IV isozymes substantially as herein described with reference to the accompanying drawings. 46. A purified preparation on a commercial scale of cod trypsin substantially as herein described with reference to the accompanying drawings.

Full Text

I

FISH SERINE PROTEINASES AND THEIR PHARMACEUTICAL AND COSMETIC USE

FIELD OF INVENTION

The present invention relates in its broadest aspect to the field of treatment and prevention of diseases and to the cosmetic field, in particular the pharmaceutical and cosmetic use of serine proteinases derived from fish. Specifically there are provided pharmaceutically or cosmetically active compositions comprising as an active component, trypsin and/or chymotrypsin isolated from Atlantic cod, and serine proteinases related to such enzymes.

TECHNICAL BACKGROUND AND PRIOR ART

U.S. Patent Nos. 4,801,451 and 4,963,491 disclose a mixture of exo- and endopeptidases isolated from Antarctic krill (Euphasia superba) and the use of this mixture as a cleaning solution. U.S. Patent No. 4,801,451 discloses the use of such enzymes to remove foreign matter and dead tissue from wounds. International patent application WO 85/04809 discloses the use of krill enzymes as a digestion-promoting agent. EP-A1-0170115 discloses the use of krill enzymes to dissolve blood clots.

However, all of these references disclose impure or poorly characterized materials. A purified peptidase or mixture of purified peptidases is desirable to provide a pharmaceutically useful product. Furthermore, no disclosure is found wherein a purified peptidase or mixture of purified peptidases is useful for treating e.g. arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid arthritis, septic arthritis, phlebitis, eczema. rash, psoriasis or infectious diseases.

International patent application WO 96/24371 discloses the use of a krill-derived multifunctional proteolytic enzyme and a family of crustacean and fish derived proteolytic enzymes having substantial structural similarity to the multifunctional enzyme derived from Antarctic krill. This application also relates to methods of purifying the multifunctional enzyme and to pharmaceutical, cosmetic and other uses of the multifunctional enzyme.. Substantial structural similarity to the multifunctional enzyme derived from Antarctic krill is

defined in that applcatior, as at least 70% homology with the killi derived multifunctional hydrolase. The cod derived trypsin and chymotrypsiri as disclosed herein have less than 70% homology with the krill derived multifunctional enzyme. Additionally, the cod derived trypsins a~-d ohymotrypsins are not multifunctior,al enzymes. Their major known activity is that of trypsin proteases and chymotrypsin proteaseu respectively.

A major objective of the present invention is to provide the use of the cod derived trypsins and chymbtrypsins ir pharmaceutical compositoni or medicaments for local and topical application, to treat internal diseases and disorders and cosmetic use of such enzymes. The topical use for treating local, internal disorders, without the enzyme having to penetrate through open wounds or mucous tissue, was found to be surprisingly effective as described In the accompanying examples. This i; contrary to prior art teachings of related use of other enzymes where, e.g., the enzyme k~ill muitifunctional hydrotase is injected for treatment of various local pain. Another objective of the present invention is the use of the cod derived trypsins and chymctiypsns for use in pharmaceutical compositions or mecicaments for the transport of other proteineous or nori-proteineous active compounds t-irou~h the skin for local an: topcal application to treat internal diseases and disorders. It has been found that such serine proteinases are pharmaceutically highly active against a variety of diseases.

SUMMARY OF THE INVENTION

Accordingly, the present invention pertains in a first aspact to a fish serine proteinase for use as a r-~edicament. In this context, particularly useful proteinases include trypsins and chymotrypsins derived from cod such as the Atlantic: cod.

In a further aspect the invention relates to the use of a fsh serine protein ase in the manufacturing of a medicament for treating and/zr ~rev~nting a disease in a human or an animal. Diseases and disorders which may be treati~d or prevented include pain, acute inflammaton, chronic inflammation. arthrit~s, inflame d jcints, oursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic artnrttis, fibromyalgia, systemic lupus erytnematosus, phlebitis, tendinitis, rash, psoliasis, acne, eczema, facial seborrheic eczema, eczema of the hands, face or neck, foreskn ln~ectlons, athlet&s foot, fistulae infections, infected topical ulcers, navel infections ir newborns, wrinkles, scars, kelloids,

boils, warts and allergic itch, hemorrhoids, wounds, wound infections, wounds from burns, a fungal infection and an immunological disorder including an autoimmune disease.

In still further aspects, the invention provides the use of a fish serine proteinase in the manufacturing of a medicament for removing dead or peeling skin from otherwise healthy skin and the use of a fish serine proteinase in the manufacturing of a medicament for treating or preventing a disease in whose pathogenesis a receptor-mediated binding is involved.

In yet further aspects, the invention relates to pharmaceutical and cosmetic compositions comprising a fish serine proteinase and to a method of treating and/or preventing a disease in a human or an animal, the method comprising administering to said human or animal a pharmaceutically effective amount of a fish serine proteinase.

In accordance with the invention there are also provided a method of preparing on a commercial scale a purified preparation of cod trypsin isoenzymes, the method comprising the steps of (i) preparing an aqueous extract of cod viscera, (ii) subjecting the aqueous extract one or more chromatography steps including an affinity chromatography step using a p-aminobenzamidine affinity ligand, and (iii) desorbing and eluting the trypsin bound to the p-aminobenzamidine affinity ligand and a purified preparation on a commercial scale of cod trypsin.

DETAILED DISCLOSURE .OF THE INVENTION

The present invention provides the use of cod derived trypsins or other related trypsins or serine proteinases such as chymotrypsins derived from animals, in particular aquatic animals such as fish including cod, in a variety of medical and cosmetic contexts.

As used herein, the term "enzyme' is used to indicate an active enzyme if not otherwise specified. Also, as used herein the term 'trypsin or other related peptidases" indicates peptidases of the trypsin type (EC 3.4.21 .4) and all peptidases that have 90% or more sequence homology with the Atlantic cod trypsins (Asgeirsson et al., Eur. J. Bioch em. 180:85-94, 1989; Gudmundsdottiret al., Eur. J. Biochem. 217:1091-1097, 1993). As used herein, the term 'chymotrypsin or other related peptidases" means all peptidases of the chymotrypsin type (EC 3.4.21 .1) and all peptidases that have 90% or more sequence

homology with the Atlantic cod chymotrypsins A and B (4sgeirsson and Bjamason, Comp. Biochem. Physiol. 998: 327-335-9~, 1991; Gudmundsdottir et al., Biochim etSlophy.s. Acta 1219:211-214, 1994).

In particular, the enzymes according to tne invention are useful for treating pain, inflammation, acute or chronic inflammation, arthritis, inflamec joints, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemic lupus eythematosus, phlebiti~, eczema, rash, psoriasis, acne, wounds and candidiasis. In useful embodiments, cod trypsin or cod chymotrypsin or a mixture of these enzymes are administered locally arid topically at the affected site in a pharmaceutical composition comprising the purified peptidase or mixture of purified peptidases and a pharmaceutically acceptable diluerit or carrier, in particular but not limited to a hydrogel. In addition, the enzynes are useful for treating viral infections such as herpes outbreaks, fungal, bacterial or parasitic infections, including colitis, ulcers, hemorrhoids corneal scarring, dental plaque and immune disorders including autoimmune d:sease.

Enzymes that are substantially structurally similar to the cod-delved trypsin and chymnotrypsin enzymes have the same utility as the cod enzymes

In particular, these trypsin and chymotrypain enzymes are useful for treating arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid ar hrit's, juvenile rheumatoid arthritis. septic arthritis and phlebitis. In such treatments, the irypsin or chymotrypsin or a mixture of both enzymes are administered locally and topically az the site of arthritis or irilammation in a pharmaceutical compc'sition comprising the trypsins or the chymotrypsins or both enzymes and a ~harmaceutically acceptable diluent or carrier, in particular bur not limited to hydrogel. n addition, the trypsin end chymotrypsin enzymes according to the invention are useft.:l for treating viral infections such as herpes outbreaks. fungal, bacterial or parasitic Infections, including colitis, ulcers, hemorrhoids, corneal scarring, dental plaque, acne, wounds, candidiasis a~d immune disorders including a~jtoimmune disease.

In particular, the invention relates to t~e medical, pharmaceutical and cosmetic uses of trypsins derived from Atl~intic cod or other animals. There are three major isoenzymes of trypsin in Atlantic cod That have been purified and characterized. They have been termed

Trypsin 1,11 and Ill (Asgeirsson et al., Eur. J. Biochem. 180:85-94, 1989). The cod trypsins have the amino terminal sequence l-V-G-G-Y-Q/E-C-E/T-K/R-H-S-Q-A-H-QV-S-L-N-S while mammalian trypsins such as bovine trypsin have the amino terminal sequence l-VG-G-Y-T-C-G-A-N-T-V-P-Y-Q-V-S-L-N-S. All three isoforms of cod trypsin have a similar molecular mass of about 24 kDa.

The invention also relates to the medical, pharmaceutical and cosmetic uses of chymotrypsins derived from Atlantic cod or other animals. There are two major isoenzymes of chymotrypsin in Atlantic cod that have been purified and characterized. They have been designated Chymotrypsin A and B (Asgeirsson and Bjarnason, Comp. Biochem. Physiol. 99B:327-335-94, 1991). The cod chymotrypsins have the dual amino terminal sequences of one of its active forms C-G-R/S-P-A-l-S/Q-P-V/Q-IiV-T-G-Y (A chain) and J-V-N-G-E-E-A-V-P-H-SFF-W-S/PIY-W-Q-V-S-LQ-D/Q (B chain) whereas mammalian chymotrypsins such as bovine chymotrypsin A have the amino terminal sequences C-G-V-P-A-l-Q-P-V-L-S-G-L (A chain) and l-V-N-G-E-E-A-V-P-G-S-W-P-W-QV-S-L-Q-D (B chain). Both isoforms of cod chymotrypsin have a similar molecular mass of about 26 kDa.

Cod trypsins and chymotrypsins are isolated and purified from cod viscera except liver, spleen and roe. The inventors have found the methods described below useful for a commercial scale production of cod trypsins and chymotrypsins. The enzymes are extracted from the whole or minced viscera by aqueous extraction with gentle stirring at any ratio of viscera to water, preferably a ratio .(w/w) of from 1:1 to 1:10 of offal to water. After separation of the crude extract solution and residual offal, the extract is clarified by sedimentation or filtration. The clarified solution is concentrated preferably by ultrafiltration and/or optionally ion exchange and then preferably microfiltered to achieve a true solution of low bioburden suitable for packed column chromatography that may include several steps. Subsequently, cod trypsin is purified by affinity chromatography preferably by amino benzamidine affinity chromatography and chymotrypsin by hydrophobic interaction chromatography preferably by Phenyl Sepharose chromatography.

As used herein, commercial scale production of cod trypsins and chymotrypsins refers to methods that are reliable and robust and allow the production of batches on the order of 1 to 10 g or more, such as 10 to 100 g or more.

The preferred method of application of the purified enzymes or mixture of purified enzymes is in a preparation of hydrogel and water containing 0 to 85% (vol/vol) of a polyvalent alcohol (polyol) such as glyceroL A suitable concentration of trypsin activity is 0.1 to 10,000 enryme units of activity for CBZ-C~ly-Pro-Arg-pNA (carbobenzoxy Gly-ProArg-para nitroanalide) per 100 milliliters of the rinal hydrogel preparation arid the appropriate concentration of chyrnotrypsin ac~ivlty is 0.1 to 10,000 enzyme units of activity for Succinyl-Ala-Ala-Pro-Phe-pNA per 100 milliliters of the final hydrogel preparation.

The invention also orovides a pharmaceutical compcsition comprising the cod trypsins and related peptidases and a pharmaceutically acceptable diluent or carrier.

The Invention further provides (a) methods relating t~ certain conditions using effective amounts of the purified enzymes described above, (o) compositions or substances for use in such methods, (c) pharmaceutical compositions containing effective amounts of enzymes for use in such methods, and (d) uses of the eizyme or enzyme composition for manufacturing a medicament for use in such methods. The methods are for:

treating or prophylactically preventing an indication ~electec~ from the group consisting of pain, inflammation, acute or chronic inflammation, arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatcdd arthritis, septic arthritis, fibromyalgia, systemic lupus erythematosus and pnl~bitis where preferably the amount is treating or preventing effective amount;

treating or prophylactically preventing derinatologca~ cc iditioris such as e.g. acne, rash, psoriasis or eczema, including facial seborrheic eczema or eczema of the hands, face, scalp or neck, hemorrhoids and the like, where prefe~abry the amount of the cod trypsins and related peptidases administered is a dermatolo~ical condition treating or preventing effective amoun.t

treating or prophylactically prevent~ng wound nfectYi and debriding wounds (by applying to the wound a microbial infection-pr3ventin~ effect..e a!T1~unt of the enzyme or by enhancing the healing of wounds by admini~:ering ~ micrc'bs inhibiting effective amount of the enzyme), when treated the wound can Le substamial~,' free of necrotic tissue;

removing dead or peeling skin from otherwise healthy skin to improve the skin's appearance, where preferably the amount of the enzyme administered is a dead skin removing effective amount;

treating or prophylactically preventing cystic fibrosis, cancer, e.g. by administering a tumor treating effective amount or a tumor metastasis preventing or inhibiting amount of enzyme, atherosclerosis, asthma, septic shock, toxic shock syndrome, tissue adhesions such as tendon-sheath, abdominal post-surgical or joint adhesions, reperfusion injury, malaria, immune disorder such as an autoimmune disease, apoptosis, colitis and enteritis such as Crohn's disease, where preferably the amount of the cod trypsins and related peptidases administered are effective for treating or preventing;

treating or prophylactically preventing a microbial infection, e.g. a viral infection such as a herpes virus infection (e.g. HSV-1, HSV-2, herpes zoster or genital herpes infection), HIV, hepatitis, influenza, coronavirus, cytomegalovirus, rhinovirus or papilloma virus infection; an infection causing a gastrointestinal disease such as ulcer or diarrhoea; a fungal infection such as systemic, skin, oral, vaginal or esophageal fungal infection, including e.g. yeast infection, including a fungal nail infection and candida infections; microbial infections of the eye, preferably treated with ocular administrations; bacterial infections including infection by Staphylococcus spp., Streptococcus spp., Kiebsiella spp., Pseudomonas spp., Neisseria gonorrheae, Haemophllus spp., Chiamydia spp., syphilis and E. co/i infections and bacterial infections causing chancroid; opportunistic microbial infections in immunocompromised patients where preferably the administered amount of the cod trypsins is a microbial infection-treating or -preventing effective amount or has inhibitory activity against cell-cell or cell-virus adhesion;

removing dental plaque, where preferably the amount of the enzyme administered is a dental plaque removing effective amount; and

lysing blood clots, where preferably the amount of the enzyme is a clot lysing effective amount.

The method comprises administering a composition comprising the

cod trypsins and/or chymotrypsins, or related peptidases as described above. The composition of the invention can also be used to remove dead or divergent cells.

The invention provides topical cosmetic and medical compositions comprising the purified cod trypsin, chymcti-ypsin or other re~ated peptidase~ described above; and gel, cream or suppository composition.

The invention also provides (a) methods for treating or prophylactically preventing a cell-cell or cell-virus adhesion related syndrome, compriiiing administering an anti-adhesion effective amount of the cod trypsins or chyrr'otrypsins or related peptidases effective to remove or inactivate a cellular or viral acceptor or receptor adhesion component that is involved In the cell-cell or cell-vIrus adhesion, (b) corn pc'sitions or substances for use In such methods, (c) pharmaceutical compositions containing effective amounts of enzyme for use in such methods, and (d) uses of me enzyme composition for manufacturing a medicament for use in such methods. Generally, the syndrome involves inflammation, arthritis, inflamed joints, bursitis, osteoarthi-itis, rneurnatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, phlebitis, eczema, rash, psoriasis, acne, wounds, candidiasis and immune disorders including autoimmure diseases, i;hock, tumor metastasis, transplantation rejection reactions or microbial infeclions. Preferably, (a) the syndrome is selected from the group consisting of r~~icrobal infection, immune disorder, cystic fibrosis, atherosclerosis, cancer, asthma, seFtic shock, toxic shock syndrome, conjunctivitis, reperfusion injury and pain, and (b~ cell surface re~eptor, associated with the cell-cell or cell-virus adhesion syndrome. semected from followirg, which cod trypsin has been shown to cleave, consisting of CD4, O')8, CD54 (ICAM-I), CD31, CDS2L, CD1O2 (ICAM-2), CDI la/CD1S, is removed or hactivated by the administered enzyme. Preferably, a microolal infection is treated ~r prevented and th~ microbial infection is herpes, HIV, hepatitis or papilloma infee:ron, an infection causing colitis or diarrhoea; a Candida infection, such as an oral, vaginal or esophageal Caridida infection; a cold or influenza infection; a StapP~yioco~cus, Streptococcus, KiebsieJa, Pseudomonas, Haemophilus or E. coli infection; a primary or secondary infection of leprosy; or an infection causing conjunctivitis or tuberculosis.

l~ one embodiment, the inventor, provides a method of inhibiting or prophylactically preventing the transmission of a pathogenic microbe: by administering the cod trypsin or chymotrypsin enzymes. preferably, the coc tr~psin 2ind/or chymotrypsin enzyme is applied to the portion of the body that comprises the primary transmission entryway for me mIcrobe in question. In one preferred embodiment, a spray, ointment or wash is

applied to a body orifice involved in sexual activity, for instance, to prevent HIV or hepatitis transmission. In another preferred embodiment, the cod trypsin or related peptidases is applied to the upper airways, for example. via an aerosol, to inhibit or prevent the transmission of a common cold virus, such as a rhinovirus or a coronavirus.

In one aspect, the method of extra-corporeally treating a tissue, body fluid or composition of cells to remove cell adhesion components reduces the immune rejection of tissue, body fluid or composition of cells that is transplanted from one individual to another. In another aspect, such treatments remove or inactivate the cell adhesion components found in the treated tissue, body fluid or composition of cells involved in a microbial infection.

In treating or prophylactically preventing septic shock or toxic shock syndrome by administering the cod trypsins or related peptidases, appropriate routes of administration include systemic administration. For vaginal infections associated with shock, vaginal flushes, creams, gels or suppositories may be used as a method of administration.

In treating or prophylactically preventing pain, inflammation, acute or chronic inflammation, arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemic lupus erythematosus, phlebitis by administering the cod trypsins or related peptidases, appropriate routes of administration would include without limitation creams, gels or suppositories, in particular but not limited to hydrogels containing glycerol or other polyols.

In treating or prophylactically preventing rash, psoriasis, acne, eczema, including facial seborrheic eczema or eczema of the hands, face, scalp or neck, foreskin infections, athlete's foot, fistulae infections, infected topical ulcers, navel infections in newborns, wrinkles, scars and kelloids, boils, warts and allergic itch, hemorrhoids and the like, wounds, wound infections, wounds from burns, removing dead or peeling skin from otherwise healthy skin to improve the skin's appearance, a fungal infection such as systemic, skin, oral, vaginal or esophageal fungal, including for example, yeast infection, including a fungal nail infection and Candida infections and immune disorders including autoimmune diseases by administering the cod trypsins, chymotrypsins or peptidases related to these enzymes, appropriate routes of administration would include creams, gels or suppositories, in particular but not limited to hydrogels containing glycerol or other polyols.

Accordingly, it is one objective of the present invention to provide a composition for the treatment of pain, inflammation, acute c- chronic inflammation, arthritis, inflamed joints, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis, fibromyalgia, systemIc lupus erythematosus, phlebitis, rash, psoriasis, acne, eczema, including facial sebortheic eczema oreozeira of the ~ands, face, scalp or neck,foreskin infections, athlete's foot, fistulae infections, nfected topical ulcers, navel infections in newborns, wrinkIes,~ scars and kelloids, boi!s, warts and allergic itch, hemorrhoids and the like, wounds, wound infections, wounds frcrn bums, removing dead or peeling skin from otherwise heal:hy skin to improve the skin a appearance, a fungal infection such as systemic, skin, oral, vaginal or esophageal fungal infE'ctic'n including e.g. yeast infection, including a fungal nail infection and Canc Ida spp. infections, and immune disorders including autoimrnune diseases. Another object is to provide a method for the preparation of a composition to be used for the treatment of said diseases. A third object is to provide a method for tie application of said coriposition for t~e treatment of said diseases, including dose levels for external arid topical applications. The invention hence is based on compositions comprising enzymes where previously n~ enzymes have been used or a better enzyme wnere enzymes have been used.

The above objectives are achieved by using a composition containing an effective amount of the cod trypsins and/or chymotryr~sins which is capable of relieving the pain, inflammation, arthritis, swelling, edema, psoriasis ecz3me, dermatitis, rash and/or other symptoms of the diseases mentior'ed in the introductcry part. The cod enzymes can be obtained at a irigh yield and in a relatively simple man ier from cod viscera. It has been found that the cod enzymes can oe purified on a commercial scale in a reistively straightforward manner wIth rea~onabie yields. T~ie pL.rified cod trypsin contains three major isozymes of the enzyme and cod chymotrypsin ~on:ains two major isozymes. One or all isozymes can be used for the purpose of the invEmtion,

It has now been established that the cod trypsin and c'iymotrypsin enzymes of the invention effectively remove or inactivate certain cell-s irface adhesion molecules, bacterial enter~toxins, cytokines, inflammatory mediators and matrix metallop roteinases (MMPs) such as CD4, CD8, C~54 (lZAM-i), CDeI, CDe2L, CDIO2 (teAM-2), CD1la/CD18, TNF-alfa, IL-i, and MMP-9, without affecting the cell viability. This adhesion site cleavage or inactivaton phenomenon of cytckines, Inflammatory mediators

and matrix metalloproteinases is believed to provide at least a partial explanation for the effectiveness of the cod trypsins and chymotrypsins against many, though probably not all, of the indications against which the cod trypsins and chymotrypsins are effective. Thus TNF-alpha is implicated in both arthritis, psoriasis, cancer and inflammation (See Cytokines, Eds. Mire-Slui and Thrope, Academic press, 1998, p 350).

The cod trypsins and chymotrypsins appear to be more effective than other conventional proteases in many respects. When a panel of 13 of the most active proteases in this respect were compared for activity towards 16 of the aforementioned certain cell-surface adhesion molecules, bacterial enterotoxins, cytokines, inflammatory mediators and MMPs, as well as their sensitivity to serum inhibition, the Atlantic cod trypsin and chymotrypsin were found to be far the most active. The proteases were scored 2 points for high to very high activity, 1 point for low to moderate activity and 0 point for very low to no activity (reverse scores for serum inhibitory activity for the proteases). On the basis of this scoring scheme the Atlantic cod trypsin scored 30 points and the Atlantic cod chymotrypsin scored 29 points, whereas the known krill multifunctional protease scored 16 points. The bovine trypsin and chymotrypsin proteases were found lacking in this respect. Furthermore, the cod trypsins and chymotrypsins are 2 to 20-fold more active than their bovine counterparts when conventional small chromogenic substrates are used to compare their activities (Asgeirsson et al., Eur. J. Biochem. 180:85-94, 1989; Asgeirsson and Bjarnason., Comp. Biochem. Physiol. 99B:327-335-94, 1991).

Cod viscera are useful sources for the cod trypsin and chymotrypsin enzymes of the invention. For instance, the enzymes can be extracted on a commercial scale from frozen cod viscera as described above. The clarified and concentrated extract solution can be optionally fractionated by commercial scale ion exchange chromatography, preferably a first cation exchange chromatography step followed by an anion exchange chromatography step to bind and thereby remove related serine proteases such as elastase and serine collagenases. In a subsequent and final step, cod trypsin is purified by commercial scale affinity chromatography preferably using p-aminobenzamidine affinity column chromatography and chymotrypsin by hydrophobic interaction column chromatography preferably using Phenyl Sepharose.

The cod trypsin used in the invention can be desorbed from the affinity matrix by applying conditions that will destabilize the interaction between the enzyme and the affinity ligand.

Such conditions include high salt followed by low pH, preferably in 30% or more glycerol. The column eluent is allowed to flow Into a neutralizing buffer to stabilize the cod trypsin after the acid elution step (Asgeirsson at al., Ear. J. ffoohem. 180:85-94, 1989). The cod chymotrypsin used in the invention can be released from the Phenyl Sepharose column by applying conditions that will destabilize the interaction between the enzyme and the hydrophobic interaction Igand. Such conditions induce high glycerol content in water or buffer, preferably 50% or more glyc&ol in water or buffer (Asgeirsson and Bjarnason, Comp; Biochem. Physio/, 99B:327-335-94, 1991). By these methods the cod trypsin and chymotrypsin enzymes with a purity in excess of about 90% can be isolated. The cod trypsin fraction thus obtained contains 3 major forms or isozyn,es of cod trypsin as manifested by SDS electrophoresis which yields one band, isoelectric focusing showing three bands and chromatofocusing chromatography which gives three peaks associated with the three ~rypsin isoforms (Fig. 1). At least four i~ozymes of trypsin IV are additionally included in the trypsin fraction if anior. exchange chromatography is not employed. These may be separated as subfracticns with subsequent mono-Q or DEAE anion exchange chromatography.

The serine proteases from otner sources than Atlantic cod can be compared to the isolated Atlantic cod trypsin enzymes for molecular mass, isoeleztric point, amino acid sequence. temperatu-~ or pH stability, temperature or pH maximum, proteclytic specificity and kinetic parameters, or for other properties of the Atlantic cod trypein enzymes exemplified in the examples (Asgeirason et al., Eu. J. Bicchem. 180:85-94, 1989). The activity of cod trypsin can be measured using tyrosirie arginine methyl este- (TAME) as substrate. Th. isolated cod trypsin from the affinity column purification (90 —130% purity) will prefera~ly have specific activity of at least 140 U/mg at 250C and pN 8.1, as compared to 60 U/ma for the krill muitifunctional ~ydrolase. Also, tryptic activity can be measured usIng CK-Gly-Pro-ArQ-p-nltrcanllide (Zbz-GPR-pNA) a~ substra:e yielding a specific acivity preferably at least about 100 U/mg. Furthermore when Bz-Arg-pNA is used as suostrete catalytic efficiency constants (Kat/Km) of 52, 20 ~,d 7 s1mM1 are obtained for cod t.ypsin 1,11 and Ill respectively as compared to 3 s1m!.K1 for bovine trypsin (Asgeirsson et al., Eur. J. Biochem. 180:85-94. 1989). Similar differences are seen for the Qd chymotrypsins and bovine chymotrypsin (Asgeirsson and Bjarnason, Cornp. Biochem. Pnys!oI. 99B:327-335, 1991).
The molecular mass of the cod trypsins is about 24 kDa, whereas their isoelectric points are 6.6, 6.2 and 5.5 for trypsin 1,11 and Ill respectively, and even lower for trypsin IV isozymes. The amino acid sequences of the isozymes of cod trypsin can be expressed with the following sequence which contains point variability due to the multiple isoforms:

l~V-G-G-Y-E-C-T-KR-H-S-Q-A-H-Q-V-S-L-N-S-G-Y-H-Y/F-C-G-G-S L-l-N-KIE-D/Q-W-VV-S-A-A-H-C-Y-K-S-V-L- R-V-R-L-G-E-H-H-l-R-V-N-E-G-T-E-Q-Y/F-l -S-S-S-S-V-l/X-R-HP -N-Y-S-S-Y-N -l -N/D-N-D-l-M -L-l-K-L-T-KIE-P-A-T-L-N-Q-Y-V-H -A-V-A-L-P-T-E-C-A-A
T-M-A-N/S-Y,

wherein X is any amino acid or no amino acid, and cod trypsin I has amino acid residue K in position 9, whereas cod trypsins II and Ill contain R in this position (Gudmundsdottir at al. 1993 and unpublished data).

Generally, the cod trypsin isoenzymes will be sufficiently stable so that at least about 500/c of the TAME activity is retained after incubation at 5 0C for 18 hours at pH 7.0 in a buffer solution containing 10mM calcium chloride. However, only about 10% of the TAME activity is retained after incubation at 5 0C for 18 hours at pH 2.0 (Figure 2).

The pH optimum of the cod trypsins foc TAME as~ substrate is preferably between about 7.0 and 8.7 more preferably about 8.0 (Figure 3). Using TAME as the substrate, the Km at about pH 8.1 and 25 0C in the presence of 10 mM calcium chloride is preferably about 0.029 mM, 0.021 mM and 0.049 mM for cod trypsins 1,11 and Ill respectively. Preferably, cod trypsin has a temperature maximum of activity for TAME as substrate between 50 and 60 0C (Figure 4).

The cod trypsin and chymotrypsin enzymes of the invention are administered topically, orally, rectally, vaginally, by instillation (for instance into the urinary tract or into fistulas), by the pulmonary route e.g. by use of an aerosol, by application of drops to the eye, or systemically, such as parenterally, including, for example, intramuscularly, subcutaneously, intraperitoneally, intraarterially or intravenously. The cod enzymes are administered in solution or combined with a pharmaceutically-acceptable carrier or

excipient according to standard pharmaceut'cal practice. For the oral mode of administratIon, the cod enzymes are used in the form of tablets, capsules, lozenges, chewing gum, troches, powders, syrups, elixirs, alucous solutions and suspensions, and the like. ~or parenteral administration, ster~e ~ of the cod enzymes are usually prepared, and the pH values of the solutions are sui~ably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. For ocular administratIon, ointments or dropable liquids may be delivered by ocular delivery systems known to the sit such as applicators or eye droppers. For pulmonary administration, diluents and/or carriers will be selected to be appropriate to allow the formation of an aerosol. Fo!- topical admlnl'~trations, the cod trypsin enzymes are typically administered in a hydrogel, containing 0 to 35% glycerol, such as about 20% to 30% glycerol and possibly up to 85 % glycerol.

For topical treatments, a suitable dose of the cod typsin enzymes per application ranges from about 0.01 ~±g/crri2 to about 1 mg/cm2, preferably from about 0.1 j4cm2 to about 0.01 mg/cm2 (using e.g. about 0.01 mg/mi enzyme gel). For systematic treatments, dosages will generally be selected to maintan a serum level of the cod trypsin enzymes between about 0.1 mg/100 ml and abzut 100 mg!100m1, preferably between about 0.5 mg/laO ml and about 2.0 mgflOO ml. In an alternative measure of preferred systematic administration amounts, preferably from about 0.1 mg/kg to about 10 mg/kg, more preferably about 1 mg/kg, will be used. For vaginal and urinary tract treatments, suitable flushing/instillation solutions of the cod trypsin enzymes will generally have concentrations from about 1 ~±g/ml to about 15 mg/mI, preferably fro,, about 100 jig/mI to about 3 mgfml. For all treatments, the enzyme compo'~itior. will generally be applied from about 1 to about 10 times per day, preferably from abcut 2 to about 5 timt~s per day. These values, of course, will vary with a number of factors including the type and severity of the disease, and the age, weight and medical condition of the pat~ent, as ~viI~ be recognized by those of ordinay skill in the medical arts. It is believed that sLlbstiintia[y higher doses can be used without substantial adverse effect.

For wound healing, the cod trypsin enzymes are preleraoly applied more often than simply at the time at which the wound is first dressec. Prefe'~ably, the cod trypsin is applied at least about every time the wounc~ dressing is ~Thanged. The cod trypsin enzymes can also be applied at least about every other day, more preferably, every day, or a 'ew times per day. For dermatological situation5 such as eczema, psoriasis and the

like, the cod trypsin and/or chymotrypsin enzyme hydrogel is preferably applied every day, more preferably twice per day. For acute or chronic inflammation, arthritis, inflamed joints, bursitis, osteoarthritis, and the like, the cod trypsin enzymes are preferably applied every day more preferably twice every day.

As illustrated in many of the clinical examples below, the cod enzyme hydrogel of the invention is effective to treat or prevent inflammatory conditions. Typically, inflammations are reduced to acceptable levels within 2 or 3 days from initiation of treatment. The examples also illustrate that the enzyme is effective to alleviate pain. Pain relief is often reported within 20 minutes to 2 hours of the start of treatment. Pain relief was not accompanied by loss of feeling in the treated tissue. More complete pain relief, such that the patient only suffered mild pain or a feeling of tenderness, was often experienced within 1 day of the start of treatment.

Generally, the cod enzymes according to the invention will be administered in an effective amount. An effective amount is an amount effective to either (1) reduce the symptoms of the disease sought to be treated, (2) induce a pharmacological change relevant to treating the disease sought to be treated, (3) inhibit or prevent infection or re-infection by an infective agent, or (4) prevent the occurrence of a non-infectious disease (for instance a disease treatable by blocking a cell adhesion phenomenon). For wound treatment, in one aspect, an effective amount includes an amount which, if regularly applied, prevents the occurrence of infection. In another aspect, for wound healing, an effective amount includes an amount effective to reduce the average time it takes for a wound to heal.

Numerous methods for determining percent homology of proteins are known in the art.

One preferred method is to use version 6.0 of the GAP computer program for making

sequence comparisons. The program is available from the University of Wisconsin

Genetics Computer Group and utilizes the alignment method of Needleman and Wunsch,

J. Mol. Biol. 48, 443 (1970) as revised by Smith and Waterman, Adv. Appi. Math., 2, 482

(1981). Another available method uses the FASTA computer program.

The invention will now be further illustrated in the following non-limiting examples and the drawings wherein
Fig. 1. shows a chromatofocusing chromatograph of the Atlantic cod trypsin fraction off the aminobenzamidine affinity c~l~mn purification stEp. The fraction eluted from the para ~rriirtuWrii.~riiidiii~ ~ulumn was ge~ filtered on a Sepl,adex G-25 column equilibrated in 25 n,M monoethanolan,ine/acetic acid pH 9.4 containin~l 10mM calcium chloride and subsequently applied to a column of ~BE-94 ion exchange material (Pharmacia) equilibrated in the same buffer. The column was then eluted with Polybuffer 94 (adjusted to pH 5.5 with acetic acid) contai,ing 10 mM calcium chloride. Activity for the substrate TAME (filled circles), absorbance at 28' nm (open circles) and pH (open diamonds) was monitored. The irst eluted fraction was termed trypsin I, the second fraction trypsin II and the third fraction trypsin Ill;

Fig. 2. illustrates the effect of pH on :~e stability of Atlantic cod trypsin purified by para aminobenzamidine as measured b~; residual TAME activity after incubation at various pH values and two different time per~ds The Atlantic ccd trypsin samples were Incubated at 5 0C for 30 minutes (open circi~s) and 18 hours (closed aquares) at varying pH values. Residual activity for the substrate TAME was subsequently measured ~t pH 8.1 and 25

Fig. 3 shows the pH dependence of the activity c~Atl antic cod trypsin purified by paraaminobenzamid~ne as measured by using the TAME substrate. The buffers used at a final concemration 0.1 M were acetate (pH 3.0-5.9), hepe~~/HCl (pi-l 6.0-8.0) and glycinate (pH 8.3-9.0), all containing 10 mM calcium chloride; and

Fig. 4 shows the temperature dependence of the activity of Atlantic cod trypsin I compared to the activity of bovine trypsin measured by u3ing the TAME substrate. The enzymes were added to a preheated therrnostated cuveti:e and after a short equilibration period the average rate of hydrolysis was determined during the following 3 minutes.

EXAMPLE 1

Preparation o' a mixture of oroteases from cod

About 100 kg of frozen cod viscera were thawed and adoed to a four-fold volume of cold potable water in an extraction tank and the pH adjustt~d to pH 8 to 9 with a sodium

hydroxide solution. The mixture was stirred for about 2-6 hours at 0 to 50C. After a brief period of crude sedimentation (about 30 minutes) the aqueous extract was run off the remaining insoluble viscera with a pump and collected in a sedimentation tank. The aqueous extract was allowed to stand in the cooled sedimentation tank to sediment for about 24 to 60 hours. The supernatant was decanted from the supernatant tank to a holding tank using a pump. The supernatant was concentrated 10 to 20-fold by ultrafiltration and diafiltered to an acceptable level of ionic strength with conductivity below about 3 mS/cm. About 10-15 liters of ultrafiltered and diafiltered protein concentrate was obtained.

EXAMPLE 2

Purification of cod try~sin from concentrated cod viscera extract

About 10 liters of ultrafiltratered and diafiltered concentrate as obtained in. Example 1 was applied to a continuous connected series of about 1 liter packed chromatography columns, the first containing a CM fast flow cation exchange resin (Pharmacia, Sweden), the second one a DEAE fast flow anion exchange resin (Pharmacia, Sweden) and the third one a p-aminobenzamidine affinity ligand coupled to a sepharose resin (Pharmacia, Sweden). The columns were pre-equilibrated with about 10 column volumes of 25 mM Tris buffer of pH 7.8, containing 2.5 mM calcium chloride (buffer A). The concentrate was pumped onto the columns at a flow rate of about 100~ml per minute. When the application of the concentrated solution onto the columns was completed, residual material was washed off the continuous column system with about 8 liters of buffer A.

After this wash was completed, the p-aminobenzamidine affinity column was disconnected from the other columns and washed with about 5 column volumes of a high salt solution of 25 mM Tris buffer pH 7.5 containing 0.5 M NaCI and 2.5 mM calcium chloride. The cod trypsins were then desorbed from the affinity ligand and eluted off the column with an acid solution of 25 mM acetic acid pH 3.2 containing 10 mM calcium chloride and 30% glycerol. The cod trypsin fraction was collected into a neutralizing buffer of 200 mM Tris pH 8.5 containing 30% glycerol.

The purified cod trypsin preparation was homogeneous by SOS PAGE electrophoresis and FPLC Mono Q chromatography and showed the three trypsin isozyme bands on isoelectric focusing. The enzyme preparation had a specific activIty of about 100 U/mg using to Cbz-GPR-pNA as substrate as previously descr:bed. The purified preparation was filter sterilized through a 0.22 micron filter and stored frozen at about -20CC.

EXAMPLE 3
Altemative commercial scale ourification of cod trvosin from concentrated cod viscera extract
About 10 liters of ultrafiltratered and diafiltered zoncentrate as obtained In Example I was applied to a p-aminobenzamidine affinity ligand coupled Ic a sepharose resin (Pharmacia, Swede-i). The column was pre-equilibrated witt- about IC' column volumes of 25 mM Tris buffer of pH 7.8, containing 2.5mM calcium chloride bufer A). The concentrate was pumped onto the column at a flow rate of about 100 r~l psr minute. When the application of the co-icentrated solution onto the column was completed, residual material was washed off the column with about 8 liters of buf~r A.

The affinity column was subsequently washed with about 5 column volumes of a high salt solution of 25 mM Tris buffer pH 7.5 containing 0.5 M NaCI and 2.5 mM calcium chloride. The cod trypsins were then desorbed from the affinity ligand and eluted off the column with an acid solutiort of 25 mM acetic acid pH 3.2 con tain!ng 10 mM calcium chloride and 30% glycerol. The cod trypsin fraction was collected into a neutralizing buffer of 200 mM Tris pH 8.5 containing 30% glycerol, and contained a~ orcxlmately 5 to 6 g of trypsin.

The purified cod trypsin preparation was homogeneous by SDS PAGE electrophoresis and showed the th-ee trypsin isozyn~e banos on isoelectric focusIng. A minor fraction ( XAMPLE 4

Preparation of a hvdroQel preparation of cod trvpsins

The purified cod trypsin preparation of Example 2 was mixed with hydrocolloid gel comprising an aqueous gel containing 0.8% w/v Carbomer 940, 30% glycerol and 0.08% paraoxybenzoate. The cod trypsin preparation was mixed in a 1:1 ratio with the hydrogel to give a final concentration of 1 enzyme unit per mg (U/mg) of the final gel-enzyme mixture (the enzyme hydrogel ointment), the enzyme unit being determined using CbzGPR-pNA as substrate as previously described. Thus, the resulting enzyme hydrogel ointment contained about 0.01 mg/mI, or 1 U/mI of the cod trypsin enzymes, 0.4% Carbomer 940, 20% glycerol and 0.04% paraoxybenzoate. In the following, this enzyme hydrogel ointment is referred to as Penzyme 100.

EXAMPLE 5

Treating osteoarthritis with Penzyme 1 00

Seventeen individuals with osteoarthtitis, some with acute or very severe symptoms, were treated with the enzyme hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied once to twice per day, always once in the evening before retiring to bed and in most cases also in the morning. About 5 milliliters of the enzyme gel was applied on each affected area, such as each knee or hip or both hands. Larger quantities may be applied in severe cases. The gel was left to dry for about 10 to 30 minutes, depending on the amount applied, time available and the needs and requirements of the patient. This drying can be accelerated by the use of an air blowing device such as a hair dryer. All these patients experienced relief from symptoms within one week, most of them within 2 to 4 days. Patient No. 71 was unable to walk stairs before treatment but has had no problems doing so after treatment. These results are summarized in the following Table
5.1:

(Table Removed)

A few of the patients (Nos. 19 and 21) have been able to discominue treatment and thus appear to have enjoyed permanent or long term reliet from the disease, but most of them keep the symptoms under control witn cons:ant or int3rrnittent use of Penzyme 100.

EXAMPLE S

Treatment of oatients sufferino from tendinitis

Eight individuals with tendinitis, some with very seve e symptoms causing problems with sleep at night, were treated with the enzyme hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied topically on the affected area once to twice per day, about 5 to 10 milliliters on each affected area, .ii~pending on the size of the area. The gel was left to dry for about 10 to 30 minutes, depending on the amount applied, time available and the needs and requirements of ~he patit~nt. All these patients experienced relief from symptoms with in two weeks, mos'~ of therm within 2 to 4 days but others took

longer, especially those with tendinitis of the elbow (tennis elbow or golfer's elbow). These results are summarized in the following Table 6.1

All of these patients have enjoyed permanent relief from the condition and have discontinued treatment.

EXAMPLE 7

Treatment of fibromyalpia

Two patients suffering from severe fibromyalgia were treated with the enzyme hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied topically on the affected area twice per day, about S to 10 milliliters on each affected area, depending on size of the area. The gel was left to dry for about 10 to 30 minutes, depending on the amount applied, time available and the needs and requirements of the patient. Both patients (No. 78, F-SO and No. 92, F-53) experienced relief from symptoms within three weeks. Both use the enzyme gel treatment when pain reappears.

EXAMPLE 8

Treatment of rheumatoid arthritis

Five patients with rheumatoid arthritis, some with vey severe symptoms, were treated with the enzyme hydrogel ointment (Penzyme 1 ~0) of Example 4. The enzyme gel was applied topically on the affected area usually at east twice per day, about 3 to 5 milliliters on each affected area, depending on the size of the area. The gel was left to dr5' for about 15 minutes, depending on the thickness of the gel applied. This drying period can be shortened by the use of an air-blowing device such ~ts a hair dryer. All five patients experienced relief from symptoms within 5 days. ThE~se results are summarized in the following Table 8.1:

Patient No. 61 could not get out of bed in tne momin~ and dress without assistance before she began the treatment, but was fully capabl'3 of doing so on her own after treatment. Patient No 94 ~ad pain in the chest when breathing due to a complIcation of pleural effusion. She was relieved from these symptoms after 3 days of treatment with Penzyme 100. All of these patients keep the symptoms under control with constant or intermittent use of the enzyme gel treatment.

EXAMPLE 9

Treatment of phlebitis

Five patients with various forms of phlebitis, such as thrombophlebitis, phlebothrombosis, chronic postphlebitic syndrome and varicose veins, in all cases in the calves, some with very severe symptoms causing pain with difficulty of sleep, were treated with the enzyme hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied topically on the calf at least once per day. i.e. in the evening and in some cases also in the morning, about 5 milliliters on each calf. The gel is left to dry for about 15 minutes. All five patients experienced relief from symptoms within a few weeks. These results are summarized in the following table 9.1:

Patient No. 2 was diagnosed with chronic postphlebtic syndrome. Patient No. 68 does not sleep properly at night due to pain if she does not apply the gel before bedtime. All of these patients keep the symptoms under control with constant or intermittent use of the enzyme gel treatment.

EXAMPLE 10
Treatment of psoriasis

Six patients with psoriasis, some chronic cases with very severe symptoms, were treated with the enzyme hydrogel of Example 4, but in the majority of the cases of psoriasis a hydrogel composition designated Penzyme 200 with double the cod trypsin concentration

or 2 U/mI was used, since Penzyme 100 (lU/mI cod trypsin) yielded rather slow and in some cases uncertain results. Patients claimed greal: improvements in treatment with the gel containing the higher cod trypsin content (Penzyrne 2D0) as compared with the gel containing the lower trypsin content (Penzyme 100). A combination of cod trypsin and cod chymotrypsin, with a concentration of 1 U/mI of each enzyme, designated Penzyme 200-C, was also tested, and gave similar posit~e results as Penzyme 200. The enzyme gel was applied once to twice per day, always once In the evening before bedtime, after which the enzyme gel was left on the akir overnight and in some cases also in the morning after bathing and before dressitig. The gel i~ applied as a thin layer or film and takes about 5 mInutes to dry. All six patients experienced relief from symptoms within two weeks. The treatment was in some cases supplemented with conventional self-help methods such as sun bathing or UV light treatment. The results are summarised in the following Table 10.1:

Patient No. 3 has suffered from chronic and very severe psoriasis since the age of 5. She has tried all available treatments with limited results. The treatment with Penzyme 200 has yielded the only long-term relief from symptoms for her. She claims that relief from symptoms is noticeable after about Z. days and total relief is accomplished within 2 to 3 weeks of treatment with one application per day before bedtime.

Patient No. 69, also with severe chronic psoriasis, has experienced the permanent disappearance of some spots. All of these patients keep tne symptoms under control with constant or intermittent enzyme gel treatment.

EXAMPLE 11

Treatment of acne and boils

Two individuals (patient Nos. 25, F-16 and no. 63, M-17) suffering from acne and one suffering from boils (No. 64, M-22) were treated with the cod trypsin hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied topically on the affected area (face) once per day, about 5 milliliters each time. The gel is left to dry for about 15 minutes after which it is left on the skin. All three individuals experienced total relief from symptoms within two weeks, and only used the enzyme gel treatment occasionally when symptoms started to reappear.

EXAMPLE 12

Treatment of eczema. dermatitis and other skin conditions

Eighteen patients suffering from eczema, dermatitis and various other skin conditions, were treated with the cod trypsin hydrogel ointment (Penzyme 100) of Example 4. The enzyme gel was applied once per day (occasionally twice), usually in the evening before retiring to bed. The gel was applied as a thin layer or film. Drying occurred after about 5-10 minutes. A higher amount may be applied in severe cases. This drying can be accelerated by the use of an air-blowing device such as a hair dryer. All these patients experienced relief from symptoms within one week, most of them in 2 to 4 days. The results are summarised in the following Table 12.1:

Some of the patients (Nos. 40,41,48,66,50,20,46, 87, 97, 100) have been able to discontinue treatment and thus appear to have enjoyed permanent or long term relief from the disease, but others keep the symptoms under control with constant or intermittent ~se of Penzyme 100.

EXAMPLE 13

Wound healing

Two individuals (patient Nos. 44, M-4 anc 85, M-9) with bums were treated with the cod trypsin hydrogel ointment (Penzyme 100) of Example 4. Two o:her patients (No. 62, F-47 and 96, M-70) were treated for post-operationa wounds that would not heal. Tne enzyme

gel was applied topically on the wounds at least twice per day and left on the affected area. All four individuals experienced good and complication free healing of their wounds within one to three weeks.

EXAMPLE 14

Treatments with cod chvmotrvpsin

For each of examples 5-13, the cod trypsin enzyme hydrogel is substituted with a cod chymotrypsin enzyme hydrogel or a hydrogel containing a mixture of cod trypsin and cod ch y m ot ryp sin.

CLAIMS
1. A fish serine proteinase for use as a medicament.
2. A proteinase according to claim 1 that is selected from the group consisting of a trypsin,
a chymotrypsin and any mixture hereof.
3. A.proteinase according to claim 1 or 2 that is not multifunctional.
4. A proteinase according to any of claims 1-3 that is derived from Atlantic cod.
5. A proteinase according to daim 1 that is a trypsin derived from Atlantic cod.
6. A proteinase according to claim 5 that is selected from the group consisting of trypsin I,
trypsin II. trypsin III, trypsin IV a, trypsin IV b, trypsin IV c, and trypsin IV d.
7. A proteinase according to claim 4 that is selected from the group consisting of
chymotrypsin A and chymotrypsin B.
8. A proteinase according to claim 1 that is a trypsin having at least 90% amino acid
sequence homology with any of trypsin I, trypsin II, tiypsin 111, and trypsin IV derived from
Atlantic cod.
9. A proteinasa according to claim 1 that is a chymotrypsin having at least 90% amino
acid sequence homology with any of chymotrypsin A and chymotrypsin B isolated from
Atlantic cod.
10. A proteinase according to claim 1 for use as a medicament for treating and/or
preventing a disease in a human or an animal, said disease is selected from the group
consisting of pain, acute inflammation, chronic inflammation, arthritis, inflamed joints,
bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic arthritis,
fibromyalgia, systemic lupus erythematosus, phiebitis, tendinitis, rash, psoriasis, acne,
eczema, facial Beborrheic eczema, eczema of the hscxmds, face or neck, foreskin infections,
athlete's foot, fistuiae infections, infected topical ulcers, navel infections in newborns,
wrinkles, scars, kelloids, boils, warts and allergic itch, hemorrhoids, wounds, wound infections, wounds from burns, a fungal infection and an immunological disorder including an autoimmune disease.
11. A proteinase according to claim 1 for use as a medicament for
removing dead or peeling skin from otherwise healthy skin.
12. Use of a fish serine proteinase in the manufacturing of a medicament for treating
and/or preventing a disease in a human or an animal, said disease is selected from the
group consisting of pain, acute inflammation, chronic inflammation, arthritis, inflamed
joints, bursitis, osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, septic
arthritis, fibromyalgia, systemic lupus erythematosus, phlebitis, tendinitis, rash, psoriasis,
acne, eczema, facial seborrheic eczema, eczema of the hands, face or neck, foreskin
infections, athlete's foot, fistulae infections, infected topical ulcers, navel infections in
newborns, wrinkles, scars, kelloids, boils, warts and allergic itch, hemorrhoids, wounds,
wound infections, wounds from burns, a fungal infection and an immunological disorder
including an autoimmune disease.
13. Use of a fish serine proteinase in the manufacturing of a medicament for removing
dead or peeling skin from otherwise healthy skin.
14. Use of a fish serine proteinase in the manufacturing of a medicament for treating or
preventing a disease in whose pathogenesis a receptor-mediated binding is involved.
15. Use according to claim 14 where the disease is caused by a pathogenic organism
selected from the group consisting of a virus, a bacterium, a fungus, a parasite and a
protozoan.
16. Use according to any of claims 12-14 where the fish serine proteinase is a proteinase
according to any of claims 2-9.
17. A pharmaceutical composition comprising a fish serine proteinase.
18. A composition according to claim 17 wherein the fish serine proteinase is a
proteinase according to any of claims 2-11
19. A composition according to claim 17 or 18 that is a composition for topical use.
20. A composition according to claim 19 that is a hydrogel.
21. A composition accordJng to claim 20 further comprising a polyvalent alcohol including
gly cerol.
22. A composition according to claim 17 comprising a further pharmaceutical!/ active
compound.
23. A cosmetic composition comprising a fish serine proteinase.

24. A composition according to claim 23 wherein the fish serine proteinase is a
proteinase according to any of claims 2-11
25. A composition according to claim 23 that is a hydrogel.
25. A composition according to claim 23 further comprising a polyvalent alcohol including glycerol.
27. A composition according to claim 23 comprising £ further cosmetically active
compound.
28. A method of treating and/or preventing a disease in a human or an animal, the method
comprising administering to said human or animal a pharnaceutically effective amount of
a fish serine proteinase.
29. A method according to claim 28 wherein the proteinase is a proteinase according to
any of claims 2-11.
30. A method according to claim 28 wherein the proteinase is administered topically.
31. A method according to claim 30 wherein the prote nase is administered
gastroenteraliy,
32. A method of preparing on a commercial scale a purified preparation of cod trypsin isoenzymes comprising trypsin I, II, III and IV isozymes, the method comprising the steps of
(i) preparing an aqueous extract of cod viscera,
(ii) subjecting the aqueous extract to one or more chromatography steps including an affinity chromatography step using a p-aminobenzamidine affinity ligand, and
(iii) desorbing and eluting the trypsin bound to the p-aminobenzamidine affinity iigand.
33. A method of preparing a purified preparation of cod trypsin isoenzymes, according to claim 32, wherein the method further comprises one or more steps using a cation exchange resin, and one or more steps using an anion exchange resin prior to the affinity chromatography step.
34. A method according to claim 32 or 33 wherein the purification is continued until the
preparation contains a specific activity of at least 50 U/mg protein using Cbz-GPR-pNA as
substrate.
35. A method according to claim 34 wherein the specific activity is 100 U/mg or more.
36. A method according to claim 32 comprising as a further step that the purified
preparation is sterile filtered using a 0.22 fim filter.
37. A purified preparation on a commercial scale of cod trypsin.
38. A purified preparation of cod trypsin according to claim 37 comprising, when it is
subjected to SDS-PAGE electrophoresis and FPLC Mono Q chromatography as the only
protein bands three to seven bands of trypsin isoenzymes.
39. A preparation according to claim 37 or claim 38 containing a specific activity of at least
50 U/mg protein using Cbz-GPR-pNA as substrate.
40. A preparation according to claim 38 where the saecific activity is 100 U/mg or more.
41. A fish serine proteinase for use as a medicament substantially as
herein described with reference to the accompanying drawings.
42. Use of a fish serine proteinase in the manufacturing of a
medicament for treating and / or preventing a disease in a human
or an animal substantially as herein described with reference to
the accompanying drawings.
43. A pharmaceutical composition substantially as herein described
with reference to the accompanying drawings.
44. A method of treating and / or preventing a disease in a human or
an animal substantially as herein described with reference to the
accompanying drawings.
45. A method of preparing on a commercial scale a purified
preparation of cod trypsin isoenzymes comprising trypsin I, II,
III and IV isozymes substantially as herein described with
reference to the accompanying drawings.
46. A purified preparation on a commercial scale of cod trypsin
substantially as herein described with reference to the
accompanying drawings.

Documents:

in-pct-2002-034-del-abstract.pdf

in-pct-2002-034-del-claims.pdf

in-pct-2002-034-del-correspondence-others.pdf

in-pct-2002-034-del-correspondence-po.pdf

in-pct-2002-034-del-description (complete).pdf

in-pct-2002-034-del-drawings.pdf

in-pct-2002-034-del-form-1.pdf

in-pct-2002-034-del-form-19.pdf

in-pct-2002-034-del-form-2.pdf

in-pct-2002-034-del-form-3.pdf

in-pct-2002-034-del-form-5.pdf

in-pct-2002-034-del-gpa.pdf

in-pct-2002-034-del-pct-101.pdf

in-pct-2002-034-del-pct-210.pdf

in-pct-2002-034-del-pct-401.pdf

in-pct-2002-034-del-pct-408.pdf

in-pct-2002-034-del-pct-409.pdf

in-pct-2002-034-del-pct-416.pdf

in-pct-2002-034-del-petition-137.pdf

in-pct-2002-034-del-petition-others.pdf

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Patent Number

223104

Indian Patent Application Number

IN/PCT/2002/00034/DEL

PG Journal Number

40/2008

Publication Date

03-Oct-2008

Grant Date

04-Sep-2008

Date of Filing

08-Jan-2002

Name of Patentee

BJARNASON Jon Bragi

Applicant Address

SKILDINGANES 29, 101 REYKJAVIK, ICELAND.

Inventors:

#	Inventor's Name	Inventor's Address
1	BJARNASON JON BRAGI	SKILDINGANES 29, 101 REYKJAVIK, ICELAND.

PCT International Classification Number

A61K 38/00

PCT International Application Number

PCT/IS00/00005

PCT International Filing date

2000-06-15

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	5086	1999-06-18	Ice Land