NZ503162A - Use of a krill- or atlantic cod-derived hydrolase with chymotrypsin, collagenase, elastase and/or exo peptidase activity to inactivate call adhesion components including ICAM-1 (CD54), CD4, CD8, CD11 and CD28. - Google Patents

Abstract

The use of a hydrolase derived from krill or Atlantic cod in a medicament to inhibit tissue transplant rejection. The tissue is treated extra-corporeally prior to transplant into the recipient. The hydrolase has substantial anti cell-cell adhesion activity by selectively removing or inactivating ICAM-1 (CD54), CD4, CD8, CD11 and/or CD28. Preferably the hydrolase also has at least one of a chymotrypsin, trypsin, elastase, collagenase and exo peptidase activity.

Description

Patents Form No. 5 Our Ref: JP802119 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION DIVISIONAL APPLICATION OUT OF NEW ZEALAND PATENT APPLICATION NO. 302984 FILED ON 8 FEBRUARY 1996 MULTIFUNCTIONAL ENZYME We, PHAIRSON MEDICAL, INC, a body corporate organised under the laws of Sweden of P.O. Box 15099, S-750 15 Uppsala, Sweden. hereby declare the invention, for which We pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: INTELLECTUAL PROPERTY OFFICE ] OF NZ PT0548114 | 2- MAR 2000 RECEIVED . MULTIFUNCTIONAL ENZYME The present invention relates to a kriil-derived multifunctional enzyme and a family of crustacean and fish derived enzymes having substantial structural similarity to the multifunctional enzyme derived from antarctic krill.

The invention additionally relates to the multifunctional enzyme, to methods of purifying the multifunctional enzyme, to purified multifunctional enzyme and to pharmaceutical, cosmetic and other uses of the multifunctional enzyme of the invention.

The enzymes that are substantially structurally similar to the krill-#10 derived multifunctional enzyme have the same utility as the krill enzyme. In particular, these multifunctional enzymes are useful for treating viral infections such as herpes outbreaks, fungal, bacterial or parasitic infections, including the primary and secondary infections of leprosy, colitis, ulcers, hemorrhoids, corneal scarring, dental plaque, acne, cystic fibrosis, blood clots, wounds, 1 5 immune disorders including autoimmune disease, such as lupus erythematosus and multiple sclerosis, and cancer.

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 the mixture as cleaning solutions. U.S. Patent No. 4,801,451 20 discloses the use of such enzymes to remove foreign matter and dead tissue ^ from wounds. Patent Application WO 85/04809 discloses the use of krill enzymes as a digestion promotor. European Application EP-A1-0170115 discloses the use of krill enzymes to dissolve blood clots. All of these references employ impure or poorly characterized materials. A purified 25 multifunctional enzyme is desirable to provide a pharmaceutical^ useful product.

SUMMARY OF THE INVENTION The present invention provides a multifunctional enzyme that has been found to be useful in numerous medical and cosmetic contexts. In particular, 30 the invention relates to an enzyme having multifunctional activity comprising - 2a - The applicant's related parent application, NZ 302984 provides a pharmaceutical composition comprising: (a) an enzyme having a purity with respect to macromolecules of at least about 95%, having a least two of a chymotrypsin, trypsin, collagenase, elastase or exo peptidase activity, a molecular weight between about 26 kD and about 32 kD as determined by SDS PAGE, and an N-terminal sequence comprising: l-V-G-G-X-E-V-T-P-H-A-Y-P-W-Q-V-G-L-F-l-D-D-M-Y-F wherein X is any amino acid; and (b) a pharmaceutically acceptable carrier or excipient.

In another preferred embodiment, the multifunctional enzyme shall have at least about 70% homology with the krill derived multifunctional hydrolase, more preferably at least about 80% homology, still more preferably at least about 90% homology, yet still more preferably at least about 95% homology. Homology measurements will score conservative substitutions as homologous. The krill-derived multifunctional hydrolase can be the multifunctional enzyme. The krill-derived multifunctional hydrolase is 100% (FOLLOWED BY PAGE 3) homologous with itself..

The invention also provides a pharmaceutical composition comprising the multifunctional enzyme of claim 1 and a pharmaceutical^ acceptable diluent or carrier. i The invention further provides (a) methods relating to certain conditions using effective amounts of the enzyme described above, (b) compositions for use in such methods, (c) pharmaceutical compositions containing effective amounts of enzyme for use in such methods, and (d) uses of the enzyme composition for manufacturing a medicament for use in such methods. The 110 methods are for: (1) treating or prophylgctically preventing a microbial infection [e.g. viral such as: a herpes (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 a systemic, skin , oral, vaginal or esophageal fungal, including, for example, yeast infection, including a fungal nail infection and Candida infections; microbial infections of the eye, preferably treated with occular 20 administrations; bacterial infections including staphylococcus, streptococcus, klebsiella, pseudomonas, gonorrhea, haemophilus, chlamydia, syphilis and E. coli infections and bacterial infections causing chancroid; opportunistic microbial infections in immunocompromised patients where preferably the administered amount 25 of the multifunctional enzyme, described below, is a microbial infection treating or preventing effective amount or has inhibitory activity against cell-cell or cell-virus adhesion; (2) treating or prophylactically preventing dermatological conditions, such as, for example, acne, psoriasis or eczema, including facial seborrheic eczema or eczema of the hands, face or neck, diarrhoea, eye disease, such as glaucoma or cataracts, and hair-thinness, where preferably the amount of the multifunctional enzyme administered is a treating or preventing effective amount; (7) removing dead or peeling skin from otherwise healthy skin to improve the skin's appearance, where preferably the amount of the multifunctional enzyme administered is a dead skin removing effective amount; (8) lysing blood clots, where preferably the amount of the multifunctional enzyme administered is a clot lysing effective amount; and (9) removing dental plaque, where preferably the amount of the multifunctional enzyme administered is a dental plaque removing effective amount.

The method comprises administering a composition comprising a the multifunctional enzyme described above. The composition of the invention can also be used to remove dead of divergent cells.

The invention provides topical cosmetic composition comprising the multifunctional enzyme described above; and cream, gel or suppository 20 composition.

The invention also provides method of purifying the multifunctional enzyme described above to obtain a composition containing substantially no other proteins that bind a selected protease inhibitor other than the multifunctional enzyme, the method comprising the steps of: 25 (a) applying a composition containing the multifunctional enzyme to an ion exchange column; (b) eluting a first adsorbed material from the column with a first aqueous solution of first ionic strength I,; and (c) eluting the multifunctional enzyme from the column with a 30 second aqueous solution of second ionic strength l2; passages or sinuses on an animal at risk of infection in a microbial infection preventing effective amount; (12) treating or prophylactically preventing a primary or secondary microbial infection in a patient having leprosy, preferably administering in a primary or secondary infection treating or preventing effective amount; (13) treating a tissue, body fluid or composition of cells to remove or inactivate a cell adhesion component comprising, wherein the enzyme is administered to the tissue, body fluid or composition ,10 of cells, preferably a cell-adhesion component removing or inactivating effective amount or an immune rejection inhibiting amount of the enzyme is administered, preferably the tissue, body fluid or composition of cells is treated extra-corporally, although they may also be treated in situ in an animal; (14) cleaning a contact lens, preferably applying to the lens a lens cleaning effective amount of the enzyme, where the application can be done while the lens is in the eye; (15) treating or prophylactically preventing coronavirus, such as the coronavirus causing feline infectious peritonitis, cytomegalovirus, rhinovirus or papilloma virus (such as human papilloma virus) infection, where preferably a viral infection treating or preventing effective amount of enzyme is administered; (16) treating or prophylactically preventing HIV infection, preferably administering an HIV infection treating or preventing effective amount of the enzyme; (17) treating or prophylactically preventing an oral or esophageal fungal, including yeast infection, preferably by administering a microbial infection treating or preventing effective amount of the enzyme; (18) treating or prophylactically preventing a pseudomonas, lichen scleroderma, preferably by administering an autoimmune disease treating or preventing effective amount of the enzyme; and (25) treating hemorrhoids (for example, post-partum hemorrhoids) preferably by administering a hemorrhoids treating effective 5 amount of the enzyme.

The method comprises administering a composition comprising a the multifunctional enzyme described above.

The invention further provides (a) methods for treating or prophylactically preventing a cell-cell or cell-virus adhesion syndrome 0 comprising administering an anti-adhesion effective amount of a hydrolase 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) compositions or substances for use in such methods, (c) pharmaceutical compositions containing effective amounts of enzyme for use in such 1 5 methods, and (d) uses of the enzyme composition for manufacturing a medicament for use in such methods. Preferably, the syndrome comprises inflammation, shock, tumor metastases, autoimmune disease, transplantation rejection reactions or microbial infections. Preferably, (a) the syndrome is selected from the group consisting of microbial infection, immune disorder, 20 cystic fibrosis, COPD, atherosclerosis, cancer, asthma, septic shock, toxic ) shock syndrome, conjunctivitis, reperfusion injury and pain, and (b) a cell surface receptor, associated with the cell-cell or cell-virus adhesion syndrome, selected from the group consisting of ICAM-1, ICAM-2, VCAM-1, CD4, CD8, CD11, CD18, CD28, CD29D, CD31, CD44, CD 49, CD62L, CD102 and 25 asialo GM1 ceramide is removed or inactivated by the administered hydrolase. Preferably, a microbial infection is treated or prevented and the microbial infection is a herpes, HIV, hepatitis or papilloma infection; an infection causing colitis, ulcer or diarrhoea; a Candida infection, such as an oral, vaginal or esophageal Candida infection; a cold or influenza infection; a pseudomonas, 30 haemophilus, staphylococcus, streptococcus, klebsiella or E. coli infection; a - 1 1 - of the identified proteolytic activities, more preferably at least three, still more preferably at least four. Vet more preferably, the enzyme has all of the identified proteolytic activities. Preferably, the multifunctional enzyme has substantial anti cell-cell and cell-virus adhesion activity. Preferably, the 5 multifunctional enzyme has substantial homology with the krill-derived multifunctional hydrolase.

In a preferred embodiment, HIV-infected patients are treated to slow the progression of the associated diseases by the process of (1) isolating T-cells from the patient, (2) treating the T-cells with a hydrolase effective to |f10 remove CD4, and (3) injecting the T-cells into the patient.

The method of treating tumors can be done by administering orally or parenterally, including but not limited to intravenously, intra-arterially, intraperitoneally, subcutaneously, intramuscularly or intra-tumorally. As a part of this embodiment, the invention provides a method of preventing or limiting 15 tumor metastatic processes.

In one embodiment, the invention provides a method of inhibiting or prophylactically preventing the transmission of a pathogenic microbe by administering the multifunctional enzyme. Preferably, the multifunctional enzyme is applied to the portion of the body that comprises the primary 20 transmission entryway for the microbe in question. In one preferred I 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 multifunctional enzyme is applied to the upper airways, for example, via an aerosol, to inhibit or prevent the 25 transmission of a cold virus, such as a rhinovirus or a corona virus.

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 a tissue, body fluid or composition of cells that is transplanted from one individual to another. In another aspect, such treatments 30 remove or inactivate the cell adhesion components found in the treated Fig. 5 shows the survival of the enzyme-treated and untreated mice containing the ovarian tumor.

Fig. 6 shows the increase in time between urinations for urinary bladder infection patients treated with a poly-enzyme preparation of Example 1A.

Fig. 7 shows the reduction in erythema/swelling in patients with viral lung infections treated with the multifunctional enzyme purified as described in Example 1B.

Fig. 8 shows the protective effect of the multifunctional enzyme, purified as described in Example 1C, against HIV.

Fig. 9 shows the pH dependence of the multifunctional enzyme, purified as described in Example 1C, when tested against different substrates.

Fig. 10 shows the titration of the activity of the multifunctional enzyme, purified as described in Example 1C, with 2 different protease inhibitors.

Fig. 11 shows the pain relief experienced by lame race horses treated with the poly-enzyme preparation of Example 1A.

Fig. 12 shows certain indicators of healing following the treatment necrotic wounds with the multifunctional enzyme purified as described in Example 1B.

DETAILED DESCRIPTION It has now been established that the multifunctional enzyme of the invention effectively removes or inactivates certain cell-surface adhesion molecules, such as ICAM-1 (i.e., CD 54), ICAM-2, VCAM-1, CD4, CD8, CD28, CD31, CD44 and the asialo GM1 ceramide, without affecting cell 25 viability. This adhesion site removal or inactivation phenomenon is believed to provide at least a partial explanation for the enzyme's effectiveness against many, though probably not all, of the indications against which the multifunctional ehzyme is effective. Other cell surface receptors have been found to be substantially resistant to removal or inactivation by the 30 multifunctional protein, such as the T-cell receptor, the Class I major of Ischemial/Reperfusion," in Adhesion Molecules, Wegner, Ed., Academic Press, 1994, pp. 163-190. ICAM-1 specific antibodies interfere with neutrophil adherence to and migration into ischemic tissue, id., thereby limiting the role of neutrophils in re-perfusion injury. These antibodies can 5 also limit the size of a myocardial infarction. Id. at 177, figure 8.17. The multifunctional enzyme interferes with ICAM-1 by removing it from the cell surface or by inactivating it.

Again not wishing to be limited by any particular theory, the anti-CD4 cell surface adhesion molecule activity of the multifunctional enzyme is 00 believed to be responsible, at least in part, for the enzyme's HlV-transmission inhibitory activity. The HIV infective pathway utilizes the CD4 cell-surface molecule. See, Lentz, "Molecular Interaction of Viruses with Host-Cell Receptors," in Adhesion Molecules, Wegner, Ed., Academic Press, 1994, pp. 223-251 at p. 229.

In Candida infections, the hyphal stage is believed to be more infective.

Ligands on the Candida fungi that possibly recognize a GalNAc/?(1-4)Gal carbohydrate structure on asialo-GM1 ceramides of the infection host, are believed to be involved in the process by which Candida infect. Such ligand molecules may also be involved in the process by which Candida yeast are 20 converted to the hypal stage. In pseudomonas infections of the lungs, a # bacterial cell-surface ligand that recognizes a GalNAcy9(1-4)Gal carbohydrate structure is believed to play a role in the lung infection pathway. Abraham, "Bacterial Adhesions," in Adhesion Molecules, Wegner, Ed., Academic Press, 1994, pp. 253-276. The same or similar glycolipid structure is believed to be 25 involved in adhering several pathogens to the lungs, including Pseudomonas aeruginosa, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae and certain isolates of E. coli. See, Krivan et al., Proc. Natl. Acad. Sci. U.S.A. 85: 6157-6161, 1988.

Wrinkled skin to a major part is caused by free radicals crosslinking 30 collagen. The formation of free radicals is believed to be caused by the enzyme of the invention. Further, such treatment of lung epithelial cells reduces the level attachment of Pseudomonas bacteria to the lung epithelial cells.

It is believed that the above discussed adhesion molecules and others 5 will prove to play a role in a number of other diseases for which the multifunctional enzyme is an effective treatment or preventative agent.

For the purposes of this application, the terms listed below shall have the following meaning: at'risk of infection • cell-cell or cell-virus adhesion syndrome • cell adhesion component involved in a microbial infection • dermatological condition • derived from fish or crustacean • drug-resistant an animal is at risk of infection if some feature of its environment or medical history is correlated with an increased risk of one or more infections. a disease in which a receptor or acceptor cell adhesion component plays a role in the etiology of the disease, for instance by playing a role in the development, transmission, growth or course of the disease. a cell adhesion component used by a microbe to facilitate adherence to a tissue or cell at risk of infection. a condition relating to lesions of the topical surface of an animal, including without limitation the skin, the vagina and the surface of the eyes. refers to an enzyme having the same sequence as an enzyme isolated from fish or crustacean. a microbe of a type that is or once was generally sensitive to given amounts of one or more antimicrobial agents but which is not sensitive to the agents or is only sensitive at significantly higher amounts.

PCT/U S96/01650 19 - • reactive with a cellular or viral acceptor or receptor adhesion component • SDS-PAGE • selectively reactive with a cell-surface protein • sexually transmitted microbial infection means removes, destroys, inactivates or disables a cell's or a virus' ability to interact with a cell, virus, ligand, group or molecule, regardless of the mechanism. means polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate. means removes, destroys, inactivates or disables certain cell-surface proteins on the surface of a cell but not others. any microbial disease that is transmitted during sexual contact. • substantial homology at least about 60% sequence homology. • substantially no other proteins that bind a selected protease inhibitor systemic administration a composition contains substantially no other proteins that bind a selected protease inhibitor if such proteins comprise no more than about 5% w/w of the total protein in the composition. an administration of a biological agent, such as the multifunctional enzyme, designed deliver the agent to the blood or other circulatory system (such as the lymphatic system) of an animal.

Crustaceans, including antarctic krill, are useful sources for the multifunctional enzyme of the invention. For instance, frozen krill can be homogenized in water or buffer, preferably containing an antimicrobial agent. The supernate, diluted if appropriate, can then be fractionated by ion exchange chromatography (preferably anion exchange chromatography), gel filtration, chromatofocusing chromatography, or other traditional separation process. Preferably, however, some part of the separation process will include affinity chromatography using a matrix having attached molecules of a trypsin inhibitor, such as soybean trypsin inhibitor. The krill-derived multifunctional hydrolase used in the invention can be desorbed from such a matrix by applying conditions that will destabilize the interaction between the hydrolase and the inhibitor. Such conditions include high salt, low pH or the PCT/U S96/01650 have specific activity at least about 260 Units per mg. Elastase activity can be measured using Boc-Ala-Ala-Pro-Ala-p-N02-anilide as the substrate. Using this substrate and the method of J. Biol. Chem., 269: 19565-19572, 1994, the multifunctional enzyme will preferably have specific activity of at least 5 about 270 Units per mg.

Protein purity is generally determined by SDS-PAGE with Coomassie blue staining. The percent staining in the appropriate band reflects the purity. Protein concentrations are generally determined by amino acid analysis or by absorbance at 280 nm.

Generally, the multifunctional enzyme will be sufficiently stabile so that at least about 50% of the proteolytic activity is retained after incubation at 50°C for 24 hours at pH 7.0 at a concentration of 5 mg/ml. Preferably at least about 50% of the proteolytic activity is retained after incubation at 60°C for 5 hours at pH 7.0 at a concentration of 5 mg/ml. 15 Preferably, the pH optimum of the multifunctional enzyme is substrate dependent. For the substrate azocasein, the pH optimum is preferably from about 3.5 to about 6.5, more preferably, from about 4.0 to about 6.0. For the substrate Benzoyl-Val-Gly-Arg-p-nitroanilide, the pH optimum is preferably in excess of about 8.0, more preferably in excess of about 9.0. For the 20 substrate Boc-Ala-Ala-Pro-Ala-p-nitroanilide, the pH optimum is preferably ' between about 6.0 and about 7.0, more preferably about 7.0.

Using Benzoyl-Val-Gly-Arg-p-nitroanilide as the substrate, the Km at about pH 9.5 in the presence of 2 mM Ca2+ is preferably between about 200 ' and about 240 /;M. Using Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the 25 substrate, the Km at pH 9.5 in the presence of 2 mM Ca2+ is preferably between about 250 and about 290 pM.

Preferably, the multifunctional enzyme has a temperature optimum for activity against casein of between about 45°C and about 60°C. Generally, the enzyme retains at least about 50% of its activity when incubated at 5 30 mg/ml for 18 hours at a pH ranging from about 5.0 to about 9.5 at 25°C. intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include 5 mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or polyvinyl alcohol, preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. For pulmonary administration, diluents and/or carriers will be selected to be appropriate to allow the formation of an aerosol. For topical 010 administrations, the multifunctional enzyme is typically administered in aqueous form or in a hydrogel.. A preferred hydrogel comprises an aqueous suspension of from about 1% (w/v) to about 10% of low molecular weight hydrolyzed starch.

Suppository forms of the multifunctional enzyme are useful for vaginal, 15 urethral and rectal administrations. Such suppositories will generally be constructed of a mixture of substances that is solid at room temperature but melts at body temperature. The substances commonly used to create such vehicles include theobroma oil, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weighty and fatty 20 acid esters of polyethylene glycol. See, Remington's Pharmaceutical # Sciences, 16th Ed., Mack Publishing, Easton, PA, 1980, pp. 1530-1533 for further discussion of suppository dosage forms. Analogous gels or cremes can be used for vaginal, urethral and rectal administrations.

Numerous administration vehicles will be apparent to those of ordinary 25 skill in the art, including without limitation slow release formulations, liposomal formulations and polymeric matrices.

For topical treatments, a suitable dose of multifunctional enzyme per application ranges from about 0.1 /yg/cm2 to about 1 mg/cm2, preferably from about 1 /yg/cm2 (for example, using about 10 /yg/ml) to about 1 mg/cm2 (for 30 example, using about 10 mg/ml), more preferably from about 5 /yg/cm2 (for / / about 5 mg. For all treatments, the enzyme composition will generally be applied from about 1 to about 10 times per day, preferably from about 2 to about 5 times 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 5 and medical condition of the patient, as will be recognized by those of ordinary skill in the medical arts. It is believed that substantially higher doses can be used without substantial adverse effect.

For treating or preventing infection, the multifunctional enzyme can be administered systemically or in a manner adapted to target the affected 10 tissue. For preventing cold or influenza transmission, the composition is preferably applied to the lungs or airways. For treating immune disorders, the composition may be applied systemically or in a manner adapted to target the affected tissue. For treating the primary and secondary infections of leprosy, the primary administration route will generally be the topical route. For 1 5 treating scar or keloid tissue, generally the composition will be injected into the scar or keloid, except that for corneal scars the composition will generally be applied ocularly without injection. For cancer treatment, the composition will generally be administered systemically by a route or in a manner adopted to target the affected tissue. For treating atherosclerosis, the composition 20 will generally be administered systemically, although the site of administration may be chosen to administer the highest dosages to the portion of the circulatory system most at risk. For asthma, the general route of administration will be pulmonary. For treating pseudomonas infections, the infection will typically be a lung infection and the administration route 25 pulmonary. For reperfusion injury, the composition will generally be administered systemically, although the site of administration may be designed to administer the highest dosages to the portion of the body that suffered an ischemic event. For treating the painful symptoms of malaria, the administration mode will generally by systemic.

For wound healing, the multifunctional enzyme is preferably be applied PCT/U S96/01650 inhibitory embodiment of the invention are sexually-transmitted diseases, such as Candida, gonorrhea, chlamydia, syphilis, trichomonas, chancroid, HIV, herpes or hepatitis infections. Among these, viral diseases are particularly preferred targets for transmission prevention; HIV is a still more preferred 5 target. For this use, the body cavity involved in sexual activity is generally rinsed or flushed with a composition containing the multifunctional enzyme, or a creme, gel or suppository designed to localize the composition to the body cavity is used. The composition can be used soon before, in conjunction with, or soon after, sexual activity, although prior or concurrent )10 use is preferred.

For herpes" infections, the viral targets include HSV-1, which primarily manifests as oral herpes, HSV-2, which primarily manifests as genital herpes, and herpes zoster.

For autoimmune diseases or diseases with autoimmune components, 1 5 treatment targets include without limitation rheumatoid arthritis, multiple sclerosis, primary biliary cirrhosis, active chronic hepatitis, ulcerative colitis, rheumatic arthritis, scleroderma, systemic lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, thyroroxicosis, pernicious anemia, Addison's disease, premature onset of menopause, autoimmune male infertility, insulin-20 dependent diabetes, type B insulin resistance of acanthosis nigricans, atopic ^ allergy, myasthenia gravis, Lambert-Eaton syndrome, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, phacogenic uveitis, sympathetic ophthalmia, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Sjogren's syndrome, discoid lupus erythematosus, dermatomyositis 25 and mixed connective tissue disease.

For adhesion disorders, the cells or viruses involved can include, without limitation, endothelial cells, lymphocytes, including T-cells, tumor cells, microbial cells, viruses, including HIV and herpes. Adhesion processes are believed to be involved in tissue invasion, for instance, by immune cells, 30 microbes, and tumor cells. 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.

Humans are the preferred subjects for treatment. However, the 5 multifunctional enzyme can be used in many veterinary contexts to treat animals, preferably to treat mammals, as will be recognized by those of ordinary skill in light of the present disclosure.

Numerous methods for determining percent homology are known in the art. One preferred method is to use version 6.0 of the GAP computer ^ 10 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. Mot. Biol. 48, 443, 1970 as revised by Smith and Waterman, Adv. Appl. Math., 2, 482, 1981. Another available method uses the FASTA computer program. 15 The multifunctional enzyme has been observed to treat infections.

However, its direct effect on the growth of microbes in vitro is small. While not wishing to be limited by any particular theory, it is believed that the enzyme attacks the mechanisms by which microbes and tumors invade tissues. These mechanisms include cell-cell or cell-virus adhesion 20 mechanisms by which a tumor or microbe may establish itself in a tissue. The importance of cell-surface adhesion molecules and adhesion processes in tumor metastasis is illustrated by the discussion in a Albelda, "Role of Cell Adhesion Molecules in Tumor Progression and Metastasis," in Adhesion Molecules, Craig Wegner, Ed., Academic Press, San Diego, 1994, pp. 71-88. 25 The disruption of other cell-cell or cell-virus adhesion reactions by the multifunctional enzyme is believed to be relevant to other conditions that are treatable with the multifunctional enzyme, including dental plaque and immune disorders.

The adhesion of HL60 cells ( a hyman lymphocyte cell line) to 30 endothelial cells is believed to model a mechanism for tumor cell invasion and pH values. Following the incubation, the pH was adjusted to about pH 7.0, and the remaining activity determined as described above. The optimum stability is achieved at about pH 7.0. However, at least 50% of the original activity is retained following about 18 h incubations in solutions having pH 5 values between about 5.0 and about 9.5.

The multifunctional enzyme can be purified from tissue homogenates of fish or crustaceans or from homogenates or supernates derived from a cell culture of transformed or normal prokaryotic or eukaryotic cells that produce the multifunctional enzyme. The preferred purification comprises the use of j)10 an affinity column comprising an inhibitor reactive with the multifunctional enzyme. After the multifunctional enzyme is eluted from the affinity column, residual enzyme inhibitor in the preparation is removed. One method of removal is to pass the preparation over an affinity matrix comprising a molecule, typically a protease, with which the inhibitor binds strongly (for 15 instance, with an affinity constant of at least about 106 M, preferably at least about 107 M). More preferably, the affinity ligand is a previously isolated preparation of the multifunctional enzyme that the procedure seeks to purify.

Some amount of traditional protein purification will preferably be done prior to the affinity chromatography step. This can include differential 20 precipitation, gel filtration chromatography, ion-exchange chromatography, ^ chromatography on weakly hydrophobic matrices such as dye matrices, chromatofocusing and reversed phase liquid chromatography. Preferably, these one or more steps will be sufficient to remove all proteins, other than the multifunctional enzyme, that bind to the affinity ligand. Alternatively or 25 supplementally, one or more traditional protein purification steps can be applied after the affinity chromatography step.

A preferred purification procedure comprises the steps of: (a) applying a composition containing the multifunctional enzyme to an ion exchange column; 30 (b) eluting a first adsorbed material from the column with a matrix comprising, in the swelled state, between about 0.05 mmol and about 0.6 mmol anion exchange sites per ml. Preferably, the matrix is a cross-linked dextran of the type sold under the tradename Sepharose. Preferably, the anion exchange groups comprise diethylaminoethyl (DEAE) or 5 quaternaryaminoethyl (QAE) groups, more preferably DEAE groups.

The affinity chromatography of steps (d)-(f) preferably further comprise, between steps (d) and (e), washing the inhibitor-containing column with a solution having ionic strength of at least about that of 0.5 M NaCI, more preferably of about 0.8 M NaCI, yet more preferably of about 1M NaCI. This ^|10 step and the prior eluting step will preferably be conducted at a pH between about 5.5 and about 7.5, preferably between about 6.0 and about 7.0. The eluting step (e) preferably comprises applying a buffer having pH of about 2 to about 4, preferably about 3. Alternately, it may preferably comprise applying a buffer having pH of at least about 8. The eluting buffer will 1 5 preferably have sufficient ionic strength to suppress weak ionic interactions with the affinity matrix.

Steps (g) and (h) of the method will preferably be conducted at a pH between about 5.5 and about 7.5, more preferably between about 6.0 and about 7.0. The buffer used in these steps will preferably have sufficient ionic 20 strength to suppress weak ionic interactions with the affinity matrix. Q The matrix used to create the affinity matrices will preferably comprise a carbohydrate matrix such as cross-linked dextran (e.g., that sold under the tradename Sepharose) or agarose (e.g., that sold by Pharmacia, Sweden as "Sephacryl"). The matrix should have pore sizes sufficient to admit both the 25 affinity ligand that will be attached to the matrix and the multifunctional enzyme of the invention. Methods of synthesizing appropriate affinity columns are well known. See, for instance, Axen et al., Nature, 214:1302-1304, 1967.

Preferably, the affinity ligand is selected to be resistant to digestion by 30 the multifunctional enzyme under the conditions used in steps (d) and (e).

PCI7US96/01650 Panaeus monodon chymotryptic - 2 Sequence reported: Tsai et al., Biochem. et Biophys. Acta, 1080: 59-67, 1991 Reported activities: chymotryptic collagenase Apparent MW: -26kd Uca pubifator (Fiddler Crab) 1 Sequence reported: Tsai et al., Biochem. et Biophys. Acta, 1080: 59-67, 1991 Reported activities: chymotryptic Apparent MW: Ph optimum 25kd 8.0 - 8.5 Uca pugilator II Sequence reported: Grant et al.. Biochemistry, 19: 4653-4659, 1980.

Reported activities: chymotryptic collagenase tryptic elastase Apparent MW: 25kd pi: 8.0 - 8.5 Kamchatka crab (at least four proteases) Sequence Reported: Grant et al., Biochemistry, 22: 354-358, 1983 Reported Activities: tryptic collagenase Apparent MW: 23-26kd Crayfish Protease Sequence reported: Titani et al.. Biochemistry, 22: 1459-1465, The sequence of the first 25 amino acids of the Krill derived multifunctional enzyme is |-V-G-G-N/M-E-V-T-P-H-A-Y-P-(W)-Q-V-G-L-F-I-D-D-M-Y-F (SEQ ID NO. 1). The parentheses indicate a weak recovery of the 14th 25 amino acid and "N/M" indicates heterogeneity at the 5th position. A herein can be used as.the basis of oligonucleotide probes for screening expression or genomic libraries to isolate the complete structural gene. See, e.g., Suggs et al., Proc. Natl. Acad. Sci. USA, 78: 6613, 1981 or Berent et al., BioTechniques, 3: 208, 1985. Alternately, known protein sequences can 5 be used to design primers for use in PCR-based amplification of nucleic acid encoding a multifunctional enzyme. See generally, Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor, 1989 and PCR Protocols, A Guide to Methods and Applications, edited by Michael et al., Academic Press, 1990. Once fully identified, these structural genes can be 10 edited and appropriately inserted into expression vectors by methods known to the art.

These structural genes can be altered by mutagenesis methods such as that described by Adelman et al., DNA, 2: 183, 1983 or through the use of synthetic nucleic acid strands. The products of mutant genes can be readily 15 tested for multifunctional enzymic activity. Conservative mutations are preferred. Such conservative mutations include mutations that switch one amino acid for another within one of the following groups: 1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; 2. . Polar, negatively charged residues and their amides: Asp, Asn, Glu and Gin; 3. Polar, positively charged residues: His, Arg and Lys; 4. Large aliphatic, nonpolar residues: Met, Leu, lie, Val and Cys; and . Aromatic residues: Phe, Tyr and Trp.

A preferred listing of conservative substitutions is the following: Original Residue Substitution Ala Gly, Ser Arg Lys Asn Gin, His Asp Glu times daily is effective to cure the hemorrhoid outbreak within two days, and usually within one day.

A double blind, placebo controlled trial with patients suffering from genital herpes has shown that 1 ml of hydrogel (of Example 1F) containing 5 500 /yg/ml of the krill-derived hydrolase of Example 1C applied three times daily is effective to heal the sores of the herpes outbreaks within an average of 5 days. In patients treated with placebo, the outbreaks lasted an average of 12 days. In the patients treated with the hydrolase, pain and itching were gone within 36 hours, while these symptoms peaked at day 3 or 4 in the J^O placebo controls.

Clinical trials with HIV patients suffering from oral Candida have shown that a lozenge (of Example 1H) containing 3 mg of the krill-derived hydrolase of Example 1C applied three times daily is effective to clear the infection within 7 to 9 days.

Clinical trials with patients suffering from recurrent vaginal candidiasis that is resistant to traditional antifungal drugs have shown that 1 ml of hydrogel (of Example 1F) containing either 100 //g/ml or 250 //g/ml of the krill-derived hydrolase of Example 1C applied three times daily is effective to clear the Candida outbreak within an average of four days and to reduce the 20 recurrence rate.

) A topical methicillin-resistant Staphylococcus aureus ("MRSA") infection characterized by necrotic tissue, erythematous and underlying tissues that were distended and edematous and affecting an approximately 100 cm2 area has been effectively treated by applying 3 ml of hydrogel (of 25 Example 1F) containing 500 //g/ml of the krill-derived hydrolase of Example 1C three times daily. After application, the infection was covered by a dressing. By the third treatment on the second day, all necrotic tissue was removed, the erythema substantially reduced, and exudate flowed from the affected tissue. By day five, the edema was gone. By day seven, the wound 30 was 40% closed, and the MRSA culture was negative. By day nine, the preparation was alloquoted into ampules at 25 Casein Units per ampule. Example 1B - Preparation of krill-derived multifunctional enzvme A poly-enzyme preparation prepared as described in Example 1A was reapplied to a Sephacryl 200 column. A fraction that was sub-stantially 5 homogeneous and displayed an apparent molecular weight of 29 kd was isolated. This fraction was found, using the proteolysis assays described below, to have multifunctional activity. The lyophilizate of this krill-derived multifunctional enzyme was placed into ampules at 15 Casein Units per ampule for sterile storage without preservatives or anti-microbial additives. ^10 Example 1C - Multifunctional Enzvme Purification A 100 kg of frozen antarctic Krill were thawed, and mixed with 100 kgs of distilled water, and stirred for 30 minutes. The krill used were harvested in the January through March period. During this period, krill are largely without pigment and are called "white" krill. (Due to dietary changes, 15 later in the season krill are harvested as "red" krill. Multifunctional hydrolase yields from red krill are 30 to 40% less than from white krill. Still later in the season, during June through August, "black" krill are harvested. Black krill yield still less enzyme.) The supernate was collected by centrifugation using a GL-sieve, starch centrifuge (Model 220, available from G. Larssons ^20 Mekaniska Verkstad, Fjalkinge, Sweden) using a 125 micron spinning cone at 1,000 rpm. The pH of the supernate was adjusted to 6.2. The supernate had turbidity of less than 4% and fat content of less than 2%.

The supernate, 100 mis, was further clarified by centrifugation at 17,000 xg and mixed with 5 kg of DEAE-sepharose gel (Pharmacia, Sweden), 25 which had been previously been equilibrated to pH 6.2. The mixture was stirred gently for 1 hour. The gel was collected on a filter bed and washed with 5 volumes of 0.4 M NaCI. The gel was then packed into a suitable column container using a 0.4 M NaCI saline solution. The column was then washed overnight with 15 bed volumes of 0.4 M NaCI.

A protein containing fraction (approximately 1.5 liters collected) was membranes and sequenced through 25 cycles of Edman degradation. See, Matsudaira, J. Biol. Chem., 262: 10035-10038, 1987. Each preparation yielded the identical sequence: I V G G M/N E V T P H A-YPWQVGLFIDDMYF. Accordingly, it is clear that all three 5 preparations are homogenous, although each is micro-heterogeneous at position 5. The proteolytic activity of each of the three preparations was tested against substrate benzoyl-val-gly-arg-p-nitroaniline. Hydrolysis of this substrate can be monitored at 210 nm, reflecting the release of p-nitroaniline. The pH-dependence of the three preparations at an ionic strength of 0.1 M is 10 shown in Fig. 9. The profile for Prep-3 (shown with filled squares), Prep-8 (shown with open diamonds) and Prep-11 (shown with filled diamonds) are identical. All three had a pH optimum for this substrate of 9.5.

With the elastase substrates succinyl-p-ala-pro-ala-p-nitroanilide and boc-ala-ala-pro-ala-p-nitroanilide, the pH optimum for Prep-8 was 7.0. See 15 the profile in Fig. 9, represented by the X's. Similar model substrate studies determined that the order of cleavage efficiencies for the krill multifunctional hydrolase is chymotrypsin ^ trypsin > elastase.

For the substrate azocasein, the profile for pH dependence was broad and in the acidic pH region. See the profile in Fig. 9, represented by the open 20 triangles.

Km values were determined using benzoyl-pal-gly-arg-p-nitroanilide in 0.1 M in CAPS buffer containing 2 mM Ca++ at pH 9.5. The Km values for Prep-3, Prep-8 and Prep-11 were 210 ± 8, 210 ± 8 and 230 ±13 //M, respectively. Against the substrate succinyl-ala-ala-pro-phe-p-nitroanilide, 25 under the same conditions, the Km values were 260 ± 50, 270 ± 50 and 270 ± 40 //M, respectively.

The effectiveness of three protease inhibitors was tested at pH 9.5 against the three hydrolase preparations. The results were as follows: Example 1E - Hvdroqel Preparation The multifunctional enzyme is mixed with a hydrogel made up of low molecular weight hydrolyzed starch containing 90% water by methods known in the art to obtain a hydrogel formulation of the enzyme.

Example IF - Hvdroael Preparation The multifunctional hydrolase according to Example 1B was mixed with hydrocolloid gel comprising an aqueous gel containing 0.8% w/v Carbopol™ (Dow Corning, Midland, Michigan) and 23.5% w/v glycerin prior to use. (Carbopol™ is a vinyl polymer with active carboxy groups described in Chem. #0 Eng. News 36:64 (1958).) Example 1G - Hvdroael Preparation The multifunctional enzyme according to Example 1C was mixed with hydrocolloid gel comprising an aqueous gel containing 0.8% w/v Carbopol™ (Dow Corning, Midland, Michigan), 23.5% w/v glycerin and 10% w/v Pluronic 15 P85 (BASF, Wyandotte, Michigan) prior to use. (Carbopor is a vinyl polymer with active carboxy groups. Chem. Eng. News 36:64 (1958).) Example 1H - Lozenge Preparation Lozenges containing 3 or 6 mg of the multifunctional enzyme according to Example 1 C were formed from 50% lactose, 20%, Avicel (microcrystalline 20 cellulose), 29% sucrose and 1% magnesium stearate.

Example 2 - Protease activity The multifunctional enzyme is compared to isolated krill multifunctional protease for molecular weight, sequence, temperature or pH stability, temperature or pH optima and proteolytic specificity.

Example 2A - Specificity To study proteolytic specificity, the following substrates are used: Substrate Type of Activity Succinyl-Ala-Ala-Pro-Phe-pN02 anilide Chymotrypsin Boc-Ala-Ala-Pro-Ala-pN02 anilide Elastase o2-macrogiobin, bovine' pancreas protein inhibitor, and soybean protein inhibitor. Conditions for conducting such studies are described in Example 1D. These inhibitors are available from Sigma Chemical Co, St. Louis, MO. K, values are determined in solutions of various pHs using the following 5 protease substrates, including TAME, Benzoyl-Val-Gly-Arg-p-N02-ani!ide, Succinyl-Ala-Ala-Pro-Phe-p-N02-anilide, Boc-Ala-Ala-Pro-Ala-p-N02-anilide and azocasein.

Example 3A - In Vitro Binding of HL60 Ceils to Endothelial Cells Endothelial cells were first passaged onto 96 well plates at a given ho concentration. The endothelial cells used in the experiment are described in Edgell et al., Proc. Natl. Acad. Sci. USA (1983) 80:3734. The cells were incubated at 37 *C under a DMEM cell culture medium containing 10% fetal calf serum and under a 5% C02 atmosphere. Then, the medium was removed and replaced with 100//I of a suspension of 200,000 HL60 cells (a 15 human lymphocyte cell line, available from the European Cell Culture Bank under ECACC Accession No. 85011431) in RPMI medium containing 10% fetal calf serum. The cells were incubated for 30 minutes. After this, the medium was removed and the adherent cells were washed two times with DMEM medium. The relative adherence of the HL60 cells was measured by 20 measuring the difference in optical density at 450nm between the plates on which the cells were co-incubated and plates having endothelial cells alone.

The effect of TNFar was measured by adding TNFor at 1 500 units/ml to the endothelial cells 4 hours before the incubation with HL60 cells. The effect of antibody to E-selectin was measured by adding 25//g/ml of monoclonal 25 antibody BBAZ (R&D Systems Europe, Oxford, England) to the HL60 cells. The results of the experiments were: Expt. No.

HL60 Cells Endoth Cells Absorbance* 6 pretreated with multifunctional enzyme for 2h pretreated with TNFcr 0.160 7 pretreated with multifunctional enzyme for 2h Four hours pretreatment: 0-4h TNFor 2-4h multifunctional enzyme 0.059 'increase over absorbance of endothelial cells alone.

To confirm these results, the number of adhering HL60 cells were counted by removing them from the plate and counting the cells. The number of HL60 cells was determined by subtracting the cell numbers for control plates having only endothelial cells. These counting results mirrored 10 the optical density results, as follows: EXPERIMENT HL60 CELL NUMBER 1 32,590 2 43,990 3 ,730 4 42,190 ,280 6 17,010 These adherence studies show that krill hydrolase destroyed the 20 cell-surface ligand and acceptor molecules that facilitate cell-adhesion. Example 3B - Activity Against Certain Cell-Surface Adhesion Molecules Freshly isolated T-cells from the thymus of a C57BL/6 mouse (bred by Institut Armaud Frappier) were washed three times with serum-free medium. 1 ml alloquots of the cells containing 5 - 10 X 106 cells were treated at 37 antibody specificity source VCAM-1 PharMingen, San Diego, CA CD28 PharMingen, San Diego, CA CD31 PharMingen, San Diego, CA asialoGMI Wako Bioproducts, Richmond, VA ^ In some cases, binding was detected with a labeled second antibody, for instance, binding of the asialo GM1 antibody was detected with FITC-labeled Fab fragments that were specific for rabbit IgG (heavy and light chains), 10 which was obtained from Caltag Laboratories, San Francisco, CA.

Example 3C - Timecourse of Cell Surface Recovery of Adhesion Molecules DO-11.10 T cell hybrids (this cell line is described by Shimonkevitz et al., J. Experimental Med. 158: 303, 1983) were treated with 500 /vg/ml of the krill-derived multifunctional hydrolase prepared according to Example 1B and 15 tested for the CD4 marker as described in Example 3B. Immediately after the treatment, well less than 1 % of the amount of CD4 found in the controls was found on the hydrolase-treated cells. 48 hours later, the levels in treated cells I were the same as those in untreated cells.

Example 4 - Cell Binding Comparisons 20 The effectiveness of various members of the multifunctional enzyme family, i.e., the non-krill multifunctional enzymes having at least about 60% homology with the krill-derived hydrolase, are compared with that derived from krill using the HL60 binding assay of Example 3A.

Example 5 - Mouse Ovarian Tumor Treatment 25 25,000 mouse ovarian tumor cells were injected into the abdominal cavity of 1 2 C3H/hsd mice). On days 1, 2 and each of days 5-9, 1 ml of either saline or 200 pg of krill multifunctional hydrolase (prepared as described in Example 1 C) dissolved in saline was injected into the ascites. In Figure 4, - 53 • o None of the treated animals showed any visible symptoms of illness or adverse reaction.

Example 7 - Toxicity on mice The toxicity of poly-enzyme preparation has also been tested on mice with s.c. implanted P388 murine leukemia (a chemically induced cancer) and compared with doxorubicin, a well known anti-cancer drug (abbreviation DOX). The results are summarized in the following Table. No mouse treated with the poly-enzyme preparation of Example 1A or in the control group died or lost weight, whereas all mice in the doxorubicin group, except for those receiving the lowest dosage, lost weight and died. The highest dose of poly-enzyme preparation (20 mg/kg) was far higher than any of the doses used in the clinical examples described below.

Toxicity of poly-enzyme preparation and DOX administered i.p. daily during 9 consecutive days to mice with P388 murine leukemia Drug Dose Toxic Body wt. (mg/kg) death chg. (g) Control 0 0/6 + 2.3 Krill 0/6 + 3.3 poly-enzyme 0/6 + 3.3 preparation 0/6 + 3.3 DOX 6/6 - 2.3 2.5 6/6 - 0.6 1.25 0/6 + 0.5 Example 8A - Infections in post-operative surgical wounds Forty patients were included in this study and they were divided into two groups of 20 patients each, representing 41 post-operative abdominal 35 (34) and thoracic (7) wounds. Preparations of krill multifunctional hydrolase prepared according to Example 1B (3 Casein-Units/ml), and krill poly-enzyme preparation of Example 1A (5 Casein-units/ml) were tested in each group.

The patient population was heterogeneous with respect to the etiology of the wounds, i.e. scheduled operations, traumas, burns, shots and diabetes patients. All patients were treated on an outpatient basis twice a day. No adverse reaction were observed from the test preparations. The results are 5 summarized in Figure 12. The points indicated by "Y" symbols are for scoring of yellow fibrinous and purulent tissue. The points indicated by "B" symbols are for black necrotic tissue. And, the points indicated by "R" symbols are for granulation tissue and epithelium. Necroses, fibrin, pus, blood clots, and plaques were effectively decomposed within a week and some wounds healed ^0 within a week. Burns, shots, and post-operative wounds in diabetes-patient initially showed very poor efficacy, but at termination of the study, 7 days, the necroses were completely decomposed from underneath and what remained was only the top surfaces of the necroses, like a lid, and the wounds were partially healed within a week.

Example 9 - Abscesses in caives Abscesses of approximately 25 to 40 ml volume were formed on the neck of each of two calves by injecting calcium-chloride solution. The abscesses were treated once daily by instillation of 10 ml of the poly-enzyme preparation of Example 1A (5 mg/ml) using a drainage tube. For each ^^0 treatment, the tube was sealed to keep the enzyme solution in place for a minimum of 4 hours. Then the tube was then opened, allowing the abscess to drain. After the third treatment the drainage fluid was clear and after the sixth treatment the tubes were removed. The "pockets" healed completely after 9 and 12 days, respectively.

Example 10 - Scleroderma The patient suffered from chronic hardening and thickening of the skin, i.e., scleroderma in his fingertips on his right hand. He was treated by twice daily applying 0.5 ml of hydrogel (described in Example 1F) containing 0.5 mg/ml of the krill multifunctional hydrolase according to Example 1B. The 30 pain associated with the condition was substantially reduced within 48 hours, Wounds were treated two times daily with 25 mg/treatment of multifunctional hydrolase. All wounds were completely free from all signs of infection within 5 days treatment. The lack of infection was confirmed by microorganism (MO) cultivation. Necrotic tissue, pus and fibrinous fibrils in the granulation tissue were effectively decomposed by both preparations and no perceptible difference in efficacy between the preparations could be observed. No adverse reactions were observed. The test results were: Example 13 - Infected- decubitus ulcera Fourteen elderly patients with a total of 18 decubitus ulcers on their heels or lower back were included in this study. Ulcers were rinsed thoroughly with saline and emptied as much as possible and irrigated with 5 5 ml, krill poly-enzyme preparation of Example 1A dissolved in saline (5 Casein Units/ml). The ulcers were then covered with semi-occlusive dressing. The procedure was repeated twice daily for 7 days and ulcera were inspected for inflammation, erythema, heat, swelling, necrotic tissue, pus, pain and possible adverse reactions. Infections were gone within 4 days of treatment. Six 10 wounds healed completely within 7 days, and a total of eleven have healed within 14 days. Seven wounds did not heal probably due to the overall condition of the patients, but the wounds showed some progress. No adverse reactions were observed.

Example 14 - Fistuiae infections 15 Krill multifunctional hydrolase according to Example 1B and the krill poly-enzyme preparation of Example 1A were used to treat anal fistuiae. Prior to use, one ampoule of the multifunctional hydrolase was reconstituted in 5 ml of hydrogel to a final concentration of 3 Casein-Units/ml. The poly-enzyme preparation was reconstituted in hydrogel to a final concentration of 5 ^20 Casein-Units/ml. The fistuiae were rinsed with sterile solution and emptied as far as possible, and then irrigated with hydrogel containing multifunctional hydrolase or poly-enzyme preparation. The procedure was repeated once daily and patients were inspected for erythema, heat, swelling, pus, pain and adverse reactions. The treatment continued until all signs of infection and 25 inflammation were gone, but for no longer than 10 days. For each gel preparation, two patients with anal fistuiae, with no passages to rectum, were used and treated with multifunctional hydrolase and poly-enzyme preparation, respectively. For both sets of patients, total pain relief was reported within 48 hours and all signs of infections and inflammations were 30 gone after 4 days. All fistuiae were healed between day 6 and day 9, and no WO 96/24371 PCT/US96/01650 in ail the cases and all- signs of infection and inflammation were gone within 4 days.

Example 17 A - Opportunistic infections Two patients with uterine cancer in stage IV with opportunistic 5 infections in non-healing post-operative wounds and one patient with an opportunistic infection of an irradiation wound were treated twice daily with dressings saturated with a solution of 5 mg/ml of the poly-enzyme preparation of Example 1A. The infections were believed to be bacterial infections. After 12, 14 and 17 days, respectively, the wounds had healed.

Example 17B - Mycoplasma infection A 55 years old man contracted an acute mycoplasma infection 3 years ago and was treated With different kinds of antibiotics. However, these treatments became ineffective when a resistant form developed. Some weeks after the infection the man contracted high fever with a very severe 15 cough as a result and in X-ray examination water in the pleura was observed. The complaints of the man manifested themselves in the form of respiration complaints, difficulty to walk longer distances than 100 meter without a break and severe tiredness as well as annoying hacking cough.

The patient was treated with 2 mis of a 2 mg/ml solution (4 mg total) 20 of the krill multifunctional hydrolase according to Example 1B. The wash solution was kept in the mouth for about 4 minutes and then was slowly swallowed. This treatment was repeated during the first two weeks every second day. During this time, 0.5 ml of an aerosol of the wash hydrolase solution was inhaled on a daily basis. During the first two weeks no 25 improvement was observed but after two weeks' treatment the lymph gland on the left side of the neck swelled resulting in pain. During this time the treatment was continued 3 times (every second day). After 1.5 weeks all problems regarding the lymph nodes had disappeared and the bronchitis complaints of the patient began to subside. After further 3 weeks' treatment 30 with mouth washes every fourth day the bronchitis complaint had completely - 63 BEFORE TREATMENT DAY 2 DAY 4 MO Status: (average) 1.4X104 4.3 X 103 < 1.0 X 102 Relative MO Status: (% of initial status) 100% 31% 1% Example 20 - Eve infections Fifteen patients with purulent eye infections were treated twice daily with eye-drops of the poly-enzyme preparation from krill. Before use, an ampoule of poly-enzyme preparation as described in Example 1A was reconstituted in 25 ml water to a final concentration of 1 Casein-Unit/ml. The 1 5 infected eye was treated morning and evening with two drops, 0.4 ml, of the solution. At each application, the eye was inspected for erythema, swelling, pus, lacrimal secretion and possible adverse reactions. The patient was treated until all signs of infection were gone, but not for longer than 10 days. All patients were free from infections within 3 days of treatment. Erythema 20 and swelling around the eyes faded away within 2 days and excess lacrimal secretion ceased within 2 days. After the first application all patients experienced a soothing feeling in the infected eye and irritation and tenderness around the eyes disappeared within a few minutes. No adverse reactions were reported.

Example 21 - Gum infections Twenty-two patients with acute or chronic gum infections/inflammations were included in this study. Three times a day (morning, mid-day and evening) one of the ampoules of poly-enzyme preparation described in Example 1A was reconstituted in 5 ml tap water 30 (yielding 5 Casein units/ml) and used to rinse a patient's mouth cavity for 5 minutes. No eating and drinking within 2 hours after treatment was allowed. nasal sprays of 0.5 mdj/ml aqueous solutions of the hydrolase (0.1 mg per nostril inhaled) every 4 hours and with 0.5 mg/ml mouth wash solutions (1.5 mg per wash) every 6 hours. The mouth wash solution was kept in the mouth for about'2-4 minutes and then swallowed. After 12 hours the 5 common cold symptoms had disappeared and the patients were free of complaints.

Example 23 - Haemophilus influenzae infections A woman of 34 had recurring sinusitis caused by infection of Haemophilus influenzae. A few hours after the appearance of the symptoms 10 of pressure and pain in the nasal sinus, her mouth was washed for 3 minutes with 4.5 ml of a 5 mg/ml solution of krill multifunctional hydrolase according to Example 1B and 0.5 ml of the hydrolase solution was sprayed into each of her nostrils. This combined treatment was repeated three times at 2 hour intervals. After which, the spray treatment was repeated every three hours 1 5 over a total of 3 days. The pressure caused by the nasal sinus infection disappeared within a few hours after the first treatment and the flow of nasal secretions strongly increased. After 3 days of treatment the woman was free of complaints.

Example 24 - Bronchitis 20 A 55 year old man having severe bronchitis complaints was treated.

The man had respiration complaints, difficulty in walking more than 100 meters, severe tiredness, and a chronic hacking cough. His physician believed his bronchitis was caused by a mycoplasma which had first infected the patient 3 years earlier and had developed antibiotic resistance. The 25 infection led to the formation of water in the pleura, as was verified by X-ray examination. The patient was treated with mouth washes containing 4 mg/ml saline of krill multifunctional hydrolase according to Example 1B. The hydrolase solution was kept in the mouth for about 4 minutes and then it was slowly swallowed. This treatment was repeated on every alternative day for 30 the first two weeks of treatment. Also during this time, small amounts (0.5 \ further adverse reactions. On days 16 and 29 she experienced gastric pain and diarrhea in connection with unusual meals. However, these episodes where shorter and less severe than her previous episodes. Through the course of the treatment, she recorded improved comfort. During the 3-4 5 months following treatment, her symptoms slowly returned to the severity reported pre-treatment.

Example 26 - Herpes genitalis A man of 62 having a long established case of Herpes genitalis was treated. Outbreaks recurred regularly every 4 months, and during acute 10 outbreaks the man abstained from sexual intercourse. During an acute outbreak, the affected area was bandaged with a bandage soaked with a solution of krill multifunctional hydrolase according to Example 1B until it contained 3 mg of hydrolase. The bandaging treatment was repeated twice a day for 2 days. His complaints disappeared after the second treatment. The 15 man had no outbreaks during the 10 months following treatment.

Example 27 - Herpes simplex infection in the mouth cavity Eight patients with relapsed herpes simplex blisters in the mouth cavity were treated twice daily with mouth-wash. Prior to use, each ampoule of krill poly-enzyme preparation (described in Example 1 A) was reconstituted in 5 ml 20 of saline to a final concentration of 5 Casein-Units/ml. The patient rinsed his or her mouth cavity with the solution for 5 minutes. No eating or drinking was allowed within 2 hours after each treatment. The procedure was repeated twice daily and clinical parameters, erythema, swelling, pain and adverse reactions were recorded once daily. The treatment was continued 25 until all signs of infection were gone, but not for longer than 10 days.

Pain relief was experienced within 2 hours after the first treatment. In some patients the pain recurred between treatments during the first two days, but never thereafter. After 5 days, all patients were free from symptoms and all blisters had healed. No adverse reactions were observed.

Example 1F) containing 2.5 Casein Units/ml of the poly-enzyme preparation of Example 1 A. Patients with dry eczema/plaque showed no signs of inflammation or infection after 2-4 treatments. The fatty type of seborrheic plaques disappeared after 6-9 days, though the associated 5 inflammations/infections had vanished within the initial 2-4 days of treatment. Patients with psoriasis plaque experienced an improved responsiveness to steroid creams, probably due to removal of plaque by the poly-enzyme preparation, resulting in better access to the skin.

Example 31B - Lichen planus - with associated infection 10 The patient suffered from lichen planus of the lower gum. The affected areas showed papules and lesions, and were covered with yeast plaque. Each day at that time 1 g of a hydrogel (described in Example 1F) containing 0.5 mg/ml of krill multifunctional hydrolase according to Example 1B was applied to the affected areas. After three days, the plaques and papules were gone. 15 On day 7, all lesions were healed and the treatment was stopped.

Example 32 - Thrombolytic /anti-embolic properties Thrombi were caused by applying an artificial stasis to the main ear vein of a rabbit until a proper thrombus had developed, krill multifunctional hydrolase according to Example 1B was injected (0.5 mg dissolved in 0.2 ml 20 of isotonic solution), into the ear vein in the direction of the thrombus, at a location 2 cm from the ischemic area. Within 30 minutes the thrombus had completely dissolved and the blood had free passage. Small necroses developed in the treated area but these were resorbed within 7 days. In the control animals the ischemic area turned necrotic within 4-5 days. 25 Example 33 - Dental plague in dogs The krill poly-enzyme preparation was used to remove dental plaque in beagles. Before use, ampoules of the poly-enzyme preparation (described in Example 1A) were reconstituted in 5 ml of saline to a final concentration of 5 Casein-Units/ml. The content from a freshly prepared ampoule was carefully 30 painted over teeth and gingiva. The tongue was fixated for 2 minutes and A. A single administration i.p. of 5 mg/kg.

B. A single administration i.t. of 5 mg/kg.

C. A single administration s.c. of 5 mg/kg.

D. Twice daily administration s.c. of 1.25 mg/kg for seven days.

E. S.c. administrations of 5 mg/kg on four alternating days.

F. S.c. administrations of 12.5 mg/kg on four alternating days. Before use, ampules of the poly- enzyme preparation powder were reconstituted in isotonic saline at a concentration of 5 mg/ml solution. 1 x 104 Yoshida Sarcoma cells were implanted subcutaneously on the back of 10 white Wistar rats. When the implanted cells had generated a 10 mm x 10 mm tumor, the rats were either"treated with poly-enzyme preparation or used as untreated controls. The rats were sacrificed 7 days after the last treatment. The size of the tumors was measured and compared with the tumors of untreated control rats.

The relative reduction of the size of the tumor was 46% for group A, 56% for group B*and 49% group C. In the group treated with repeated doses of 1.25 mg/kg twice a day over 7 days (group D), the reduction was 72%. In the groups receiving 5 mg/kg and 12.5 mg/kg s.c. every second day (groups E & F), the tumor reductions were 53% and 69%, respectively. In all treated |20 rats, the portion of the tumors that was necrotic was substantially higher than for tumors from untreated rats. Also, the treated rats gained weight more rapidly than did untreated rats.

One rat in the group receiving 12.5 mg/kg every second day showed an absolute loss of tumor mass (rather than a relative loss). The degree of 25 metastasis in the treatment groups was very small compared to the control rats. The treated rats showed normal behavior regarding drinking and eating, in contrast to the control rats, which were subdued and exhibited no appetite. No adverse reactions could be observed during this trial.

\ WO 96/24371 PCT/US96/01650 days both eyes looked'very clear and the treatment was terminated. No adverse reactions were observed.

Example 38 - Cataract in human A woman in her 80's with day/night vision on one eye due to gray 5 cataract was treated every three days with 0.4 ml of solution a 0.1 mg/ml of the poly-enzyme preparation of Example 1 A. After 5 treatments the study had to be terminated due to other medical reasons but an obvious reduction in opacity was observed and the lady reported improved vision on the affected eye. No irritation, pain or other discomfort could be observed during the # 10 treatment or 1 month post-treatment.

Example 39 - Glaucoma A man, age 74, experienced a slight pain/discomfort in the right eye and it was found that he suffered from an increased intraocular pressure that was found to be permanent and not caused by acute reasons. Every four 1 5 days a solution containing 0.1 mg of the poly-enzyme preparation of Example 1A was dropped into the affected eye for a total of three times. Two weeks post-treatment the intraocular pressure was normal and remained normal 4 months post-treatment. The man experienced immediate pain relief, within 20 minutes, at the first treatment. No adverse reactions were observed. 20 Example 40 - General Purpose Eve Drops Twelve patients with no record of eye diseases, allergic reactions to air pollutants or eye infections/inflammation were treated for "tired and irritated eyes". Two drops of a 0.1 Casein Unit/ml solution of the poly-enzyme preparation of Example 1A were dropped into the eyes whenever needed. All 25 patients experienced an alleviation of tension and pain within 30 minutes. No one reported any change in light sensitivity, focusing or any dilatation of the pupils. Occasionally a transitory dryness was experienced. No irritation, increase in lacrimal secretion or other adverse reactions were observed. Example 41 - Tendon-sheath adhesions 30 The left Achilles tendon of each of two rabbits was ruptured and scar/keloid was inspected for erythema, swelling, heat, bleeding, necroses and adverse reactions. The treatment was terminated at 80% decomposition of scar/keloid but for no longer than 7 days. Fibrinous scars were reduced to 25% of their initial volumes and collagenous keloids to 70% after 7 days of 5 treatment. No adverse reactions were observed during this trial.

Example 44 - Wrinkle reduction A 33 year old woman was treated each night for 60 days by applying for 30 minutes a gauze bandage moistened with a solution of the multifunctional hydrolase according to Example 1B. The total amount of '10 multifunction hydrolase used per application was 0.15 mg. The area below one of the patient's eyes was treated, while the other side was served as a control. After 15 days of treatment one could observe a difference in the elasticity of the skin and after 60 days of treatment there was a visually apparent difference in wrinkles. The treated skin area was very soft and 15 elastic and the number and depth of the wrinkles had decreased considerably compared to the untreated skin area.

Example 45A - Polyps A man of 62 having a polyp in his anus was treated. The man had suffered from the polyp for 3 years and had been treated by a physician. He k20 had been treated with different kinds of medicines but no improvement was observed. He was treated with a gauze bandage containing about 5 mg of the multifunctional hydrolase according to Example 1B dissolved in 5 ml of saline. The treatment was repeated for a total of 5 times. All trouble disappeared and at the next visit to the physician it was observed that the 25 polyp had disappeared. Eight months after the treatment, the man is still free of complaints.

Example 45B - Warts A 35 years old man having a wart on his neck was treated for one week with a plaster ("Hansaplast") containing a solution of the multifunctional 30 hydrolase according to Example 1B (0.1 mg/plaster). The treatment was WO 96/24371 PCT/US96/01650 infection, in contrast to the majority of the babies who were examined on the same occasion.

Example 47 - Allergic itch A woman, age 28, had allergic problems in the form of intense itch 5 with nettle rashes above one of the knees and also on and below the chin. The rash exhibited white, somewhat spread elevations on the knee whereas the chin was totally spotted with small rash of the same color as the skin. A gauze bandage was wetted with a solution containing 2 mg of the krill multifunctional hydrolase according to Example 1B. The gauze was applied to ^10 the rash on the area above the knee. After 10 minutes the gauze bandage was removed. 45 minutes later the itch began to fade out and had completely disappeared after 1.5 hours. The white elevations had also disappeared and the nettle rash had faded.

One day later the area under and on the chin was treated. This area 15 now itched intensively and was more irritated than before owing to the woman's scratching. Directly upon application of the gauze bandage with the solution of krill multifunction hydrolase, the itch increased and the gauze bandage was removed after 7 minutes owing to a very intense itch. During the following 1.5 hours the itch declined and completely disappeared after ^^^0 2.95 hours. Two days after the initial treatment to the knees, no adverse reactions were observed on the treated areas.

Example 48 - Prostatitis A man of 52 years suffered from prostatitis complaints each winter since the age of 20. During the last 4 years the complaints became more 25 acute resulting in extremely severe abdominal pain. In every acute phase he was treated with different kinds of antibiotics but as soon as the antibiotic treatment was completed, the complaints recurred within about a week. During one episode, the man received acid-resistant capsules containing 5 mg/capsule of the dry form of the multifunction hydrolase according to 30 Example 1 B. He took 2 capsules/day for a week. All symptoms disappeared horses were back on easy training.

Example 51 - Tourist diarrhoea A 41 years old man acutely developed food poisoning (probably from Staphylococcus) with diarrhoea and vomiting. One hour after the man had fallen ill he was treated with a solution containing 5 mg of the krill multifunction hydrolase according to Example 1B by keeping it in the mouth for about 3 minutes, whereupon it was slowly swallowed. This procedure was repeated 3 times every other hour. After the fourth treatment the stomach pains had disappeared and the vomiting and severe diarrhoea had ended.

Example 52 - Hair thinness Two men, 55 years old and 62 years old, respectively, were treated. They both had suffered from hair thinness for the last 10 years. The treatment was carried out by soaking the entire scalp with a solution containing 5 mg of the krill multifunction hydrolase according to Example 1B. To maintain the humidity in the scalp, it was covered with a shower cap for 30 minutes. The treatment was repeated once a week for about 3 months. After this time of treatment fresh hair began to grow out.

Example 53 - Treatments with non-krill enzymes For each of Examples 8-52, the krill-derived multifunctional hydrolase is substituted with the multifunctional enzyme from another source and is comparably effective. intellectual property office of n.z.

Claims (9)

CLAIMS: - < OCT 2001 RECEIVED

1. The use of a hydrolase derived from krill or Atlantic cod, effective to selectively remove or inactivate at least one cell adhesion component selected from ICAM-1 (i.e , CD54), CD4, CD8, CD11 and CD28, in the manufacture of a medicament to inhibit transplantation rejection by extra-corporeally treating a tissue, body fluid or composition of cells to selectively remove or inactivate at least one said cell adhesion component, wherein the extra-corporeally treated tissue, body fluid or composition of cells is thereafter transplanted into or onto a mammal.

2. The use of a hydrolase as recited in claim 1 wherein the hydrolase is derived from Atlantic cod.

3. The use of a hydrolase as recited in claim 1, wherein the hydrolase is derived from krill and has chymotrypsin and trypsin activity, a molecular weight between about 26 kd and about 32 kd as determined by SDS PAGE, and an N-terminal sequence comprising: IVGGXEVTPHAYPWQVGLFIDDMYF-, where X is any amino acid.

4. The use of claim 1 or 2, wherein the hydrolase has chymotryptic activity.

5. The use of claim 4, wherein the hydrolase has chymotryptic and tryptic activity.

6. The use of claim 1 or 2, wherein the hydrolase has tryptic activity and at least one of a chymotrypsin, collagenase, elastase or exo peptidase activity.

7. The use of a hydrolase as claimed in any one of claims 1-6 wherein the hydrolase is effective to remove at least two of the cell adhesion components.

8. The use as claimed in claim 7 wherein the hydrolase is effective to remove at least three of the cell adhesion components.

9. The use as claimed in claim 8 wherein the hydrolase is effective to remove at least four of the cell adhesion components. 10 The use as claimed in claim 9 wherein the hydrolase is effective to remove all of the cell adhesion components 1 1 The use of a hydrolase derived from krill or Atlantic cod as claimed in claim 1 and substantially as herein described with reference to the accompanying examples