WO1999041361A1 - Protease-based dietary supplementation for decreasing recovery time from soft-tissue injury - Google Patents

Protease-based dietary supplementation for decreasing recovery time from soft-tissue injury Download PDF

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WO1999041361A1
WO1999041361A1 PCT/US1999/001690 US9901690W WO9941361A1 WO 1999041361 A1 WO1999041361 A1 WO 1999041361A1 US 9901690 W US9901690 W US 9901690W WO 9941361 A1 WO9941361 A1 WO 9941361A1
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parts
weight
mixtures
composition
protease
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PCT/US1999/001690
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French (fr)
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Devin B. Houston
Danielle Harrison
John Davidson
Jack Harris
Tony Collier
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National Enzyme Company
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46Hydrolases (3)
    • A61K38/48Hydrolases (3) acting on peptide bonds (3.4)
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A23B - A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16Inorganic salts, minerals or trace elements
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46Hydrolases (3)
    • A61K38/48Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4873Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Abstract

A composition and method of use thereof for promoting recovery from soft-tissue injury is disclosed. The composition contains a mixture of fungal, plant, and bacterial proteases, antioxidants, vitamins, minerals, and excipients. The composition can also include a non-prescription analgesic.

Description

PROTEASE-BASED DIETARY SUPPLEMENTATION FOR DECREASING RECOVERY TIME FROM SOFT-TISSUE INJURY

BACKGROUND OF THE INVENTION This invention relates to a dietary supplement. More particularly, the invention relates to a composition and method of use thereof for ingestion as a dietary supplement for decreasing the recovery time from soft-tissue injuries such as may occur from accidents, sports injuries, and certain surgical procedures. In preferred embodiments, the composition comprises a mixture of selected proteases, vitamins, antioxidants, and minerals.

Injury is an accepted, albeit unwelcome, companion to life's activities. Indeed it seems that minor injuries such as bumps and bruises, occur during even mundane activities. An equally ubiquitous experience of life is the onset of tissue- swelling secondary to an injury. The magnitude of tissue-swelling is somewhat dependent on the fluid which is the cause of the swelling. One example of this type of fluid-caused swelling is a subepidermal hematoma, or a bruise. A bruise that involves a broken blood vessel causes the flow of blood from the broken vessel into the surrounding tissue. The presence of the blood in the tissue presents both a source of discomfort and a potential biological problem for the individual thus affected. The characteristics of inflammation are such that host defensive and reparative processes can be inimical to the body's welfare. It is well known that defensive mechanisms in inflammation can bring about the release of products toxic to the host or destruction of some of its tissues. Tissue necrosis follows hypersensitivity reactions of the Arthus or Swartzman variety, for example. Additional detrimental consequences of inflammation include fibrin deposition and reduction in vascularity causing changes in tissue permeability creating additional morphologic barriers to the penetration of antibodies or pharmacological agents into the injured area. Some of the autolysis products released by necrosis often constitute a good medium for microorganisms and can even antagonize the antimicrobial activity of many pharmaceutical agents, thereby exacerbating the injury and prolonging the recovery process. 2

A biological repair process involving inflammation, when unbridled, can distort and damage tissue. For example, cicatricial obstruction may follow caustic burns of the esophagus. Additionally, Dupuytren's contracture, uveitis, scarring of the heart valves secondary to rheumatic fever, derangement of articular and perioticular structures in rheumatoid arthritis, and pulmonary fibrosis secondary to silicosis are all potential consequences of unchecked inflammation. Also, postphlebitic syndrome often follows a prolonged bout of acute thrombophlebitis. Indeed, even though host-induced inflammation is part of the repair process of the body in postphlebitic syndrome, the inflammation can cause vascular scarring and damage if not controlled and treated.

Because inflammation is a predominant biological reaction to myriads of injurious agents and events, specific measures aimed solely at the etiologic agent will often prove inadequate. On the other hand, a therapeutic agent directed at the host response level and capable of modifying defensive and reparative processes of localized inflammation without depriving a body of its benefits would prove valuable.

Chemical reactions in biological systems rarely occur in the absence of a catalyst. These biological catalysts are specific proteins called enzymes. Some characteristics of enzymes include their catalytic power and specificity regarding catalyzed reactions. An enzyme may accelerate a biochemical reaction by as much as a million-fold. Indeed, an enzyme may promote a biochemical reaction that would not otherwise occur to any significant degree under physiological conditions in the absence of the enzyme. A subset of enzymes, referred to as proteolytic enzymes or proteases, is specifically adapted to the degradation (hydrolysis) of proteins. These proteases selectively cleave peptide bonds between amino acid residues, depending upon the protease 's specificity of action. Proteases are distinguished by substrate specificity, kinetic reactions, pH, temperature reaction optima, inhibitors, cofactors, and composition of the proteolytic active site.

Proteases are ubiquitous in living organisms. Proteases fulfill a wide variety of regulatory and functional roles in almost every cellular process. Repair and healing processes are no exception. The discovery of the biological impact of 3 proteases on biological functions such as tissue repair inspired a good deal of experimentation into their source and mode of action. One common source for commercially available proteases is the pancreas of pork and beef animals slaughtered for meat. The pancreases of these slaughtered animals provide a mixture of proteases often called pancreatin. Pancreatin contains such proteases as trypsin, chymotrypsin, carboxypeptidase, and elastase. Early in the study of proteases it was observed that the administration of animal-derived proteases could accelerate the healing of an inflamed site. As soon as proteases were available commercially, in purified form, and their roles in the inflammatory process were beginning to be ascertained, a logical step was to administer proteases from exogenous sources to accelerate or enhance healing of the injured site. During the 1950s a large number of clinical trials was conducted with intravenous or intramuscular injections of proteases, usually trypsin, chymotrypsin, or other crude preparations of pancreatic proteases. However, when the adverse reactions to repeated injections of proteinaceous material were encountered, injectable use of proteases was abandoned.

Oral administration soon followed, and a large database of articles on human subjects was accumulated regarding the mechanisms, bioavailability, and clinical effects of oral proteases as reasonable therapeutic agents. Some of these medical uses of proteases include digestive enzyme replacement, debridement, mucous or exudate liquification, fibrinolysis, and anti-inflammatory effects.

An important use and beneficial effect of protease administration is its effect in causing anti-inflammation of an injured tissue site. The anti-inflammatory mechanisms of proteases are caused in part by: (1) destruction and activation of cell surface enzymes involved in activation of eicosanoids, thromboxane synthetase, cyclooxygenase, decreased pro-inflammatory eicosanoids and increased anti- inflammatory eicosanoids; (2) reduction or inactivation of bradykinins, causing a reduction in pain and prevention of progression of inflammation; (3) reduction in viscosity of extracellular fluid and increased nutrient and waste transport to and from the injured site, thereby causing a reduction of edema; (4) activation of endogenous proteases (plasmin) and prevention of excess thrombin clot formation; (5) introduction of anti-proteases that mediate inflammation and inhibit progression of 4 inflammation, thereby causing a reduction of acute phase reactants; (6) replacement of endogenous proteases, activation of endogenous systems for resolution of inflammation, and formation of regulatory peptides; and (7) molecular debridement, renewal of necrosing tissue and proteins that aid phagocytic functions thereby aiding in reducing edema. Proteases produced by the human body are also involved in digestion of food, removal of denatured or damaged proteins, conception, and termination of certain biochemical reactions.

In addition to endogenous proteases, further protease supplementation is provided by eating uncooked meats, fruits, and vegetables. With the advent of food processing techniques using high heat and/or pressure, however, enzymes present in food are destroyed. This substantially decreases the ingestion of proteases through a normal nutritive diet. Oral enzyme supplements, supplied individually as extracts from animal pancreas and plant or microbial products, have demonstrated utility as aids to digestion and in the alleviation of certain pathological conditions such as pancreatitis. External application of enzymes has also been used in wound debridement. These applications led to the experimental use of proteolytic enzymes administered intramuscularly or intravenously to the circulation for systemic treatment. Many clinical trials in the 1950s indicated limited efficacy of intravenous or intramuscular injection of enzymes in alleviating thrombolytic occlusions. However, adverse reactions to repeated injections of proteins were also common.

The Schwartzman Phenomenon, first described in 1953, is characterized by hemorrhagic necrosis and thrombosis of small blood vessels. In treatment of this phenomena it was reported that intravenous administration of animal-derived trypsin and streptokinase derivatives reversed the severity of the Schwartzman Phenomena. Indeed, Condie et al. observed reversal and inhibition of the Schwartzman

Phenomenon in rabbits after intravenous administration of streptokinase. R.M. Condie et al., Reversal of Lesions of Generalized Schwartzman Phenomenon by Treatment of Rabbits with Streptokinase, 50 J. Lab & Clin. Med. 803-804 (1957). Condie et al. concluded from their studies that deposits of fibrinoid material, which occur in the course of a Schwartzman Phenomenon reaction, are reversible by 5 treatment with streptokinase and that such treatment prevents the progression of the lesion to hemologic necrosis.

Numerous animal and human studies have shown a reduction in inflammatory edema following administration of proteases. Indeed, due to their activity in eliminating inflammatory sequelae, proteases administered transdermally and buccally have proven to be a useful adjunct to surgery, both therapeutically and prophylactically. A reduction in edema, induration, and inflammation permit better approximation of tissue, less scar formation, faster healing, and less pain and discomfort for the patient. However, buccal protease therapy has limited application because it requires a localized inflammatory indication. Such localized inflammations include boils, abscesses, thrombo-phlebitis, respiratory tract infections, traumatic edema, hematomas, acne vulgaris, and other conditions. I. Innerfield, Physiological and Clinical Effects of Buccally Given Proteases, 170 J. Am. Med. Ass'n 925 (1959). Early studies with potent proteolytic enzymes, such as trypsin and chymotrypsin, did not prove to be promising, in part because of first pass metabolic destruction of the protease. The first pass metabolism of the protease caused a complete inactivation of the enzyme secondary to plasma inhibitory decomposition.

Moreover, while low doses of these enzymes are readily inactivated by plasma inhibitors, doses sufficiently high to overcome the plasma inhibitory decomposition induce a state of general plasma proteolysis whose hazards, particularly in reaction to the coagulation system, by far out weigh the deleterious thrombolytic effects connected with inflammation.

The absorption of proteases secondary to oral administration in animals and humans has been extensively studied. The prevailing finding of these studies is that proteases can be absorbed intact, with activity preserved, into the circulation. However, these findings presuppose the protease' s survival of the gastric environment. Obviously, where the acidic pH and digestive enzymes of the alimentary canal destroy the protease' s activity there can be no meaningful absorption into the circulatory system. Therefore, a method whereby proteases can be ingested and absorbed without digestive destruction is lacking in prior art. 6

Even in view of the difficulties involving oral administration, many papers describing well-controlled studies have been published demonstrating the ability of a variety of proteases to enhance healing in sports related injuries. The most striking results were observed in those injuries in which inflammatory processes predominated such as sprains and blunt trauma injuries. However, the injuries investigated involved not only soft-tissue contusions and cuts, but muscle strains and bone fractures. Protease supplementation in the treatment of strains, sprains, and deep contusions resulted in athletes returning to active play sooner than placebo- treated controls. Several reports have come from German studies on the effects of

Wobenzym®, which is a mixture of papain, bromelain, trypsin, chymotrypsin, pancreatin, and rutin. Results indicated that this enzyme supplement reduced pain, edema, and bruising resulting from trauma and surgical procedures. Better results were observed when supplements were taken prior to and after surgical procedures, rather than only following surgery. In most cases, enzyme supplementation was in addition to standard medical care. It must also be noted that a large quantity of Wobenzym® was required to observe beneficial results, sometimes as many as 30 tablets per day. This large quantity was required in part to overcome the first pass metabolic destruction of the protease. Protease supplementation for providing anti-inflammatory effects is not utilized today on a regular basis for a number of reasons: (1) most research using proteases occurred in the 1960s, a time in which steroids were considered a first-line agent for inflammation relief, (2) non-steroidal anti-inflammatory drugs (NSAIDs) were introduced in this same time period, and offered advantages over enzymes in terms of patient compliance and dosing, and (3) prevailing medical dogma at the time taught that aU large proteins were degraded in the stomach, therefore, enzymes could not be absorbed in an active form. Subsequent research has shown that although substantial types and quantities of proteins are destroyed in the digestive system, it is possible to effect the absorption of many proteases into the circulatory system. In addition, several well-controlled studies have demonstrated that proteins, 7 including proteolytic enzymes, can be detected in the circulation in active whole form after oral administration.

The utility of the present invention in improving upon known compositions and methods can be better appreciated through an understanding of the physiology of inflammation. The introduction into tissues of microorganisms, antigen-antibody complexes, or precipitates of crystalline materials provokes local tissue injury and inflammation. Vasodilation and hyperemia, edema from exudation of fluid from vessels with increased permeability, accumulation of specialized blood cells known as granulocytes and phagocytes, deposition of fibrin, and damage to cells and connective tissue may result. Although direct effects of invading microorganisms undoubtedly contribute to such findings, damage stems primarily from the effects of mediators involved in the body's defense mechanisms. The effectors of phagocytosis and microbicidal killing are also the effectors of inflammation. Of major importance are preformed neutral proteases that may be released into the tissues from the lysosomal granules of phagocytes, and two classes of materials, toxic oxygen products (such as superoxide) and lipid mediators (arachidonic acid metabolites), which are generated when the surface membrane of the granulocyte is stimulated.

The interrelationship between phagocytosis and inflammation works both to the body's advantage and disadvantage. Release of materials from phagocytes into the surrounding tissues may facilitate the phagocytic attack upon microorganisms, particularly pathogens such as fungi and larval forms of parasites that are too large for single phagocytes to destroy. However, inflammatory tissue damage caused by products of phagocytes also represents an important mechanism of disease, as evidenced by arthritis produced by immune complex deposition into joints.

Inflammation as an acute response to injury, however, may be more beneficial than harmful and should be accepted as a normal physiological response. Current drug treatment which incorporate steroids and the NSAIDs is aimed at complete inhibition of inflammation. The goal of using proteases in treating soft-tissue injury is to minimize, not inhibit the inflammation process by accelerating the normal enzymatic processes involved in removal of bruises and thrombi. 8

The present invention is unique and offers improvement upon past products in that unlike other enzyme supplements, primary benefit is derived from the use of a protease combination derived from fungi, such as Aspergillus oryzae and Aspergillus niger. These proteases have been extensively characterized over the past 40 years as to their use in the food industry. They have been used extensively to hydrolyze protein or food mixtures to improve the taste, texture, or solubility of certain foods. Fungal proteases are derived from fermentation of fungi on sterilized mats of koji (wheat or rice bran). The enzymes are isolated through the use of standard purification techniques, which result in a clean, dry powder product that has very low microbe counts compared to other food products such as fluid, pasteurized milk. The broad spectrum of protein hydrolysis and working physiological range, and increased potency of the fungal proteases present in the invention allows for fewer capsules to be taken compared to other similar products. Because of the high potency provided by fungal enzymes, the amounts of plant proteases in this invention can be reduced significantly compared to known compositions. Bromelain and papain, being derived from plants, are more allergenic than fungal proteases to many in the population. The invention does not contain animal-derived products, such as pancreatin (another source of proteolytic enzymes), and so is acceptable to those who object to the ingestion of animal products. Today, un-desired side effects from usage of steroid and NSAIDs dictate a need for safer, but still effective, alternatives for pain and inflammation relief. The present method and composition is directed to provide this relief. The present invention is a mixture of fungal, plant, and microbial proteases, vitamin C, bioflavonoids, proanthocyanidins, and minerals encapsulated in a dry powder form. Additionally, a goal of the present invention is to provide a method whereby the stability of plant proteases is maintained by the inclusion of specific antioxidants in the supplement. BRIEF SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a nutritional supplement for decreasing the time required for healing of soft-tissue injuries resulting from accidents, sports injuries, various surgical procedures, and the like. 9

It is another object of the invention to provide a composition for human consumption that utilizes mixtures of proteases to increase the hydrolysis of fibrin protein and other protein products present in clots and bruises.

It is also an object of the invention to provide a method of prophylactic and follow-up administration of the protease formulation that results in decreased edema and recovery time resulting from certain surgical procedures and contact sports injuries and the like.

It is still another object of the invention to provide a composition and method of use thereof for limiting inflammatory conditions by the addition of agents for inhibiting inflammation through the scavenging of free radicals.

It is yet another object of the invention to provide a composition and method of use thereof whereby the activity of susceptible proteases is protected from damaging oxidative processes.

These and other objects can be addressed by providing a dietary supplement composition for reducing the time for healing of soft-tissue injuries comprising in parts by weight (a) from about 2.5 x 10"2 to 5 x 10"1 parts of a fungal protease; (b) from about 1 x 10"3 to 1.1 x 10"1 parts of a plant protease; (c) from about 1 x 10"3 to 1.1 x 10" ' parts of a microbial protease; (d) from about 8 x 10"3 to 1.6 x 10"1 parts of an antioxidant selected from the group consisting of rutin, quercitin, and mixtures thereof; and (e) from about 1.4 x 10"2 to 1 x 10"1 parts of ascorbic acid, acceptable salts thereof, and mixtures thereof. Preferably, the fungal protease is a member selected from the group consisting of exo- and endo-peptidases from Aspergillus oryzae, Aspergillus niger, Aspergillus sojae, Aspergillus flavus, Aspergillus awamori, and mixtures thereof. It is also preferred that the plant protease is a member selected from the group consisting of bromelain, papain, and mixtures thereof. Preferably, the microbial protease is a neutral protease derived from strains of Bacillus subtilis.

In a preferred embodiment, the composition also preferably comprises a mineral selected from the group consisting of about 1.4 x 10~2 to 5.4 x 10"2 parts by weight of calcium, about 5.4 x 10"7 to 5.4 x 10"3 parts by weight of zinc, about 1.6 x

10"7 to 5.4 x 10"3 parts by weight of copper, about 3.2 x 10"7 to 5.4 x 10"3 parts by 10 weight of boron, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of selenium, and mixtures thereof. The composition can also preferably comprise about 1.4 x 10"3 to 1 x 10"1 parts by weight of procyanidins and about 1 x 10'2 to 1 x 10 1 parts by weight of citric acid, acceptable salts thereof, and mixtures thereof. The composition can still further comprise fillers, flavors, coloring agents, formulation aids, and mixtures thereof.

In yet another preferred embodiment, the composition contains an effective amount of a non-prescription analgesic, such as acetaminophen, ibuprofen, ketoprofen, salicylates such as acetylsalicylic acid, indomethacin, and the like. A preferred analgesic is acetaminophen, and a preferred amount is about 1 x 10"3 to 2 x 10"' parts by weight.

A method for promoting recovery from soft tissue injury comprises orally administering to an individual in need thereof an effective amount of a composition comprising in parts by weight (a) from about 2.5 x 10"2 to 5 x 10"1 parts of a fungal protease; (b) from about 1 x 10"3 to 1.1 x 10"1 parts of a plant protease; (c) from about

1 x 10"3 to 1.1 x 10"1 parts of a microbial protease; (d) from about 8 x 10"3 to 1.6 x 10" 1 parts of an antioxidant selected from the group consisting of rutin, quercitin, and mixtures thereof; and (e) from about 1.4 x 102 to 1 x 10"1 parts of ascorbic acid, acceptable salts thereof, and mixtures thereof. DETAILED DESCRIPTION

Before the present composition and method of use thereof for decreasing recovery time in soft-tissue injuries are disclosed and described, it is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antioxidant" includes 11 a mixture of two or more antioxidants, reference to "a vitamin" includes reference to one or more of such vitamins, and reference to "a fungal protease" includes references to two or more of such fungal proteases.

As used herein, "effective amount" means an amount of a composition according to the present invention that is nontoxic but sufficient to provide the selected local or systemic effect and performance at a reasonable benefit/risk ratio attending any dietary supplement. An effective amount of an analgesic is an amount sufficient to provide a selected level of pain relief.

As used herein, "acceptable salts" means salts of, for example, citric acid or ascorbic acid, wherein such salts are generally regarded as safe for human consumption.

In preferred embodiments, the present invention comprises a mixture of proteases from sources other than animals combined in a fiber base with naturally occurring antioxidants, vitamins, minerals, and bioflavonoids. The use of fungal proteases in this formulation as anti-inflammatory agents and promoters of wound healing constitutes an improvement upon known compositions. Three fungal proteases, Fungal Protease A, Fungal Protease B, and acid Fungal Protease C, are complementary to each other in action. Fungal Protease A, extracted from Aspergillus oryzae, is commercially available and is comprised of several enzymatic activities. The predominant activity is characterized by having a pH optimum in the range of 6.0 to 9.0 and broad substrate specificity, with a slight preference for cleaving the carboxyl side of hydrophobic amino acid residues. Fungal Protease B, from Aspergillus oryzae, is also commercially available. This enzyme has a broad pH range, but is more active at a pH below 6.0. This extract exhibits endopeptidase activity and an exopeptidase activity observed as preferential cleavage of leucine residues from the amino terminus of peptides. Fungal Protease C, from Aspergillus niger, is also commercially available and is advantageous for its stability in acidic environments. The pH optimum range is about 2.0 to 7.0. These enzymes can also be obtained by published methods, e.g. S. Schwimmer, Source Book of Food Enzymology 89-122 (Avi Publishing, 1981) (hereby incorporated by reference).

These enzyme preparations are complementary in that they provide overlap in pH 12 optima, substrate specificity, and mode of cleaving the peptide backbone of proteins. As an example, the acid stability of Fungal Protease C allows its proteolytic activity to predominate in the acidic environment of the stomach. As the pH increases through food ingestion and movement into the more alkaline environment of the intestine, proteases A and B become more active. The broad substrate specificity exhibited by these proteases decreases the time for protein breakdown, thus allowing hydrolyzed proteins to be absorbed into circulation more quickly. The inclusion of all three of these fungal proteases increases the potential for hydrolytic cleavage of specific protein substrates over a broader range of pH and physiological conditions. Unlike many other types of proteins, these proteases are more stable in the presence of stomach acid and, therefore, are less likely to be destroyed by the process of digestion. Of the total quantity of enzymes ingested, approximately 10% to 40% is absorbed from the small intestine into the general circulation. The broad range of physiological conditions under which these proteases remain active is necessary, since microenvironmental conditions within the body may include pH values much different from pH 7.4, the norm for human serum. Interestingly, the proteolytic activity of human serum has been observed to increase after ingestion of enzyme- containing preparations, indicating gastric survival of preoteases.

It is also preferred that the mixture of proteases of the present composition include plant-derived proteases, such as bromelain and papain. These two proteases have been studied more extensively than the fungal enzymes, and possess potent anti-inflammatory and analgesic properties. These proteases are referred to as thiol proteases and contain a cysteine residue at the active site. Under oxidizing conditions, such as exposure to air, the thiol group of this cysteine loses a hydrogen atom and may crosslink with another thiol group, forming a disulfide bridge or, alternatively, crosslinking with another residue through the same oxidative process. In this oxidized state, the protease loses activity. Therefore, bromelain and papain lose activity under oxidative conditions, such as exposure to air. The present invention solves this problem through the inclusion of an antioxidant. For example, calcium ascorbate as a protective agent for papain and bromelain by means of its activity as a free radical scavenger, thus minimizing the oxidative effect caused 13 thereby. Fungal protease molecules have different active sites and are not labile under oxidative conditions, thus providing superiority over known compositions that relied solely or primarily on the actions of plant thiol proteases. The inclusion of fungal proteases in the composition of the present invention greatly increases the stability of the composition because of their resistance to inactivation through oxidation.

It is also preferred to include a microbial protease, such as a neutral bacterial protease, in the supplement. Such neutral bacterial protease has an active pH optimum in the range of pH 6 to pH 8, which corresponds to the pH found in the small intestine. This protease provides additional hydrolysis of protein substrates at physiological pH of 7.4. This commercially available protease is isolated from the microbe, Bacillus subtilis, and is active in the narrow pH range of about 6.0 to 8.0.

Animal studies have shown that multiple protease combinations are more effective at reducing inflammation than an equivalent amount of a single protease. Therefore, this invention has combined plant, fungal, and microbial proteases in a synergistic manner. Since each protease has different and specific sites of cleaving amino acid residues, maximal hydrolysis of the protein components of injury are obtained. Specifically, because the plant, fungal and microbial proteases act on different sites of the unwanted protein, the actions of one protease do not interfere with the actions of any other protease. In this manner the composition of the present invention is able to cause the desired hydrolytic cleavage of the unwanted protein in a minimum of time. Moreover, because each included protease has a preferred active site and a preferred pH range, the composition of the present invention is able to produce the desired result through a diverse pH range, more diverse than previously available. The composition and method of the present invention further provide a benefit over previous compositions and methods through a lower dosing requirement. The composition of the present invention is able to accomplish this through the inclusion of at least one plant protease, at least one fungal protease and at least one microbial protease. Furthermore, encapsulation as a dry powder prevents self-hydrolysis of the proteases or hydrolysis of other formula components. 14

Bromelain is derived from the stem of the pineapple plant, Ananas comosus. The preparation of bromelain is well known in the art, and bromelain is commercially available from a variety of sources. Papain is purified from the fruit of the tropical melon tree, Carica papaya. The preparation of papain is also well known in the art, and papain is also commercially available from a variety of sources. Fungal and microbial proteases can be obtained commercially or by methods published in S. Schwimmer, Source Book for Food Enzymology 89-122 (Avi Publications, 1981).

Antioxidant compounds such as vitamin E have been studied for over three decades, and are now accepted as efficacious in preventing oxidation of low-density lipoproteins (LDL). Oxidized LDL may adversely affect endothelial cells and may also be vasoconstrictive. Recently, other potent antioxidants have been found in the form of the bioflavonoids, quercetin, and rutin. Quercetin (3,3',4',5,7- pentahydroxyflavone) has been shown to inhibit certain mediators of the inflammatory process, while rutin (3-rhamnoglucoside of 5,7,3',4'- tetrahydroxyflavonol) has been shown to reduce chemically-induced inflammation in clinical studies. The addition of grape seed extract to the present invention also enhances the antioxidant potential of the formulation. Research has shown that the active antioxidants in grape seeds, known as procyanidins, are anti-inflammatory agents capable of inhibiting the process of edema (swelling resulting from inflammation).

The combination of proteolytic enzymes, bioflavonoids, and ascorbate has been shown to be as effective as non-steroidal anti-inflammatory drugs (NSAIDs) with fewer side effects and a much larger margin of safety. Unlike the NSAIDs, administration of proteases also enhances and promotes wound healing.

Vitamin C, a bioessential organic acid, is preferably included in the present invention in the form of calcium ascorbate. Vitamin C is well known as an antioxidant, as essential in the biosynthesis of collagen, and for supporting immune system functions, thereby improving the healing of damaged tissues. The calcium allows for buffering towards neutrality of the ascorbic acid (vitamin C), and also provides a mineral necessary for human nutrition. Calcium plays an important role 15 in musculoskeletal healing, and has been shown to improve the healing rate of fractures in older populations and in individuals with calcium deficiencies. In addition, the calcium ascorbate protects the activity of bromelain and papain proteases by providing a source of transferable hydrogens capable of keeping the active sulfhydryl group of those proteases from being oxidized. Ascorbate is well characterized as moving the equilibrium of reduction-oxidation reactions away from the oxidized state. Through the inclusion of ascorbic acid, the oxidation of the sulfhydryl group of thiol proteases is prevented. Importantly the shifting of the equilibrium in favor of the reduced sulfhydryl group guarantees that the activity of the bromelain and papain proteases is protected. This protection of the active form of the bromelain and papain proteases facilitates the absorption and transfer into the circulatory system of the desired active form. Importantly, ascorbic acid and its metal salts are essential components of a healthy diet. Thus the composition of the present invention preferably includes ascorbic acid in furtherance of two goals that of the protection of bromelain and papain proteases and to supplement the diet with additional portions of the bioessential organic acid, vitamin C.

It is also preferred that the presently described composition include minerals for use as electrolytes, enzyme cofactors, and the like. In addition to calcium, discussed above, the present composition also preferably includes the trace minerals zinc, copper, boron, and selenium. Zinc is a cofactor needed for maximal activation of the fungal and microbial proteases. Zinc is also essential for wound healing and for the activation of metalloenzymes necessary to cell growth and healing including RNA and DNA polymerases, nucleotide transferases, and the antioxidant enzyme superoxide dismutase. Zinc also plays a critical part in protein and collagen synthesis. Copper is essential for the formation and maintenance of connective tissues and is important in the mineralization of bone. Copper is also involved in the cross-linking of collagen and elastin, thereby contributing to the structural integrity of connective tissues. Copper is also important as a component of superoxide dismutase, and preliminary studies indicate that copper contributes to the reduction of inflammation. Boron is integral to the healing and mineralization of bone tissue and also has nutritive healing effects for degenerative joint diseases. Selenium is a 16 component of the antioxidant enzyme, glutathione peroxidase. Preliminary reports indicate that selenium plays a beneficial role in the improvement of chronic inflammatory conditions and degenerative joint conditions. Zinc and boron have been shown to improve rates of healing of soft-tissue injuries. These trace minerals can be supplied as an inorganic salt, such as a chloride, phosphate, and the like.

Preferably, however, such minerals are provided through the addition of marine kelp (Laminaria spp.) and Irish moss (Chondrus crispus) to the composition.

In another preferred embodiment, calcium citrate, a bioessential organic acid, is added to provide nutritional support for bone growth and to improve healing, as well as being a cofactor in many biochemical reactions. Because citric acid and corresponding salts are essential organic acids in metabolic functions and are also involved in numerous host defense mechanisms their inclusion in the composition of the present invention is of additional benefit.

It is also preferred that the present composition contain an effective amount of a non-prescription analgesic, such as acetaminophen, ibuprofen, ketoprofen, salicylates such as acetylsalicylic acid (aspirin), and indomethacin. Dosages of such analgesics should not exceed federal regulations. For example, for adults the oral dosage of products containing acetaminophen, aspirin, or sodium salicylate is 325 to 650 milligrams every 4 hours or 325 to 500 milligrams every 3 hours or 650 to 1,000 milligrams every 6 hours while symptoms persist, not to exceed 4,000 milligrams in 24 hours. In accordance therewith, a preferred dosage range for acetaminophen is up to about 200 mg per capsule, with a preferred dose of about 80 mg per capsule.

The composition of the present invention may optionally include fillers, flavors, coloring agents, formulation aids, and the like. For example, cellulose-based fillers (such as FIBREX 595 and ELCEMA G-250) and light mineral oil can be added to the composition to provide optimized flow properties to the dry powder during the formulation process.

The composition of the present invention is provided in capsule, tablet, or powder form and dissolves easily in the stomach. But one skilled in the art will perceive other physical forms of the composition that will be equally as useful. 17

Enteric coatings may also be used to provide protection from stomach acidity. In a preferred embodiment, the composition is prepared by blending together the stated raw material ingredients in an agglomerator so as to result in a product having a uniform composition with the precise proportions of the components as indicated. The agglomerated material is then placed in gelatin or other capsules, pressed into tablets, or packaged in a suitable container. In preferred embodiments, the formula comprises the following ingredients:

I II III IV V VI VII VIII aFungal protease 56 70 75 75 60 200 A. ( g) bFungal protease 16 20 24 20 22 200 B (mg) cFungal protease 5 6 5 6 100 5 C (mg) dBromelam (mg) 4 5 60 5 5 0.5 5

"Papain (mg) 0.8 1 50 1 1.2 0.5

Neutral 6 7.5 110 8 7 7 1 7.5 bacterial protease (mg)

Calcium 24 30 100 30 36 30 14 30 ascorbate (mg)

Calcium citrate 100 60 50 60 70 14 (mg)

Rutin (mg) 20 25 25 80 20 4

Quercitin (mg) 6.4 8 80 8 10 4 10

£Procyanidms 4 5 5 100 (mg)

Kelp (mg) 48 60 65 50 65 1 60

Irish moss (mg) 24 30 30 30 35 1 35

Filler (mg) 222.6 209.3 116.8 89.8 319.8 284.3 gMmeral oil 3.2 3.2 3.2 3.2 3.2 3.2 (mg)

Figure imgf000019_0001

500,000 HUT/g 410,000 HU/g 1070 SAPU/g 25,600 FCC PU/mg 50,000 FCC PU/mg grape seed extract U.S. P. light 18

The amounts of protease added to the formulation are based on activity units per dry weight of raw material; therefore, minor adjustments in the dry weight of proteases per capsule may occur depending upon the activity of the raw protease material. Activity units are based on assays published in the Food and Chemicals Codex or standard assays established by the industry. In addition, essential trace minerals are provided by the inclusion of kelp (Laminaria spp.) and algae (Chondrus crispus).

The recommended usage of the protease-based dietary supplement is as follows: for anticipated surgical procedures, sports participation, and other physical activities with a high likelihood of physical contact the supplement should be taken on the day prior to the procedure or anticipated activity with continued supplementation while symptoms persist. For unanticipated injury, the supplement should be taken as soon as possible with continued supplementation while symptoms persist. Dosage levels will vary according to the extent and type of injury and size of person. An acceptable starting dosage is 3 to 4 capsules every 4 hours; however, more may be taken without fear of toxicity. Since the presence of food can delay absorption of the proteases, it is recommended that the supplement be taken on an empty stomach between meals. Wliile benefit may be observed with daily administration of the nutritional supplement, it is to be used primarily for short-term nutritional support to hasten recovery from injury.

A method of use of the composition of the present invention and exemplary results are provided in the following examples. These examples are understood to be examples only and do not limit the scope of the present document which is limited only according to the included claims. Example 1

Two females were admitted for treatment of varicose veins. Treatment was to consist of injection of sodium morrhuate, a sclerosing agent, into the effected veins of each leg. One leg was to be treated at each visit with the visits separated by one month. This particular procedure lent itself well for study, as one leg could be used as the control (normal post-operative treatment) and the other as the dependent variable (normal post-operative treatment plus the protease-based 19 nutritional supplement). Follow-up visits were made at one, two and four weeks after each procedure. The first leg was treated with standard medical care. At the week-one follow-up examination, the injected vessels were observed to be clotted and palpable. The more superficial vessels contained dark blue clots. Upon palpation of the vessels, a noticeable amount of pain was reported by both patients.

At two weeks post-procedure, tissue bruising extended up to 3 mm on either side of the sclerosed vessel. Skin discoloration had changed from dark blue to yellow- green. The larger sclerosed vessels were still palpable but no longer painful. At four weeks post-procedure, bruising was apparent only in the largest vessels. The surrounding skin had a brown appearance which was due to staining by hemosiderin, a breakdown product of blood hemoglobin. The second leg was treated in an similar manner with the exception that the protease-based dietary supplement according to the present invention (Formulation II, above) was added to the treatment regimen. The patients took the supplement immediately after the procedure and four days following. The week-one post-procedure examination revealed bruising in only

50%) of the vessels treated, and complete clearance of clots from 25% of the smaller superficial vessels. Pain was present over the largest vessels only upon deep palpation. Two weeks after treatment, 80%> of all bruising and discoloration has resolved, and in the remaining areas only yellow-green bruising was observed. At four weeks after treatment, all bruising was resolved and no brown hemosiderin staining was noted. Both patients stated that they had fewer subjective problems following the second procedure. Example 2

A 35 year old male underwent lipoplasty of the right and left flank. Approximately 400 cc were aspirated from each flank. Observations on day 3 post- lipoplasty revealed significant bruising of the flanks extending to the genitalia. Edema was noted throughout the area and the patient with unable to rest without analgesia. On day 10 little change was noted in bruising and edema, but discomfort was diminishing. Twenty days post-lipoplasty, minimal yellowing of the skin was observed with pain and edema resolved. Twelve weeks later the patient underwent a 475 cc aspiration of the lower abdomen. In addition to normal post-operative 20 management, the protease-based dietary supplement according to the present invention (Formulation II, above) was added to the treatment regiment. Discomfort from the procedure was present only on the first night following surgery and no analgesia was required after the first post-surgical day. Day 10 examination showed complete resolution of bruising and edema was minimal. Examination on day 20 showed complete resolution of all symptoms. Example 3

A 39 year old female underwent lipoplasty of the lower abdomen, from which approximately 900 cc was aspirated. On days 3 and 10 post-surgery, severe (4+ on a 0 to 5 point scale) bruising, edema, and discomfort were noted. Analgesics were required for three weeks following the surgery. Discomfort was minimal but still present. Ten weeks following the first surgery, an additional lipoplasty was performed with 1700 cc removed from the flanks. Post-operative care included the protease-based dietary supplement according to the present invention. Examination 3 days post-surgery showed moderate bruising and edema with severe discomfort level. In the day 10 examination, complete resolution of bruising was noted while edema and pain levels were unchanged. Complete resolution of pain was noted by the 20-day post-operative examination with edema present only on deep palpation. Example 4 A 38 year old female underwent lipoplasty with 1000 cc aspirated from the lower abdomen. Day 3 revealed significant bruising, edema, and discomfort. On day 10 bruising and edema were reduced to moderate levels while discomfort was unchanged. Some residual bruising and edema were present on day 20, while the discomfort has resolved. Twelve weeks later the patient had 1200 cc aspirated from her flanks. The protease-based dietary supplement according to the present invention was added to the normal treatment protocol. Examination on day 3 showed low to moderate bruising and edema. Any discomfort had completely resolved. The day 20 exam revealed resolution of bruising and discomfort while edema was noted only on deep palpation. Example 5 21

In this example, the following ingredients are combined, mixed in an agglomerator, and placed in gelatin capsules:

Ingredient Parts by weight

Fungal Protease A 70 Fungal Protease B 20

Fungal Protease C 6

Bromelain 5

Papain 1

Neutral bacterial protease 7.5 Calcium ascorbate 30

Calcium citrate 60

Rutin 25

Quercitin 8

Grape seed extract 5 Kelp 60

Irish Moss 30

Acetaminophen 80

Filler 129.3

Mineral oil 3.2

While the invention has been described with reference to a preferred embodiment, it is to be understood by those skilled in the art that the invention is not limited thereto. Rather the scope of the invention is to be inteφreted only in conjunction with the appended claims

Claims

22CLAIMS We claim:
1. A dietary supplement composition for reducing the time for healing of soft-tissue injuries comprising in parts by weight (a) from about 2.5 x 10"2 to 5 x 10"1 parts of a fungal protease; (b) from about 1 x 10"3 to 1.1 x 10"1 parts of a plant protease; (c) from about 1 x 10"3 to 1.1 x 10"' parts of a microbial protease; (d) from about 8 x 10"3 to 1.6 x 10"1 parts of an antioxidant selected from the group consisting of rutin, quercitin, and mixtures thereof; and (e) from about 1.4 x 10"2 to 1 x 10"1 parts of ascorbic acid, acceptable salts thereof, and mixtures thereof.
2. The composition of claim 1 wherein said fungal protease is a member selected from the group consisting of exo- and endo-peptidases from Aspergillus oryzae, Aspergillus niger, Aspergillus sojae, Aspergillus flavus, Aspergillus awamori, and mixtures thereof.
3. The composition of claim 2 wherein said fungal protease is a member selected from the group consisting of Fungal Protease A from Aspergillus oryzae,
Fungal Protease B from Aspergillus oryzae, and Fungal Protease C from Aspergillus niger, and mixtures thereof.
4. The composition of claim 1 wherein said plant protease is a member selected from the group consisting of bromelain, papain, and mixtures thereof.
5. The composition of claim 1 wherein said microbial protease is neutral bacterial protease from Bacillus subtilis.
6. The composition of claim 1 further comprising a mineral selected from the group consisting of about 1.4 x 10"2 to 5.4 x 10"2 parts by weight of calcium, about 5.4 x 10"7 to 5.4 x 10"3 parts by weight of zinc, about 1.6 x 10'7 to 5.4 x 10"3 parts by weight of copper, about 3.2 x 10"7 to 5.4 x 10"3 parts by weight of boron, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of selenium, and mixtures thereof.
7. The composition of claim 2 further comprising a mineral selected from the group consisting of about 1.4 x 10"2 to 5.4 x 10"2 parts by weight of calcium, about 5.4 x 10"7 to 5.4 x 10"3 parts by weight of zinc, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of copper, about 3.2 x 10"7 to 5.4 x 10"3 parts by weight of 23 boron, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of selenium, and mixtures thereof.
8. The composition of claim 4 further comprising a mineral selected from the group consisting of about 1.4 x 10"2 to 5.4 x 10"2 parts by weight of calcium, about 5.4 x 10'7 to 5.4 x 10"3 parts by weight of zinc, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of copper, about 3.2 x 10"7 to 5.4 x 10"3 parts by weight of boron, about 1.6 x 10"7 to 5.4 x 10 "3 arts by weight of selenium, and mixtures thereof.
9. The composition of claim 5 further comprising a mineral selected from the group consisting of about 1.4 x 10"2 to 5.4 x 10"2 parts by weight of calcium, about 5.4 x 10"7 to 5.4 x 10"3 parts by weight of zinc, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of copper, about 3.2 x 10"7 to 5.4 x 10"3 parts by weight of boron, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of selenium, and mixtures thereof. 10. The composition of claim 1 further comprising about 1 x 10"3 to 1 x
10"1 parts by weight of procyanidins.
11. The composition of claim 1 further comprising about 1 x 10"2 to 1 x
10"' parts by weight of a member selected from the group consisting of citric acid, acceptable salts thereof, and mixtures thereof.
12. The composition of claim 1 further comprising a member selected from the group consisting of fillers, flavors, coloring agents, formulation aids, and mixtures thereof.
13. The composition of claim 1 further comprising an effective amount of a non-prescription analgesic.
14. The composition of claim 13 wherein said non-prescription analgesic is a member selected from the group consisting of acetaminophen, ibuprofen, ketoprofen, salicylates, indomethacin, and mixtures thereof.
15. The composition of claim 14 wherein said non-prescription analgesic is acetaminophen and said effective amount is about 1 x 10"3 to 2 x 10"' parts by weight. 24
16. A method for promoting recovery from soft tissue injury comprising orally administering to an individual in need thereof an effective amount of a composition comprising in parts by weight (a) from about 2.5 x 10"2 to 5 x 10"1 parts of a fungal protease; (b) from about 1 x 10"3 to 1.1 x 10"' parts of a plant protease; (c) from about 1 x 10"3 to 1.1 x 10"' parts of a microbial protease; (d) from about 8 x 10"3 to 1.6 x 10"' parts of an antioxidant selected from the group consisting of rutin, quercitin, and mixtures thereof; and (e) from about 1.4 x 10"2 to 1 x 10"1 parts of ascorbic acid, acceptable salts thereof, and mixtures thereof.
17. The method of claim 16 wherein said fungal protease is a member selected from the group consisting of exo- and endo-peptidases from Aspergillus oryzae, Aspergillus niger, Aspergillus sojae, Aspergillus flavus, Aspergillus awamori, and mixtures thereof.
18. The method of claim 17 wherein said fungal protease is a member selected from the group consisting of Fungal Protease A from Aspergillus oryzae, Fungal Protease B from Aspergillus oryzae, and Fungal Protease C from Aspergillus niger, and mixtures thereof.
19. The method of claim 16 wherein said plant protease is a member selected from the group consisting of bromelain, papain, and mixtures thereof.
20. The method of claim 16 wherein said microbial protease is neutral bacterial protease from Bacillus subtilis.
21. The method of claim 16 wherein said composition further comprises a mineral selected from the group consisting of about 1.4 x 10"2 to 5.4 x 10"2 parts by weight of calcium, about 5.4 x 10"7 to 5.4 x 10"3 parts by weight of zinc, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of copper, about 3.2 x 10"7 to 5.4 x 10"3 parts by weight of boron, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of selenium, and mixtures thereof.
22. The method of claim 17 wherein said composition further comprises a mineral selected from the group consisting of about 1.4 x 10"2 to 5.4 x 10"2 parts by weight of calcium, about 5.4 x 10"7 to 5.4 x 10"3 parts by weight of zinc, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of copper, about 3.2 x 10"7 to 5.4 x 10"3 parts by 25 weight of boron, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of selenium, and mixtures thereof.
23. The method of claim 19 wherein said composition further comprises a mineral selected from the group consisting of about 1.4 x 10"2 to 5.4 x 10"2 parts by weight of calcium, about 5.4 x 10"7 to 5.4 x 10"3 parts by weight of zinc, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of copper, about 3.2 x 10"7 to 5.4 x 10"3 parts by weight of boron, about 1.6 x 10"7 to 5.4 x 10 "3 parts by weight of selenium, and mixtures thereof.
24. The method of claim 20 wherein said composition further comprises a mineral selected from the group consisting of about 1.4 x 10'2 to 5.4 x 10"2 parts by weight of calcium, about 5.4 x 10"7 to 5.4 x 10"3 parts by weight of zinc, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of copper, about 3.2 x 10"7 to 5.4 x 10"3 parts by weight of boron, about 1.6 x 10"7 to 5.4 x 10"3 parts by weight of selenium, and mixtures thereof.
25. The method of claim 16 wherein said composition further comprises about 1.4 x 10"3 to 1 x 10"' parts by weight of procyanidins.
26. The method of claim 16 wherein said composition further comprises about 1 x 10"2 to 1 x 10"1 parts by weight of a member selected from the group consisting of citric acid, acceptable salts thereof, and mixtures thereof.
27. The method of claim 16 wherein said composition further comprises a member selected from the group consisting of fillers, flavors, coloring agents, formulation aids, and mixtures thereof.
28. The method of claim 16 wherein said composition further comprises an effective amount of a non-prescription analgesic.
29. The method of claim 28 wherein said non-prescription analgesic is a member selected from the group consisting of acetaminophen, ibuprofen, ketoprofen, salicylates, indomethacin, and mixtures thereof.
30. The method of claim 29 wherein said non-prescription analgesic is acetaminophen and said effective amount is about 1 x 10"3 to 2 x 10"' parts by weight.
PCT/US1999/001690 1998-02-13 1999-01-27 Protease-based dietary supplementation for decreasing recovery time from soft-tissue injury WO1999041361A1 (en)

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GB2423928A (en) * 2004-11-16 2006-09-13 Efflux Technology Inc Methods and compositions for treating pain
GB2440117A (en) * 2006-07-19 2008-01-23 Elemis Ltd Skin care composition comprising a trienzyme activity
EP2321640A1 (en) * 2008-08-21 2011-05-18 Alvine Pharmaceuticals, Inc. Formulation for oral administration of proteins

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US3509024A (en) * 1965-08-27 1970-04-28 Astra Ab Aspergillopeptidase for use in therapy and a process for the preparation thereof

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Publication number Priority date Publication date Assignee Title
US3509024A (en) * 1965-08-27 1970-04-28 Astra Ab Aspergillopeptidase for use in therapy and a process for the preparation thereof

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2423928A (en) * 2004-11-16 2006-09-13 Efflux Technology Inc Methods and compositions for treating pain
GB2423928B (en) * 2004-11-16 2008-04-09 Efflux Technology Inc Methods and compositions for treating pain
GB2440117A (en) * 2006-07-19 2008-01-23 Elemis Ltd Skin care composition comprising a trienzyme activity
GB2440117B (en) * 2006-07-19 2008-07-16 Elemis Ltd Skincare compositions comprising a trienzyme activity
EP2321640A1 (en) * 2008-08-21 2011-05-18 Alvine Pharmaceuticals, Inc. Formulation for oral administration of proteins
EP2321640A4 (en) * 2008-08-21 2013-07-17 Alvine Pharmaceuticals Inc Formulation for oral administration of proteins
US8980254B2 (en) 2008-08-21 2015-03-17 Alvine Pharmaceuticals, Inc. Formulation for oral administration of proteins
US9993531B2 (en) 2008-08-21 2018-06-12 Immunogenics Llc Formulation for oral administration of proteins

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