Compositions comprising protease, amylase and lipase for use in the treatment of staphylococcus aureus infections

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Descriptive Report of the Invention Patent for PHARMACEUTICAL COMPOSITION FOR USE IN THE TREATMENT OR PREVENTION OF S. AUREUS INFECTION IN A BIRD OR A MAMMAL, METHOD FOR SANITIZING OR DISINFECTING A SURFACE FOR

REDUCE THE AMOUNT OF S. AUREUS IN IT OR ERADICATE S. AUREUS FROM IT, DISINFECTANT, AND COMPOSITION.

CROSS-REFERENCE TO RELATED ORDERS

This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application Nos. 61/142,714, filed January 6, 2008; 61/153,274, filed February 17, 2009; and 61/170,915 filed April 20, 2009, the contents of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to compositions, including pharmaceutical compositions such as antibiotic compositions, and methods of using the same for treating or preventing Staphylococcus aureus (S. aureus, also referred to herein as SA) infections in humans and other animals. The present invention also relates to compositions such as disinfectants, sanitizers, antiseptics and detergents and methods of using the same for eradicating or reducing the presence of S. aureus on surfaces, including inanimate/non-living and biological surfaces (e.g. skin, wounds) and/or for attenuation of S. aureus infectivity in order to prevent and/or reduce the spread of S. aureus infections.

BACKGROUND

Staphylococcus aureus, often referred to simply as staph, is a bacterium commonly carried on the skin or in the nose of healthy people. Staph bacteria are one of the most common causes of skin infections in the United States. Most of these skin infections are minor (such as pimples and blisters) and can be treated without antibiotics. However, staph bacteria can also cause serious infections such as surgical wound infections, bloodstream infections, and pneumonia.

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Staphylococcus aureus are spherical Gram-positive bacteria that occur in microscopic clusters resembling grapes. In 1884, Rosenbach described the two pigmented colony types of Staphylococcus and proposed the appropriate nomenclature: Staphylococcus aureus (yellow) and Staphylococcus abus (white). The latter species is now called Staphylococcus epidermis. Staphylococcus aureus are facultative anaerobes that grow by aerobic respiration or by fermentation that results primarily in lactic acid. The bacteria are ketiase-positive and oxidase-negative. Staphylococcus aureus can grow in a temperature range of 10-15 to 45 degrees and in a NaCl concentration as high as 15 percent.

Some staph bacteria are resistant to antibiotics. Methicillin-resistant Staphylococcus aumus (MRSA) is a type of staph that is resistant to antibiotics called beta-lactams. Beta-lactam antibiotics include methicillin and other more common antibiotics such as oxacillin, penicillin, and amoxicillin. Most MRSA infections occur among patients in hospitals or other healthcare settings (referred to as hospital-acquired MRSA or HA-MRSA infections); however, Staph and MRSA can also cause illness in people outside of hospitals and healthcare settings. MRSA infections that are acquired by people who have not recently been hospitalized (within the past year) or had a medical procedure (such as dialysis, surgery, catheters) are known as community-acquired infections (CAMR8A). About 75 percent of CA-MRSA infections are located in the skin and soft tissue and can usually be treated effectively. However, CA-MRSA strains show increased virulence, spread more rapidly, and cause more severe disease than traditional HA-MRSA infections, and can affect vital organs leading to disseminated infection (sepsis), toxic shock syndrome, and pneumonia.

It is not known why some healthy people develop treatable CA-MRSA skin infections while others infected with the same strain develop severe, fetal infections. Studies have shown that CA-MRSA infection rates are increasing.

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In 1999, four children in Minnesota and North Dakota were reported to have died from fulminant CA-MRSA infections. A study of children in southern Texas found that cases of CA-MRSA increased 14-fold between 1999 and 2001. In 2007, CA-MRSA was the 5th most common cause of skin and soft tissue infections seen in emergency departments in the United States.

Hospital strains of Staphylococcus aureus are generally resistant to a variety of different antibiotics. Some strains are resistant to all clinically useful antibiotics except vancomycin, and vancomycin-resistant strains (VRSA) are increasingly reported. Resistance to methicillin is widespread (MRSA), and most methicillin-resistant strains are also multidrug resistant. Furthermore, S. aureus exhibits resistance to antiseptics and disinfectants, such as quaternary ammonium compounds, which may aid in its survival in the hospital environment.

More people in the United States now die from MRSA infection than from AIDS. MRSA was responsible for an estimated 94,000 life-threatening infections and 10,650 deaths in 2005, as reported by the CDC in the October 17, 2007, issue of The Journal of the American Medical Association. The national estimate is more than double the prevalence of invasive MRSA reported five years earlier. In the same year. Approximately 16,000 people have died in the United States from AIDS, according to the CDC.

SUMMARY

The present invention relates to the prevention and/or treatment of S. aureus infections, including MRSA and VRSA infections, with the use of a pharmaceutical composition comprising one or more digestive enzymes, such as pancreatic enzymes or other digestive tract enzymes (e.g., porcine paricreatic enzymes) or plant, fungal or microorganism derived enzymes that break down food components. As used herein, a pharmaceutical composition may be used for human or veterinary indications. Thus, the compositions

4/75 pharmaceutical preparations may be useful for prophylactic and/or therapeutic treatment of human populations and other mammals (e.g., pig, horse, cow, sheep, goat, monkey, rat, mouse, cat, dog) or bird populations (e.g., duck, goose, chicken, turkey).

The pharmaceutical compositions may be used alone or in combination with other antibacterial or antibiotic regimens (e.g.,_: aníi-S. aureus) and/or with other therapeutic agents or post-infection antibiotics to treat S. aureus infections.

Also provided herein are bacteriostatic and/or bacteriocidal compositions comprising one or more digestive enzymes for use as or in disinfectants, sanitizers, detergents, and antiseptics, for example, in hospitals, daycare centers, nursing facilities, day care facilities, schools, workplaces, food service environments, public transportation, and restroom facilities, to reduce, attenuate, and/or destroy S. aureus present in such environments. The surfaces treated with the described compositions may be large (e.g. operating room tables, doors, changing tables, ventilation systems) or small (e.g. medical devices, door handles); inanimate or non-living tissue (tables) or living (hands, e.g. hand washing detergents;

wounds, for example surgical wounds or wounds resulting from accidents/traumas). The compositions may then be useful for treating surfaces to reduce or eradicate S. aureus thereon or to attenuate or reduce the infectivity of S. aureus and thereby prevent or reduce the spread of S. aureus.

Accordingly, it is an object of the present invention to provide a method for treating or preventing S. aureus infection in a bird or a mammal comprising administering to the bird or mammal a therapeutically effective amount of a pharmaceutical composition comprising one or more digestive enzymes. In some embodiments, the one or more digestive enzymes comprise one or more enzymes selected from the group consisting of proteases, amylases, celluloses, sucrases, maltases, papain, and lipases. In some embodiments, the one or more

5/75 digestive enzymes comprise one or more pancreatic enzymes. The one or more digestive enzymes are, independently, derived from an animal source, a microbial source, a plant source, a fungal source, or are synthetically prepared. In some embodiments, the animal source is a pig pancreas.

In some embodiments, a pharmaceutical composition comprises at least one amylase, a protease mixture comprising chymotrypsin and trypsin, and at least one lipase. In some embodiments, a pharmaceutical composition comprises at least one protease and at least one lipase and wherein the ratio of total proteases to total lipases (in USP units) ranges from about 1:1 to about 20:1.

In some embodiments, the pharmaceutical composition is a dosage formulation selected from the group consisting of: pills, tablets, capsules, lozenges, sprays, creams, lotions, aerosols, emulsions, powders, liquids, gels, and a combination of any of them.

In some embodiments, the pharmaceutical composition is formulated for oral administration or for topical administration or for intranasal or transmucosal administration.

Also provided is a method of treating a mammal or bird exhibiting one or more symptoms of a Staphylococcus aureus infection comprising administering to the mammal or bird a therapeutically effective amount of a composition comprising one or more digestive enzymes,

Further provided is a method for promoting wound healing and/or scar reduction in a subject with a wound comprising administering a pharmaceutical composition comprising one or more digestive enzymes to the subject. The wound may be, for example, a surgical wound or a traumatic wound.

The invention also relates to a method for sanitizing or disinfecting a surface to reduce the amount of S. aureus thereon or to eradicate S. aureus thereon comprising applying to the surface a

6/75 composition comprising one or more digestive enzymes. The surface may be a living surface (e.g., skin, wound) or an inanimate or nonliving surface (e.g., medical device such as a scalpel, knife, scissors, spatula, dilator, clamp, forceps, speculum, retractor, suture, valve, surgical mesh, chisel, drill, level, file, saw, splint, caliper, clamp, forceps, hook, lancet, needle, cannula, curette, depressor, dilator, elevator, articulator, extractor, probe, clamp, catheter, sphenoid tubing, bowl, tray, sponge, cord, spoon, syringe, pacemaker, screw, plate and pin.

Also provided herein is a method for reducing the amount of S. acari present in a skin, tissue or wound region of a mammal or bird comprising applying to the skin, tissue or wound region a composition comprising one or more digestive enzymes.

Also described is a disinfectant comprising one or more digestive enzymes, where the disinfectant has a phenol coefficient of >1 to about 20 for S. aureus or E. coli/Z.

The invention also provides an antibiotic comprising one or more digestive enzymes, wherein the antibiotic is bacteriocidal and/or bacteriostatic to S. aureus,

Similarly, a detergent comprising one or more digestive enzymes is also provided, wherein the detergent is bactericidal and/or bacteriostatic to S. aureus,

Also provided is an antiseptic comprising one or more digestive enzymes, wherein the antiseptic is bacteriocidal and/or bacteriostatic to S. aureus.

The invention also provides a disinfectant comprising one or more digestive enzymes, wherein the disinfectant is bacteriocidal and/or bacteriostatic to Staphylococcus aureus.

Details of one or more embodiments of the invention are shown in the accompanying drawings and in the specification below. Any reference made herein, such as to a patent, patent application, report, book, article or scientific publication, is incorporated by reference in

7/75 its totality. Other features, objects and advantages of the invention will be apparent from the specification and drawings and from the claims.

DETAILED DESCRIPTION

The term Administration⁷' or Administering” refers to a method of giving a dosage of a pharmaceutical composition or composition to a vertebrate or invertebrate, including a mammal, a bird, a fish or an amphibian, where the method is by any route, for example, intrarespiratory, nasal, topical* oral, intravenous, intraperitoneal, intramuscular, transmucosal, buccal, rectal, vaginal or sublingual. The preferred method of administration may vary depending on several factors, for example, the components of the pharmaceutical composition, the site of the disease, the disease involved and the severity of the disease, the term “mammal” is üsãdó in its ordinary biological sense. 15 Thus, it specifically includes humans, cattle, horses, dogs and cats, but also includes many other species.

The term “pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except in the event that any conventional media or agent is incompatible with the active ingredients, their use in therapeutic compositions is understood. Supplemental active ingredients, such as antibiotics, antifungals, and antimicrobials, may also be incorporated into the compositions. In addition, various adjuvants such as those commonly used in the art may be included. These and other such compounds are described in the literature, for example, in the Merck Index, Merck & Company, Rahway, NJ. Considerations for the inclusion of various components in pharmaceutical compositions are described, for example, in Gilman et al. (Eds.) (2006): Goodman and Gilman's The Pharmacology/ Basis of Therapeutics, 11th Ed., The McGraw-Hill Companies,

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Subject or “patient” or “individual” as used herein means a human or non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.

Therapeutically effective amount or pharmaceutically effective amount” is typically one that is sufficient to achieve the desired effect and may vary with the nature and severity of the disease condition, the nature of the individual, and the potency of the composition. 10 It will be understood that concentrations employed for prophylaxis may be different from those for the treatment of active disease. This amount may also depend on the height, weight, sex, age, and medical history of the patient.

A therapeutic effect relieves, to some extent, one or more of the 15 symptoms of a disease and includes a cure of a disease. "Cure means that the symptoms of the active disease are eliminated. However, certain long-term or permanent effects of the disease may exist even after a cure is achieved (such as tissue damage).

“Treat”, treatment or “treating”, as used herein, refers to the administration of a therapeutic composition for therapeutic purposes. The term “therapeutic treatment” refers to the administration to a patient already suffering from a disease, thereby causing a therapeutically beneficial effect, such as amelioration of existing symptoms, prevention of further symptoms, amelioration or prevention of the underlying metabolic causes of the symptoms, delaying or preventing further development of a disorder and/or reducing the severity of symptoms that will develop or are expected.

The present invention provides compositions comprising one or more digestive enzymes and methods of using the same for the treatment and/or prevention of S. aureus infections, including antibiotic-resistant forms of S. aureus such as MRSA and VRSA. The present invention also provides compositions comprising one or more digestive enzymes and methods of using the same as antiseptics, detergents, disinfectants and sanitizers, for example, as bactericidal and/or bacteriostatic compositions, for eradicating or attenuating or reducing the infectivity thereof. The compositions described herein include one or more digestive enzymes, which are postulated to aid in the reduction, weakening or eradication of S. aureus and then prevent getting S. aureus infections or treat S. aureus infections (e.g., improve symptoms or shorten the time course of the infection).

Compositions

A composition for use as described herein may include one or more digestive enzymes. While not being limited by theory, it is believed that the digestive enzyme(s) in the composition may degrade the cell wall, membrane structures and/or protein of SL auraus, leading to bacteriostatic and/or bacteriocidal activity. The compositions 15 demonstrate species-specific bactericidal/bactericidal activity against S. aureus and H. coli, but not against enfeuca, possibly demonstrating that the vulnerability of the two organisms derives from proteolytic degradation of a similar protein sequence present in both organisms.

A digestive enzyme as described here is an enzyme that can break down one or more food components (e.g., proteins, fats, carbohydrates). Digestive enzymes may be animal-derived (e.g., pancreatic or other digestive enzymes) or plant, fungal, or microorganism-derived enzymes, or they may be synthetically prepared. Many digestive enzymes are commercially available or can be isolated and purified from other sources by methods well known to those skilled in the art. Enzymatic activity of the enzymes can also be assessed using standard assays.

The digestive enzymes may be used in any combination of enzyme type and any combination of enzyme sources. In some embodiments, the one or more digestive enzymes comprise one or more enzymes selected from the group consisting of proteases,

10/75 amylases, celluloses, sucrases, maltases, papain (e.g., from papaya), bromelain (e.g., from pineapple), hydrolases, and lipases. In some embodiments, the one or more digestive enzymes comprise one or more pancreatic enzymes. In some embodiments, the composition comprises one or more proteases, one or more lipases, and one or more amylases. In some embodiments, the one or more proteases comprise chymotrypsin and trypsin. In some embodiments, the composition as described herein consists essentially of, or consists of, one or more digestive enzymes.

In certain embodiments, the composition can comprise at least one amylase, at least two proteases, and at least one lipase. In certain embodiments, the composition can further include one or more hydrolases, papain, bromelain, papaya, celluloses, pancreatin, sucrases, and maltases.

As indicated, the one or more digestive enzymes can be derived from an animal source. In some embodiments, the animal source is a pig, e.g., a pig pancreas. Pig pancreatic enzyme extracts and formulations are known to those skilled in the art and are commercially available or can be prepared using known methods. For example, a pancreatic enzyme composition can be purchased from Scientific Protein Laboratories (designated PEC). A pancreatic enzyme composition, or any such composition, may be adjusted to modify the amount of one or more digestive enzymes contained therein, e.g., the lipase, amylase or protease content, such as by production and/or processing methods or by selective addition of exogenous enzymes, activators or inhibitors to the composition.

In certain circumstances, it may be desirable to have relatively greater activity of proteases than lipases. Thus, in some embodiments, a composition comprises at least one protease and at least one lipase, wherein the ratio of total proteases to total lipases (USP units) ranges from about 1:1 to about 20:1, including 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1,

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15*1, 16:1, 17*1, 18:1, 19*1, and 20:1, along with all values ​​in between. In some embodiments, the ratio of protease to lipase ranges from about 4:1 to about 10:1, including 4:1, 5:1, 6:1,_: 7:1, 8:1, 9:1, and 10:1, along with all values ​​in between.

In certain circumstances, it may be useful to modify the amount of a particular enzyme activity in a given composition. The activity of one or more digestive enzymes can be adjusted in a variety of ways known to one of skill in the art, for example, by increasing the amount of the particular enzyme or by adjusting the components of the composition, for example, through the use of stabilizers, inhibitors, and activators. In some embodiments, the composition described herein includes one or more proteases having an activity of from about 0.05 to about 400 USP Units per mg of the composition, or any value therebetween (e.g., 0.1; 0.2; 0.25; 0.5; 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 100, 150, 200, 250, 300, 350 USP Units per mg). In some embodiments, a composition described herein includes one or more lipases having an activity of from about 0.005 to about 50 Units per mg of the composition, or any value therebetween (e.g., 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.06, 0.08, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 0, 10, 12, 14, 16, 18, 20, 22, 25, 28, 30, 35, 38, 40, 45 USP Units per mg). In some embodiments, a composition described herein includes one or more amylases having an activity of from about 0.05 to about 400 USP Units per mg of the composition, or any range therebetween (e.g., 0.1; 0.2; 0.25; 0.5; 1.2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 100, 150, 200, 250, 300, 350 USP Units per mg). In some embodiments, a composition described herein includes one or more proteases in the above activity range, one or more lipases in the above activity range, and one or more amylases in the above activity range; An exemplary embodiment includes one or more proteases having an activity in the range of about 150-250 USP units/mg; one or more lipases having an activity in the range of about 20-40 USP units/mg; and one or more amylases having an activity in the range of about 200-300 USP units/mg.

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USP/mg.

In some embodiments, a composition can be formulated in a manner to stabilize the one or more digestive enzymes, for example, to preserve the enzyme activity of the enzymes. Stabilization techniques can limit or prevent self-degradation of the one or more enzymes in a composition and help maintain enzyme activity, increase half-life, and aid in tolerating the activity of the compositions to changes in temperature, humidity, and storage conditions. For example, in some embodiments, one or more enzymes in the composition are encapsulated, e.g., encapsulated in lipid. In other applications, variations in excipients, pH, enzyme inhibitors, etc., may be employed to aid in stabilizing the enzymes. Appropriate stabilization techniques will depend on the intended application of the composition (e.g., antibiotic versus detergent), the route of administration, the form of the composition, the intended site of application/enzymatic activity, and other factors, and can be determined by those skilled in the art.

Certain useful enzyme activity stabilizers include compounds that provide a source of free calcium in solution such as, for example, calcium salts; alkyl or branched alcohols such as, for example, ethanol and isopropyl alcohol; alkanolamines such as, for example, triethanolamine; acids such as organic acids; and mixtures of petroleum distillates.

In certain embodiments, an enzyme activity stabilizer 25 can be a composition selected from (1) compositions known to be effective in stabilizing enzymes in liquid aqueous solutions, including enzyme stabilizing compounds and systems, (2) selected micelle inhibitors, and mixtures of (1) and (2). In some embodiments, the activity stabilizer is a suitable concentration of boron anions. 30 In some cases, the activity stabilizer is solvated in a polyol and may be combined with enzyme stabilizing synergists or adjuvants to form an enzyme stabilizing system. “Enzyme stabilizing inhibitors”

Preferred micelle inhibitors include species known to modify as well as inhibit micelle formation and can be selected from water-miscible solvents such as C1-C2 alkanols, C1-C2 diols, C2-C24 alkyl glycol ethers, alkylene glycol alkyl ethers and mixtures thereof. A highly preferred micelle inhibitor is di-(propylene glycol) methyl ether (“DPM”) and its analogues which modify micelle formation.

An example of an "enzyme stabilization system" is a boron compound (e.g., boric acid) that has in the past been used alone or with other selected adjuvants or synergists (e.g., polyphenolic amino compounds, antioxidants, etc.) to protect proteolytic and other enzymes in storage and in various products.

An activity stabilizer may be chosen to substantially minimize the Minimum Inhibitory Concentration (“MIC”) of digestive enzyme in the formulation. MIC is a measure of the minimum concentration of the biocide that is successful in preventing bacterial growth in a culture over a specified period of time, e.g., 24 hours. Details of MIC testing are given in Bailey & Scott, “Diagnostic Microbiology,” 8^the edition, 1990, on page 177.

In some embodiments, a composition described herein may be coated with a variety of natural or synthetic coatings* for example, to provide timed release of the enzymes, to provide taste or odor masking, or to stabilize the enzymes. Coated enzyme preparations* including lipid-coated or lipid-encapsulated enzyme compositions, comprising one or more digestive enzymes useful for the methods & compositions described herein, are described in US Serial No. 12/386,051 filed April 13, 2009, incorporated herein by reference in its entirety. Such coated preparations can provide desired characteristics, including increased shelf stability, reduced aerosolization of powder or solid formulations, flavor and taste masking, enzyme stabilization, and delayed or timed release of enzymes.

Other additives for inclusion in the compositions described herein

14/75 may be determined by those skilled in the art and will be based on various characteristics, including intended application, e.g., human versus veterinary applications; desired release profile; desired pharmacokinetics; safety; stability; and physical characteristics (smell, color, taste, brightness, aerosolization). Formulation ingredients, excipients, binders, bulking agents, flavorings, colorants, etc. suitable can be determined and evaluated by methods known to those skilled in the art.

Family Compositions and Antibiotics for Human or Veterinary Use

The compositions described herein may be formulated as pharmaceutical compositions, for example, they may include a composition as described above formulated with one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical compositions 15 are useful for treating or preventing S. aureus infections in humans and other animals, such as mammals (e.g., cows, horses, pigs, sheep, goats, monkeys, cats, dogs, mice, rats) and birds (chickens, turkeys, ducks, geese). A pharmaceutical composition for treating B. aureus infections may also be referred to herein as an antibiotic or an antibiotic composition.

The susceptibility of S. augietis, including MRSA and VRSA, to an antibiotic composition described herein can be determined by methods known to those skilled in the art. A rapid procedure uses commercial filter paper discs that have been impregnated with a specified amount of the antibiotic composition. These discs are placed on the surface of agar plates that have been layered with a culture of S. aureus being tested, and the plates are observed for zones of inhibition of growth. The broth dilution susceptibility test involves preparing test tubes containing serial dilutions of the composition in liquid culture medium, then inoculating the tubes with the organism being tested. The lowest concentration of drug that inhibits growth of the bacteria after an adequate incubation period is reported

15/75 as the minimum inhibitory concentration (MIC).

The resistance or susceptibility of S. aureus to an antibiotic described herein can be determined based on clinical outcome, that is, whether administration of this antibiotic to an individual infected with that organism will successfully cure the individual. Alternatively, to facilitate identification of antibiotic resistance or susceptibility using in vitro test results, the National Committee for Clinical Laboratory Standards (NCCLS) has formulated standards for antibiotic susceptibility that relate clinical outcome to in vitro determinations of the minimum inhibitory concentration of antibiotic.

Administration of the present pharmaceutical compositions may be via any of the accepted modes of administration for agents serving similar utilities including, but not limited to, orally, subcutaneously, intravenously, intranasally, topically, transdermally, transmucosally, intraperitoneally, intramuscularly, intrapulmonarily, vaginally, rectally or intraocularly. Transmucosal, topical and parenteral administrations, for example, are common in the treatment of indications of S. aureus infection.

In the pharmaceutical compositions, effective concentrations of one or more digestive enzymes are admixed with a suitable pharmaceutical excipient or carrier. The concentrations of the digestive enzymes in the compositions are effective for administering an amount, upon administration, that is useful in reducing or eradicating S. aureus bacteria and/or treating or ameliorating one or more of the symptoms associated with S. aureus infection.

Antibiotic compositions may be formulated for single dosage administration. To formulate a composition, a weight fraction of digestive enzymes is dissolved, suspended, dispersed or otherwise mixed in a selected carrier in an effective concentration so that bacteria are reduced or eradicated, the condition treated is alleviated or one or more symptoms are improved.

Digestive enzymes are included in the acceptable carrier farms16/75 ceütfcamerite in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects in the patient treated. The therapeutically effective concentration can be determined primarily by in vitro and in vitro digestive enzyme testing and then extrapolated from there to human dosages.

The concentration of digestive enzymes in the pharmaceutical composition will depend on the absorption, inactivation and excretion rates of the enzymes, the physicochemical characteristics of the enzymes, the dosage schedule, the dosage form and the quantity administered as well as 10 other factors known to those skilled in the art.

The pharmaceutical composition may be administered all at once or may be divided into several smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment are a function of the disease being treated and may be determined empirically using known test protocols or through extrapolation of in vitro or in vitro test data. It should be noted that concentration and dosage values ​​may also vary with the severity of the condition to be alleviated. It should further be understood that for any particular individual, specific dosage regimens should be adjusted over time according to individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges shown herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

When digestive enzymes are mixed or added, the resulting mixture may be a solution, suspension, gel, powder, emulsion or similar. The form of the resulting mixture depends on several factors, including the intended mode of administration and the solubility of the digestive enzymes in the carrier or vehicle selected.

Compositions intended for pharmaceutical use may be administered as crystalline or amorphous products. Pharmaceutically acceptable compositions include solid, semi-solid,

17/75 liquid, gel, powder and aerosol forms such as, for example, tablets, capsules, lozenges, sprays, powders, liquids, suspensions, emulsions, gels, suppositories, aerosols or the like. They can be obtained, for example, as films by methods such as precipitation, crystallization, freeze-drying, spray-drying or evaporation-drying. Microwave or radiofrequency drying may be used for this purpose. The compositions may also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, pills, specialized coatings (e.g., enteric coatings), in oral dosage forms, transdermal patches (including ethertransport), and the like, for prolonged and/or timed, pulsed administration at a predetermined rate. In some embodiments, the compositions are provided in unitary dosage forms suitable for single administration of a precise dose.

The compositions may be administered either alone or more typically in combination with a pharmaceutically acceptable carrier, excipient or the like. The term “excipient” is used herein to describe any ingredient other than the compound(s) (enzymes) used in the composition. Pharmaceutically acceptable excipients include, but are not limited to, iodine exchangers, alumina, alumina stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as di-tocopheryl polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins such as serum albumin, buffering substances such as phosphates, glycine, ascorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicon, pyrrolidone polyvinyl, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylenepolypropylene block polymers and wool grease. Cyclodextrins such as ο-, β- and γ-cyclodextrin

18/75 or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2 ©3 hydroxypropyl-b-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance the delivery of compositions described herein. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art; for example, see The Science and Practice of Pharmacy, 2i³ Edition (Lippincott Williams & Wilkins, 2005).

In a preferred embodiment, the compositions will take the form of a unit dosage form such as a pill or tablet and then the composition may contain, together with the active ingredient, a diluent such as lactose, sucrose, dicalcium phosphate or the like; a lubricant such as magnesium stearate or the like and a binder such as starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose, cellulose derivatives or the like. In another solid dosage form, a powder, solution or suspension (for example, in propylene carbonate, vegetable oils or triglycerides) is encapsulated in a gelatin capsule. Unit dosage forms where two or more ingredients are physically separated are also included, for example, capsules with granules of enzyme(s) and granules of other ingredients; two-layer tablets; two-chamber gel capsules, etc.

Liquid pharmaceutically administrable compositions may, for example, be prepared by dissolving, dispersing, etc., one or more digestive enzymes and optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like) to form a solution or suspension. If desired, the pharmaceutical composition may also contain small amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g. sodium acetate, sodium citrate, cyclodextrin derivatives, sarbitan monolaurate; triethanolamine acetate; triethanolamine tea and the like); Injectables can be prepared in conventional ways, or as liquid solutions or suspensions, such as emulsions.

19/75 sions or in solid forms suitable for dissolution of the suspension in liquid prior to injection.

Solid compositions may be provided in a variety of different dosage forms, depending on the physicochemical properties of the rhizomes, the desired dissolution rate, cost considerations, and other criteria. In one embodiment, the solid composition is a single unit. This implies that a unit dose of the drug is comprised in a single, physically shaped solid form or article. In other words, the solid composition is coherent, which is in contrast to a multiple unit dosage form where the units are incoherent.

Examples of single units that can be used as dosage forms for the solid composition include tablets, such as compressed tablets, film-type units, sheet-type units, wafers, lyophilized matrix units, and the like. In one embodiment, the solid composition is a highly porous lyophilized form. Such lyophilizates, sometimes also called freeze-dried wafers or tablets, are particularly useful for their rapid disintegration, which also allows for rapid dissolution of the active compound.

On the other hand, for some applications the solid composition 20 may also be formed as a multiple unit dosage form. Examples of multiple units are powders, dusts, granules, microparticles, microcapsules, pellets, beads, freeze-dried powders, and the like. In one embodiment, the solid composition is a freeze-dried powder. Such a dispersed freeze-dried system comprises a multiplicity of powder particles, and due to the freeze-drying process used in the formation of the powder, each particle has an irregular, porous microstructure through which the powder is able to absorb water very rapidly, resulting in rapid dissolution. In another embodiment the solid composition is a spray formulation_s

Another type of multiparticulate system that is also capable of achieving rapid drug dissolution is that of powders, granules or pellets of water-soluble excipients that are coated with the ingredients of the composition (e.g., enzymes), so that the enzymes are located

20/75 coated onto the outer surface of the individual particles. In this type of system, a water-soluble low molecular weight excipient is useful for preparing the cores of such coated particles, which may subsequently be coated with a coating composition comprising the enzyme(s) and, preferably, one or more additional excipients. such as a binder, a pore former, a saccharide, a sugar alcohol, a film-forming polymer, a plasticizer or other excipients used in pharmaceutical coating compositions.

Appropriate dosages for treatment or prevention of S. aureus infections will depend on the patient (species, age, weight, health), the severity of the disease, the strain of S. aureus present, the type of formulation (e.g., liquid or ointment), and other factors known to those of ordinary skill in the art. It should be noted that concentrations and dosage values ​​may vary with the severity of the condition to be alleviated. It should further be understood that for any particular patient, specific dosage regimens must be adjusted over time according to individual need and the professional judgment of the person administering or supervising the administration of the compositions.

In some embodiments, the pharmaceutical composition comprises per dose: amylases of from about 10,000 to about 60,000 USP, including 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, and 60,000 LLS.P. together with all values ​​between them, proteases from about 10,000 to about 70,000 USP, including 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, and 70,000, together with all values ​​between them, and lipases from about 4,000 to about 30,000 USP, including 4,000, 5,000. 10,000, 15,000, 20,000, 25,000 and 30,000, along with all the values ​​in between. A pharmaceutical composition may include one or more of: chymotrypsin of from about 2 to about 5 mg and including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mg^ together with all values ​​in between; trypsin of from about ©Õ to about 100 mg including 50, 65, 70, 75, 80, 85, 90, 95 and 100 mg including all values ​​in between;

/75 papain from about 3,000 to about 10,000 USP units including 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, and 10,000 USP, along with all values ​​in between; and papaya from about 30 to about 60 mg, including 30, 35,40, 45, 50, 55, and 60 mg, along with all 5 values ​​in between.

Additional information on particular dosage forms of the compositions is provided below.

1. Compositions for oral administration

Oral pharmaceutical dosage forms are solid, gel or liquid. Solid dosage forms are tablets, capsules, granules & raw powders. Types of oral tablets include chewable tablets, compressed tablets, and lozenges, which may be enteric coated, sugar coated or film coated. Capsules may be hard capsules or powders, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art,

a. Solid compositions for oral administration

In certain embodiments, the formulations are solid dosage forms, in one embodiment, capsules or tablets. The tablets, pills, capsules, troches and the like can contain one or more of the following ingredients or compounds of a similar nature: a binder; a lubricant; a diluent; a glidant; a disintegrating agent; a coloring agent; a sweetening agent; a flavoring agent; a wetting agent; an emetic coating, and a film coating.

Examples of binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polyvinylpyrrolidone, povidone, crospovidone, sucrose, and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium, and stearic acid. Oily fluids include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide, disintegrating agents include crascarmellose sodium, sodium starch glycolate, alginic acid, sodium starch glycolate,

22/75 corn, potato starch, bentonite, methyl cellulose, agar and carboxymethyl cellulose. Colouring agents include, for example, any of the approved certified water-soluble FD and C dyes, mixtures thereof, and water-insoluble FD and Ç dyes suspended in alumina hydrate.

Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin and any number of spray-dried flavorings. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic mixtures of compounds that produce a pleasant sensation, such as, but not limited to, peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monolaurate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Emetic coatings include fatty acids, fats, waxes, shellac, shellac adhesive, and cellulose acetate phthalate. Film coatings include hydroxyethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

The digestive enzymes could be provided in a composition that protects them from the acidic environment of the stomach. For example, the composition could be formulated in an enteric coating that maintains its integrity in the stomach and releases the digestive enzymes in the intestine. The composition could also be formulated in combination with an antacid or other similar ingredient.

When the dosage unit form is a capsule, it may contain, in addition to material of the above type, a liquid carrier such as a fatty oil. Further, dosage unit forms may contain various other materials which modify the physical form of the dosage unit, for example, sugar coatings and other enteric agents. Digestive enzymes may also be administered as a component of an elixir, suspension, syrup, wafer, spray, chewable gum or the like. A syrup may contain, in addition to active digestive enzymes, 30 sucrose as a sweetening agent and certain preservatives, dyes and colors and flavors.

Digestive enzymes can also be mixed together

23/75 other active materials that do not impair the desired action or with materials that supplement the desired action, such as antacids, H2 blockers and diuretics. Higher concentrations, up to about 98% by weight of digestive enzymes, may be included.

In all embodiments, tablet and capsule formulations may be coated as known to those skilled in the art in order to modify or sustain the dissolution of digestive enzymes. Thus, for example, they may be coated with a conventional enteric-digestible coating, such as phenylsalicylate, ceres 10, and cellulose acetate phthalate.

b. Liquid compositions for oral administration

Liquid oral dosage forms include solutions, emulsions, aqueous suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, e.g. sucrose, and may contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed as small globules in another liquid. Pharmaceutical carriers used in emulsions include non-aqueous liquids, emulsifying agents, and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-effervescent granules to be reconstituted into a liquid oral dosage form include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable substances used in effervescent granules to be reconstituted into a liquid oral dosage form include organic acids and a carbon dioxide source. Coloring and flavoring agents are used in all dosage forms: above.

Solvents include glycerin, sorbitol, ethylene glycol and syrup.

24/75

Examples of preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate, and alcohol. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate.

Suspending agents include sodium carboxymethyl cellulose, pectin, tragacanth, Veegum and acacia. Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric and tartaric acids. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Colouring agents include any of the approved certified FD and C water-soluble dyes and mixtures thereof. Flavouring agents include natural flavours extracted from plants such as fruits and synthetic mixtures of compounds that produce a pleasant taste sensation.

For a solid dosage form, the solution or suspension, in, for example, propylene carbonate, vegetable oils or triglycerides, is in one embodiment encapsulated in a gelatin capsule. Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Patent Nos. 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, the solution, e.g., in a polyethylene glycol, may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be readily metered for administration.

Alternatively, liquid or semisolid oral formulations can be prepared by dissolving or dispersing the digestive enzymes in vegetable oils, glycols, triglycerides, propytene glycol esters (e.g., propytene carbonate), and other such carriers, and encasing these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those shown in U.S. Pat. Nos. RE23,819 and 4,358,603. In summary, such formulations

25/75 include, but are not limited to, those containing digestive enzymes provided herein, a mono- or dialkylated polyalkylene glycol, including, but not limited to, 1,2-dimethoxymethane, diglyman, triglyman, tetraglyman, polyethylene glycol-350-dimethyl ether, polyethylene glycol-SSO-dimethyl ether, polyethylene glycol-750-dimethyl ether where 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol and one or more antioxidants, such as butylated hydroxytoluene (NHT), butylated hydroxyanisole (BHA), propylene glycol, vitamin E, hydroquinone, hydroxycoumarin, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid 10 and its esters and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholic solutions including a pharmaceutically acceptable acetal. Alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to, di(lower alkyl)acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.

2. Injectable solutions and emulsions

Parenteral administration, in a form characterized by injection, either subcutaneously, intramuscularly or intravenously, is also encompassed herein. Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Injectable solutions and emulsions may also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. Furthermore, if desired, the pharmaceutical compositions to be administered may also contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents,_: stabilizers, solubility enhancers and other such agents, such as, for example, sodium acetate, sorbitan monooleate, triethanolamine oleate and ethylhexyldextrins,

26/75

Implantation of a tentative or sustained release system, such that a constant dosage level is maintained (see, e.g., U.S. Patent No. 3,710,795), is also encompassed herein. In short, digestive enzymes provided herein are dispersed in a solid matrix, e.g., polymethyl methacrylate, polybutyl methacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubbers, polymethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as acrylic and methacrylic acid ester hydrogels, Golagen, cross-linked polyvinyl alcohol and cross-linked partially hydrophilic polyvinyl acetate, which are surrounded by an outer polymer membrane, e.g., polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polymethyl siloxanes, neoprene rubber, golden polyethylene, polyvinyl chloride, vinyl chloride-vinyl acetate copolymers, vinylidene chloride, ethylene and propylene, polyethylene terephthalate ionomer, butyl rubber, epoxy hydride rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer and ethylene/vinyloxyethanol copolymer, which is insoluble in body fluids. The digestive enzymes diffuse through the outer polymeric membrane in a release rate-controlling step. The percentage of digestive enzymes contained in such parenteral compositions is highly dependent on their specific nature, as well as the activity of the digestive enzyme or mixture thereof and the needs of the individual.

Parenteral administration of the compositions includes intravenous, subcutaneous and intramuscular administrations. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent shortly before use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle or powder before use, and sterile emulsions. The solutions may be either aqueous or non-aqueous.

If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose, and Lactated Ringer's Injection. Nonaqueous parenteral vehicles include fixed vegetable oils, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations should be added to parenteral preparations packaged in multiple-dose containers and include phenols and cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, beriziaconium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethyl cellulose, hydroxypropylmethyl cellulose, and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). Metal chelating or sequestering agents include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

28/75

Single-dose parenteral preparations are packaged in a blister, vial or syringe with a needle. All preparations for parenteral administration must be sterile, as is known and practiced in the art.

Intravenous or intraarterial infusion of a sterile aqueous solution containing digestive enzymes is an effective mode of administration. Another modality is a sterile aqueous or oily solution or suspension containing digestive enzymes that can be injected as needed to produce the desired pharmacologic effect.

ü Injectables are designed for local and systemic administration.

In one embodiment, a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1% w/w to about 90% w/w or greater, in certain instances greater than 1% w/w, of the digestive enzymes for the liver tissue(s).

The digestive enzymes may be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product. The form of the resulting mixture depends on several factors, including the intended mode of administration and the solubility of the digestive enzymes in the carrier or vehicle selected. The effective concentration is sufficient to ameliorate the symptoms of the condition and can be determined empirically.

3. Freeze-dried powders

Of interest here are also lyophilized powders, which can be reconstituted for administration as solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels.

The sterile, lyophilized powder is prepared by dissolving digestive enzymes as provided herein in a suitable solvent. The solvent may contain a stability-enhancing excipient or other macrologic component of the powder or a prepared solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other

29/75 suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other buffer known to those of skill in the art, at: one embodiment, pH around neutral. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution will be divided into vials for lyophilization. Each vial will contain a single dosage or multiple dosages of the digestive enzymes. The freeze-dried powder can be stored under appropriate conditions, such as in about 4^andC to room temperature.

Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends on the digestive enzymes selected. Such amount can be determined empirically.

4, Topical administration

Topical admixtures may be prepared as described for local and systemic administration. The resulting mixture may be a solution, suspension*, emulsion or the like and are formulated as creams*, gels, ointments, emulsions, powders, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigants, sprays, suppositories, bandages, dermal patches or any formulation suitable for topical administration.

Digestive enzymes can be formulated as aerosols for topical applications, such as by inhalation (See, e.g., 25 U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for administering a steroid useful for treating inflammatory diseases, particularly asthma). Such formulations for administration to the respiratory tract may be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will, in one embodiment, have diameters of less than 50 microns, in another embodiment less than 100 microns, or in another embodiment less than 100 microns.

30/75 microns.

Digestive enzymes may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as the eye, in the form of gels, creams and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration includes transdermal administration and also for administration to the eye or mucosa or for inhalation therapies. Nasal solutions of digestive enzymes alone or in combination with other pharmaceutically acceptable excipients may also be administered.

These solutions, particularly those intended for ophthalmic use, may be formulated as isotonic solutions 0_:,Ó1%-1Ô%, pH about 5-7, with appropriate salts.

Powders may be formed with the aid of any suitable powder base, for example, talc, lactose* starch and the like. Solutions may be formulated with an aqueous or non-aqueous base and may include one or more dispersing agents, suspending agents, selubilizing agents and the like. Topical gels are prepared using polymers having a molecular weight and concentration level effective to form a viscous solution or colloidal gel from an aqueous or nonaqueous solution or suspension of digestive enzymes. Polymers from which topical gels can be prepared include polyphosphoesters, polyethylene glycols, high molecular weight poly(lactic) acids, hydroxypropyl cellulose, chitosan, polystyrene sulfonates and the like.

Ointments* creams and lotions are formulated, for example, with an aqueous or oily base and the addition of a suitable thickening agent, gelling agent, stabilizing agent, emulsifying agent, dispersing agent, suspending agent or consistency-regulating agent or the like. Bases include water, an alcohol or an oil such as liquid paraffin, mineral oil or vegetable oil such as peanut or castor oil. Thickening agents that can be used depending on the nature of the base include soft paraffin, stearate,

31/75 aluminium, cetostearyl alcohol, propylene glycol, polyethylene glycols, polyphosphoesters, poly(lactic) acids, hydroxyethyl cellulose, hydroxypropyl cellulose, cellulose gums, acrylate polymers, hydrophilic gelling agents, chitosan, polystyrene sulfonate, petrolatum, wool fat, hydrogenated lanolin, beeswax and similar.

Ointments, pastes, creams, gels and lotions may also contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, zinc oxide and mixtures thereof. Powders and sprays may also contain excipients such as silicic acid, aluminium hydroxide, calcium silicates and polyamide powder or mixtures of these substances. Solutions, suspensions or dispersions may be converted to aerosols or sprays by any of the known means routinely used for the manufacture of aerosols for topical application. In general, such methods comprise pressurizing or providing a pressurizing means to a container of a solution, suspension or dispersion, usually with an inert carrier gas, and passing the pressurized gas through a small orifice. Sprays and aerosols may contain common preservatives, for example, chlorofluorohydrocarbons or unsubstituted volatile hydrocarbons such as butane and propane.

Excipients include compounds that promote skin absorption, such as dimethyl sulfoxide (DMSO), partial fatty acid glycerides, and the like, present at levels up to about 10% by weight of the total formulation weight. Examples of partial fatty acid glycerides include, but are not limited to, IMWITOR 742 and IMWITOR 308 available from SASOL North America, Inc. of Houston, Tex. Topical formulations may also optionally include inactive ingredients to enhance cosmetic acceptability, including, but not limited to, humectants, surfactants, fragrances, coloring agents, emollients, fillers, and the like.

Topical compositions may also include other antibiotic agents, examples of which include bacitracin, neomycin, polymyxin, beta32/75 lactams including penicillin, methicillin, moxalactam and cephalosporins such as cefaclor, cefadroxil, cephamandole nafate, cepazolin, cepoxime, cefigenaz, cefazole, cefoperazone, cefazole, cefotanide, cefotaxime, cefotetan, cefoxitin, cefpodoxime proxetil, ceftazidime, ceftizoxime, ceftriaxone, cefriaxone, cefuroxime, cephalexin, cephalosporin C, cephalosporin C sodium salt, cephalothin, cephalothin sodium salt, cephalothin hydrate, cephalothin, cephapirin, cephradine, cephaloxine, loracarbe and the like. Essentially any anti-infective/antibiotic agent that is effective when applied topically may be used. Thus, the methods 10 of the present invention for both treating active infections and decolonizing skin pathogen populations include methods where digestive enzymes are applied singularly or in combination, either without any other anti-infective agent or at least one other anti-infective agent.

Topical compositions may be administered directly by dusting a powder, spraying an aerosol, or by spreading a film of ointment, cream, lotion, solution, or gel onto the desired area of ​​skin using the patient's or a caregiver's fingertips or other application such as a cotton swab or tissue. The product may be first applied to the skin and spread with the fingertips or an applicator or applied to the fingertips and spread onto the skin. The compositions may also optionally be first coated onto the surface of a topical application, such as a moistened bandage, cotton swab, woven or nonwoven tissue, and the like, which is then applied to the portion of skin to receive the composition.

The topical compositions of the present invention may be prepared with base formulations which are essentially conventional to one skilled in the art with respect to the ingredients employed, their amounts, and methods of preparation, all of which require no further description. Topical compositions according to the present invention may also be prepared as a cream or lotion based on an emulsion formulation having as yet no bactericidal activity.

33/75 recognized, in addition to its good skin compatibility and wound healing properties it is particularly well suited for formulation with digestive enzymes.

As discussed above, the present invention is not limited to topical cream or lotion formulations. Topical formulations based on sprays, mists, aerosols, lotions, creams, aqueous or non-aqueous solutions or liquids, oils, gels, ointments, pastes, salves, emulsions and suspensions will contain an amount of digestive enzymes, and optionally one or more other anti-infective agents, in a total concentration of between about 0.125 and about 10% by weight or more, recognizing again that optimum dosages may differ by as little as 0.05% by weight. so that representative cream and lotion embodiments will include each 0.05% by weight concentration increase within this range.

The topical compositions of the present invention are used to treat skin infections and wound infections such as superficial wounds and penetrating wounds. Suitable wounds for treatment include skin abrasions, skin or superficial cuts, pressure sores, burns and surgical wounds. The topical compositions of the present invention can also be used to de-colonize populations of S. 20 aureus to prevent secondary infections, including pretreatment of areas prior to surgery or catheter insertion.

Mucosal administration formulations may include digestive enzymes as described herein combined or co-administered with a suitable carrier or vehicle for mucosal administration. As used herein, the term^t>transporter^,t means a pharmaceutically acceptable solid or liquid filler, diluent or encapsulating material. A liquid carrier containing water may contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and/or viscosity-increasing agents, tunicity agents, wetting agents or other

34/75 biocompatible materials. A tabulation of ingredients listed by the above categories can be found in the US Pharmacopoeia National Formulary, pp. 1857-1859, 1990. Some examples of materials that may serve as pharmaceutically acceptable fillers are sugars such as lactose, glucose, and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository sugars; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propiphen glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; — buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution;

Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Wetting, emollients and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring and perfuming agents, preservatives and antioxidants may also be present in the compositions, according to the formulator's wishes. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite and the like; olefin-soluble antipyretics such as sodium benzoate palmitate, butyl hydroxyanisole (BHA), butyl hydroxytoluene (BHT); lecritin, propyl gallate, alpha-tocopherol and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like. The amount of digestive enzymes that can be combined with the carrier materials to produce a single dosage form will depend on the particular mode of administration.

Mucosal formulations are generally sterile, particle-free, and stable for pharmaceutical use. As used herein, the term “particle-free” means a formulation that meets the requirements of the USP specification for small-volume parenteral solutions. The term “stable” means a formulation that meets all chemical and physical specifications with respect to identity, strength, quality and purity that have been established in accordance with the principles of Good Manufacturing Practice, as determined by appropriate government regulatory bodies.

Within mucosal delivery compositions, various delivery enhancing agents may be employed with increased delivery of digestive enzymes to and across a mucosal surface. As used herein, mucosal delivery enhancing agents include agents that increase release or solubility (e.g., from a formulation delivery vehicle), diffusion rate, penetration capacity and timing, residence time, stability, effective half-life, peak or sustained concentration levels, clearance, and other desired mucosal delivery characteristics (e.g., as measured at the site of administration or at a selected target site of activity such as the bloodstream or central nervous system) of digestive enzymes or other biologically active compound(s). Increased mucosal delivery may then occur through any of a variety of mechanisms, for example, through increased diffusion, transport, persistence or stability of digestive enzymes, increased membrane fluidity, modulation of the availability 25 or action of calcium and other ions that regulate intracellular or paracellular permeation, solubilization of mucosal membrane components (e.g., lipids), changing non-protein and protein sulfhydryl levels in mucosal tissues, increasing water flux across the mucosal surface, modulating epithelial junctional physiology, reducing the viscosity of mucus lining the mucosal epithelium, reducing mucociliary clearance rates, and other mechanisms.

Although the mechanism of absorption promotion may vary

36/75 with intranasal delivery enhancing agents other than those of the invention, reagents useful in this context will not adversely affect subsophageal merit of the mucosal tissue and will be selected according to the physicochemical characteristics of the particular digestive enzymes or other active or delivery enhancing agent. In this context, Delivery enhancing agents that increase the penetration or permeability of mucosal tissues will often result in some alteration of the protective permeability barrier of the mucosa. For such delivery enhancing agents to be of value within the invention, it is generally desired that any significant changes in mucosal permeability be reversible within a time frame appropriate to the desired duration of drug administration. Furthermore, there should be no substantial, cumulative toxicity, nor any permanent detrimental changes induced in the mucosal barrier properties 15 with long-term use.

In some embodiments, absorption promoting agents for coordinated administration or combinatorial formulation with the digestive enzymes as described herein are selected from small hydrophilic molecules, including, but not limited to, dimethyl sulfoxide (DMSO), dimethylformamide, ethenol, propylene glycol, and 2-pyridines. Alternatively, long chain amphiphatic molecules, e.g., desacetylmethyl sulfoxide, azone, sodium lauryl sulfate, pylori acid, and bile salts, can be employed to enhance mucosal penetration of the digestive enzymes. In additional aspects, surfactants (e.g., polysorbates) 25 are employed as adjunctive compounds, processing agents, or formulation additives to enhance intranasal delivery of the digestive enzymes. Penetration-enhancing agents typically interact at either the polar head groups or the hydrophilic tail regions of molecules comprising the lipid bilayer of epithelial cells lining the nasal mucosa (Barry, Pharms Chem. of the Sci., Vol. 1, pp. 121-137; Shropt et al., Eds., Karger, Basel, 1987; Barty, J., Cochrane Retéase 6:85-97, 1987). Interaction at these sites may have the effect of

37775 disruption of the lipid molecule coating, increasing the fluidity of the bilayer, and facilitating the transport of digestive enzymes across the mucosal barrier. Interaction of these penetration enhancers with the polar head groups may also cause or allow the hydrophilic regions of adjacent biolayers to absorb more water and separate, thus opening the paracellular tract for transport of digestive enzymes. In addition to these effects, certain enhancers may have direct effects on the bulk properties of the aqueous regions of the nasal mucosa. Agents such as DMSO, polyethylene glycol and ethanol may, if present in sufficiently high concentrations in the administration environment (e.g., through pre-administration or incorporation into a therapeutic formulation), enter the aqueous phase of the mucosa and alter its solubilization properties, thereby increasing the partitioning of digestive enzymes from the vehicle to the mucosa,

Additional mucosal delivery enhancing agents that are useful within the coordinated delivery and processing methods and combinatorial formulations of the invention include, but are not limited to, mixed micelles; enamines; nitric oxide modulators (e.g., Snrtroso-N-acetyl-DL-pericyclamine, NORI, NOR4, which are preferably coadministered with a NO scavenger such as carboxy-PITO or diclofenac sodium); sodium salicylate; glycerol esters of acetoacetic acid (e.g., glyceny-1,3-diacetylglycerin-3-acetoacetic acid or 1,2-isopropylideneglycerin-3-acetoacetic acid); and other intra- or transepithelial penetration-promoting or delivery agents that are physiologically compatible for mucosal administration. Other absorption-promoting agents are selected from a variety of carriers, bases and excipients that enhance the mucosal delivery, stability, activity or transepithelial penetration of: digestive enzymes. These include, inter alia, cyclodextrin and β-Gyclodextrin derivatives (e.g., 2-hydroxypropyl-β-Gyclodextrin and heptahydroxy-β-methyl-β-Gyclodextrin). These compounds, optionally conjugated with one or more of the active ingredients and optionally further formulated in an oleaginous base, enhance the

38/75 bioavailability in the mucosal formulations of the invention. Additional absorption enhancing agents adapted for mucosal administration include medium chain fatty acids, including mono and diglycerides (e.g., coconut oil sodium caprylate extracts, Capmul) and triglycerides (e.g., amylodextrin, Estraram 299, Miglyol 810).

The mucosal therapeutic and prophylactic compositions may be supplemented with any suitable penetration promoting agent that facilitates the absorption, diffusion or penetration of digestive enzymes across mucosal barriers. The penetration promoter may be any pharmaceutically acceptable promoter. Thus, in more detailed aspects of the invention there are provided compositions incorporating one or more penetration promoting agents selected from sodium salicylate and salicylic acid derivatives (e.g., ethyl salicylate, choline salicylate, salicylamide); amino acids and salts thereof (e.g., monoaminocarboxylic acids such as glycine, alanine, phenylalanine, prophin, hydroxyproline; hydroxyamino acids such as serine; acidic amino acids such as aspartic acid, glutamic acid; and basic amino acids such as lysine — including its alkali metal or alkaline earth metal salts); and N-acetylamino acids (N-acetylalanine, N-acetylphenylalanine, N-acetylserine, N-acetylglycine, N-acetylysine, N-acetylglutamic acid, N-acetylproline, N-acetylhydroxyproline, etc.) and their aliquots (alkali metal salts and alkaline earth metal salts). Substances are also provided as penetration promoting agents within the methods and compositions of the invention that are generally used as emulsifiers (e.g., sodium oleyl phosphate, sodium lauryl phosphate, sodium laureth sulfate, sodium myristyl sulfate, polyoxyethylene alkyl ethers, alkyl: polyoxyethylene esters, etc.), caproic acid, lactic acid, malic acid and citric acid and alkali metal salts thereof, pyrrolidonecarboxylic acid, alkylpyrrolidonecarboxylic acid esters, N-alkylpyrrolidonecarboxylic acids, acyl proline esters and the like.

5. Compositions for other routes of administration

Other routes of administration, such as transdermal patches, including iontophoretic and electrophoretic devices, and administration

39/75 rectal, are also included here.

Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those skilled in the art. For example, such patches are described in U.S. Patent Nos. 6,267,983 , 6,261,595 , 6,256,533 , 6,167,301 , 6,024,975 , 6,010,715 , 5,985,317 , 5,983,132 , 5,948,433 and 5,860x957 .

For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories as used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more formally or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories are bases or carriers and additives to increase the melting point. Examples of bases include cocoa butter (theobroma cacao oil), glycerin, carbowax (polyoxyethylene glycol), and appropriate mixtures of mono-, di-, and triglycerides of fatty acids. Combinations of the various bases may be used. Agents to raise the melting point of suppositories include sperm and wax. Rectal suppositories may be prepared either by the compression method or by milling. The weight of a rectal suppository, in one embodiment, is about 2 to 3 g.

Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as the formulations for oral administration.

6. Sustained Release Formulations

Sustained release formulations are also provided to deliver the digestive enzymes to the desired target. It is understood that the levels of the digestive enzymes are maintained for a period of time as desired and can be readily determined by one skilled in the art. Such sustained release and/or timed release formulations can be made via sustained release delivery devices that are well known to those skilled in the art, such as those described in U.S. Patent Nos. 3,845,770;

40/75

3,916.899; 3.536.809; 3.598,123; 4008.719; 4710.384; 5.674.533;

5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556 and 5,733,566, the descriptive reports of which are incorporated herein by reference. These pharmaceutical compositions can be used to provide slow or sustained release of one or more digestive enzymes using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres or the like. Suitable sustained release formulations known to those skilled in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions provided herein. Thus, single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gelcaps, lozenges, powders and the like, which are adapted for sustained release are encompassed herein.

In one embodiment, the sustained release formulation contains an active compound such as, but not limited to, microcrystalline cellulose, maltodextrins, ethyl cellulose, and magnesium stearate. As described above, all known methods for encapsulation that are compatible with the properties of the digestive enzymes described are encompassed herein. The sustained release formulation is encapsulated by coating particles or granules of the pharmaceutical compositions provided herein with varying thicknesses of slowly soluble polymers or by microencapsulation. In one embodiment, the sustained release formulation is encapsulated with a coating material of varying thickness (e.g., about 1 micron to 200 microns) that allows for dissolution of the pharmaceutical composition about 48 hours to about 72 hours after administration to a mammal. In another embodiment, the coating material is an approved food additive.

In another embodiment, the sustained release formulation is a matrix dissolution device that is prepared by compressing the drug with a highly soluble polymer carrier.

41/75 a tablet. In one embodiment, the coated particles have a size range of between about 0.1 to about 300 microns, as described in U.S. Patent Nos. 4,710,384 and 5,354,556, which are incorporated herein by reference in their entireties. Each of the particles is in the form of a micromatrix, with the active ingredient uniformly distributed throughout the polymer.

The digestive enzymes provided herein may be formulated as a sustained and/or timed release formulation. All sustained release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-sustained release counterparts. Ideally, the use of an optimally designed sustained-release preparation in medical treatment is characterized by a minimum of digestive enzymes being employed to cure or alleviate the condition. Advantages of sustained-release formulations may include: 1) prolonged oral activity, 2) reduced dosage frequency, and 3) increased patient compliance. Furthermore, sustained release formulations may be used to affect the time to onset of action or other characteristics, such as blood levels of the compound, and thus may affect the occurrence of side effects.

The sustained release formulations provided herein are designed to initially release an amount of the therapeutic composition that promptly produces the desired therapeutic effect, and gradually and continuously release further amounts of the composition to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level in the body, the therapeutic composition may be released in dosage form at a rate that will replace the composition being metabolized and excreted from the body.

Sustained release of an active ingredient can be stimulated by various inducers, e.g. pH, temperature, enzymes, water or 30 other physiological conditions or compounds.

Preparations for oral administration may be suitably formulated to give controlled release of digestive enzymes. In a

42/75 embodiment, the digestive enzymes are formulated as controlled release powders of discrete microparticles that can be readily formulated into liquid form. The sustained release powder comprises particles containing an active ingredient and, optionally, an excipient having a molecular weight of at least one non-toxic polymer.

The powder may be dispersed or suspended in a liquid vehicle and will maintain its sustained release characteristics for a useful period of time. Such dispersions or suspensions have both chemical stability and stability in terms of dissolution rate. The powder may contain an excipient comprising a polymer, which may be soluble, insoluble, permeable, impermeable or biodegradable. The polymers may be polymers or copolymers. The polymer may be a natural or synthetic polymer. Natural polymers include polypeptides (e.g., zein), polysaccharides (e.g., cellulose), and alginic acid. Representative synthetic polymers include those described, but are not limited to, in column 3, lines 33-45 of U.S. Pat. No. 5,354,556, which is hereby incorporated by reference in its entirety. Particularly suitable polymers include those described, but are not limited to, in column 3, lines 46, column 4, line 8 of U.S. Pat. No. 5,354,556, which is incorporated herein by reference in its entirety.

The sustained release compositions provided herein may be formulated for parenteral administration, for example, via intramuscular injections or implants into subcutaneous tissues and various body cavities and transdermal devices. In one embodiment, intramuscular injections are formulated as aqueous or oily suspensions. In an aqueous suspension, the sustained release effect is due in part to a reduction in solubility of the digestive enzymes upon complexation or a decrease in the rate of dissolution. A similar approach is used with oil suspensions and solutions, where the release rate of digestive enzymes is determined by separating the digestive enzymes from the oil into the surrounding aqueous medium. Only digestive enzymes that are oil-soluble and have desired separation characteristics are used.

43/75 suitable. Oils that can be used for intramuscular injection include, but are not limited to, sesame, olive, peanut*, corn, almond, soybean, cottonseed, and castor oil.

Gòadmfmstration with other Fàrfiiãcêiitfcás Compositions

The pharmaceutical compositions may be used alone and/or in combination with other therapeutic agent or antibiotic regimens (e.g., anti-S. aureus). For example, a patient may be administered with other therapeutic agents, such as anti-inflammatory drugs or anesthetics, to address other aspects of an S. aureus infection (e.g., pain, tissue damage) or other conditions that the patient may be suffering from. In other embodiments, a patient may be administered a pharmaceutical composition as described herein and one or more additional antibiotics. The one or more additional antibiotics may be effective against S. aureus or other bacteria, or both (e.g., if the patient has multiple infections), and may be in the same or a different form as the present pharmaceutical compositions (e.g., one may be a liquid antibiotic and one may be a topical). The major classes of antibiotics are (1) the β-lactams, including the penicillins, cephalosparins, and monobactams; (2) the aminoglycosides, e.g., gentamicin, tobramycin, netilmicin, and amikacin; (3) the tetracyclines; (4) the sulfonamides and trimethoprim; (5) the fluoroquinolines, e.g., ciprofloxacin, norfloxacin, and ofloxacin; (6) vancomycin; (7) macrolides, which include, e.g., erythromycin, azithromycin, and cyarithromycin; è (8) other antibiotics, for example, polymyxins, chlorophenicol and lincosamides.

In some embodiments* the additional antibiotic can be a beta-lactam antibiotic (e.g., a perinicin or penicillin derivative, a cephalosporin, a monobactam, a penam, a penem, an oxapenam, a carbapenem or a cabapenam, a cefen, a carbacepem, an oxacefen, a monobactam). In some embodiments, the additional antibiotic can be a beta-lactamase inhibitor. In some embodiments, the additional antibiotic can be an aminoglycoside antibiotic.

44/75

In some embodiments, the additional antibiotic is selected from penicillin or penicillin derivative, oxacillin, amoxicillin, nafcillin, cloxacillin, methicillin, temicillin, ampicillin, coamoxicillin, azlpcillin, carbenicillin, tigarcillin, meslocillin, piperacillin, cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefamandole, cefotetan, cefexitine, ceftriaxone, cefotaxime, cefpodoxime, ceftazidime, cefepime, cefpirome, vancomycin, teloplanin, telavancin, bleomycin, ramoplanin, decaplanin, oritavancin and dalbavancin.

An antibiotic composition described herein and an additional antibiotic may be administered separately or in a single dosage form. If separately, they may be administered in any order and relative frequency.

Disinfectants and Sanitizers

Compositions comprising one or more digestive enzymes as described herein may also be used as disinfectants and sanitizers, for example, to disinfect inanimate objects and surfaces in, without limitation, hospital, health care, home and community settings by eradicating, attenuating or reducing S. auraus in such settings. Disinfectants are antimicrobial agents that are applied to non-living objects to destroy microorganisms. Disinfectants should generally be distinguished from antibiotics, which destroy microorganisms within the body, and from antiseptics, which destroy microorganisms in living tissue. Sanitizers are disinfectants that reduce the number of microorganisms to a safe level. One definition of a sanitizer states that a sanitizer must be capable of killing 99.999%, known as a 5 log reduction, of a specific bacterial test population, and do so within 30 seconds. The main difference between a sanitizer and a disinfectant is that at a specified use dilution, a disinfectant must have a greater ability to kill pathogenic bacteria compared to that of a sanitizer.

A disinfectant or sanitizer as described herein may include one or more digestive enzymes, in various embodiments as otherwise described herein, and may optionally include other ingredients.

45/75 active and inactive ingredients, including stabilizers (e.g., enzyme stabilizers), other disinfectants known to those skilled in the art, formulation excipients, colorants, perfumes, etc. A person skilled in the art can select additional active or inactive ingredients to include in a disinfectant. Examples of additional disinfectants include: active chlorine sources (i.e., hypochlorites, doramines, dichloroisocyanurate and trichloroisocyanurate, wet chlorine, chlorine dioxide, etc.); active oxygen sources (peroxides, such as peracetic acid, potassium persulfate, sodium perborate, sodium percarbonate, and urea perhydrate); iodophor iodine solutions (povidone-iodine (povidone-iodine, Betadine), Lugo's solution, tincture of iodine, iodinated nonionic surfactants); concentrated alcohols (mainly ethanol, 1-propanol, also called π-propanol and 2-propanol, also called ispprppanol and mixtures thereof; also, 2-phenoxyethanol and 1- and 2-phenoxypropanols); phenolic substances (such as phenol (also called "carbic acid"), cresols (called TysoF in combination with liquid potassium soaps), halogenated (brominated, golden) phenols, such as hexachlorofen, triclosan, trichlorophenol, tribromophenol, pentachlorophenol, dibromophenol and salts thereof); cationic surfactants, such as some quaternary ammonium cations (such as benzalkonium chloride, cetyl trimethylammonium bromide or chloride, didecyldimethylammonium chloride, cetylpyridinium chloride, benzethonium chloride) and others; non-quaternary compounds, such as chlorhexidine, glucoprostamine, octenidine biohydrochloride; strong oxidants, such as ozone and permanganate solutions; heavy metals and their salts, such as colloidal silver, silver nitrate, mercuric chloride, phenylmercuric salts, copper sulfate and copper oxide-chloride; concentrated strong acids (phosphoric, tritric, sulfuric, amide sulfuric, toluenesulfonic acids) and alkalis (sodium, potassium, calcium hydroxides) such as pH <1 or >13, particularly at elevated temperatures (above 60 °C). In some cases, a disinfectant as described herein will consist essentially of one or more digestive enzymes. In some cases, a disinfectant will consist essentially of one or more digestive enzymes and will not include additional disinfecting agents.

46/75

A disinfectant composition comprising one or more digestive enzymes as described herein may be incorporated with other ingredients to form a variety of disinfectant products including, but not limited to, hand cleaners, mouthwashes, surgical washes, body lotions, hand sanitizing gels and foams, disinfectant wipes, and similar personal care products. Additional types of products include disinfectant foams, creams, mousses and the like, and compositions containing organic and inorganic filler materials, such as emulsions, lotions, creams, pastes and the like. The compositions can be used as a bactericidal cleaner for hard surfaces, e.g., sinks and countertops in: hospitals, food service areas and game processing plants. The disinfectant compositions may also be used as disinfectant mists and disinfectant vapors. The present digestive enzyme compositions may be manufactured as diluted ready-to-use compositions or as concentrates that are diluted prior to use. The various products in which the disinfectants are used may also include fragrances, depending on the nature of the product. For example, a pine or lemon fragrance may be desirable for use in kitchen cleaning wipes because of their appealing association with cleanliness for many consumers. Additionally, gels or sprays may also be scented for similar or other reasons.

In one embodiment, the disinfectant compositions can be used to make disinfectant wipes. A disinfectant wipe can be used to clean a variety of hard and other surfaces, including, for example, human hands and feet, medical instruments and devices, countertops, floors, walls, and windows. Wipes can be made from a variety of fabrics. Fabrics are defined to include fabrics and papers, as well as woven and nonwoven materials. Woven or nonwoven fabrics may be made of suitable materials such as ralen, nylon or cotton and combinations thereof. Examples of nonwoven fabrics are described in U.S. Patent Nos. 3,786,615; 4,395,454; and 4,199,322; which are incorporated herein by reference. The fabrics or papers may be impregnated

47/75 with the disinfectant solution by any method known in the art. The wipes may be packaged in any manner known in the art including individual blister packs or rolled or stacked multi-packs.

In another embodiment, the disinfectant composition comprising one or more digestive enzymes may be formulated into a gel or gelatinous sanitizing composition. In addition to the disinfectant compositions, the gel sanitizers may include a thickening or gelling agent, where “thickening agent” and “gelling agent” are used interchangeably. As used herein, the terms “gel” or “gelatinous” sanitizing compositions refer to a liquid disinfectant substance that may have a viscosity of from about 1,000 centipoise to about 100,000 centipoise or from 2,000 centipoise to 50,000 centipoise in another embodiment, although these ranges are not intended to be limitative. For example, a hand gel may be considerably less viscous than a gel used for industrial cleaning or disinfection purposes. Examples of gelling or thickening agents include, but are not limited to, a natural gum such as guar and guar derivatives, a synthetic polymer, a clay, an oil, a wax, water vera gel, an acrylate homopolymer, an acrylate copolymer, a carbomer, cellulose, a cellulose derivative, algin* an algin derivative, an alcohol C₈-W₂g insoluble in water, carrageenan, fumed silica, mixtures thereof and the like. The gelling agent may be present in the gelatinous sanitizing composition in an amount of from about 0.1% by weight to 50% by weight of the composition gelatinous composition. In another embodiment, the gelling agent is present in an amount of from 0.25% by weight to 10% by weight of the gelatinous composition. The amount of gelling agent may be dependent on a variety of factors including the type of gelling agent and the desired viscosity of the gel. Gel sanitizers can be used for a variety of applications including sanitization of human skin, e.g., gel hand sanitization and hand sanitizers.

48/75 hard surface application. In a particular embodiment, the disinfectant composition may be mixed with natural soap gel to form a disinfectant soap formulation. Such a formulation would be useful for application to burns, skin infections, and other irritations. The soap may act as a thickening agent or may also include another thickening or gelling agent as described above, depending on the desired viscosity of the disinfectant gel.

In another embodiment, a disinfectant composition comprising one or more digestive enzymes may be formulated into a disinfectant foam or foaming composition. Disinfectant foam or foaming compositions include the disinfectant composition and foaming agents. Any foaming agent known in the art may be used depending on the application and desired characteristics of the resulting disinfectant foam. As with the disinfectant composition, the disinfectant foams of the present invention can be used in both human (e.g., hand washing) and industrial applications.

In another embodiment, the disinfectant composition comprising one or more digestive enzymes may be in the form of a disinfectant aerosol or mist. Misting, also referred to as thermal fogging, is the process by which disinfectants are aerosolized. Aerosol particles of the disinfectant are suspended in the air for a period of time in order to disinfect both the air itself and surfaces, including inaccessible parts of a structure such as air vents. The aerosolized disinfectant particles may have a particle size of from about 5 μm to about 200 μm. In another embodiment, the aerosolized particle may have a particle size of from about 20 μm to about 150 μm.

Methods for evaluating the disinfectant ability of a particulate composition are known to those skilled in the art. Typically, the relative effectiveness of a disinfectant can be measured by comparing its disinfectant ability to a known disinfectant. Phenol is a known disinfectant standard, and the corresponding rating system is called the “Tenor Coefficient.” The disinfectant to be tested is compared to a standard microbe, e.g., E. goiter or E. coli. Disinfectants that are more effective than phenol have a coefficient >1. Those that are less effective have a coefficient <1. ​​To calculate the phenol coefficient, the concentration of the test compound at which the compound kills the test organism in 10 minutes, but not in 5 minutes, is divided by the concentration of phenol that kills the organism under the same conditions. The phenol coefficient can be determined in the presence of a standard amount of added organic matter or in the absence of 10 organic matter. A particular phenol coefficient test uses the method

Rideál-Wlaker. The US Department of Agriculture also has a method that gives a US Department of Agriculture coefficient. Other methods are known to those skilled in the art. A disinfectant as described herein may have a coefficient for S. aureus that is > 1, e.g., greater than 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or more. In some cases, the phenol coefficient is in the range of from about 2 to about 20, for example, about 4 to about 10, about 2 to about 6, about 6 to about 12, or about 10 to about 15.

A disinfectant as described herein may have a phenol to E. coli coefficient that is >1, e.g., greater than, 1.05, 1.1, 1.2, 1.3, 1.4,1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 12, 14, 16. 1& In some cases, the phenol coefficient is in the range of from about 2 to about; 20, e.g., about 4 to about 10, about 2 to about 6, 25 about 6 to about 12, or about 10 to about 15.

A disinfectant or sanitizer as described herein may be bacteriocidal and/or bacteriostatic for S. aureus. In some embodiments, a disinfectant or sanitizer as described herein may be bacteriocidal and/or bacteriostatic against MRSA or VRSA, or both.

Detergents

A disinfectant composition comprising one or more digestive enzymes described herein may also be formulated as a

50/75 detergent. A detergent is a material intended to aid in cleaning. A detergent may contain one or more digestive enzymes, as described hereinafter, in a suitable formulation to maintain its disinfecting ability, and may contain optional active or inactive ingredients, for example, enzyme stabilizers, additional disinfectants, bleaches, soaps, surfactants, dyes and perfumes, abrasives, pH modifiers, acids, alkalis or caustic compounds, water softeners, oxidizers, suspending agents, fabric softeners, foaming agents and antifoaming agents, viscosity modifiers, corrosion inhibitors and optical brighteners. A detergent as described herein can be bacteriostatic and/or bacteriocidal for Staphylococcus aureus and in some embodiments can be bacteriostatic and/or bacteriocidal against MRA or VRSA or both.

A detergent composition comprising one or more digestive enzymes, especially those made for use with water, may include additional components such as surfactants to 'cut' (dissolve) grease and moisten surfaces, abrasives for scrubbing, substances to modify pH or to affect performance or stability, acids to remove excess calcium or caustics to break down organic compounds, water softeners to counteract the effect of “hardness” ions, oxidants for bleaching, disinfecting and breaking down organic compounds, non-surface-active materials to keep dirt in suspension, enzymes to digest proteins, fats or carbohydrates in dirt or to modify the feel of the fabric, ingredients that modify the foaming properties of cleaning surfactants, to either stabilize or combat foam, ingredients to increase or decrease the viscosity of the solution or to keep other ingredients in solution, in a detergent supplied as an aqueous solution or gel, ingredients that affect the aesthetic properties of the item to be cleaned, or of the detergent itself before or during use, such as optical brighteners, fabric softeners, dyes and perfumes, ingredients such as corrosion inhibitors to combat damage to equipment with which the detergent is used, ingredients to reduce harm or produce benefits to; the skin, when_p

51/75 Detergent is used by bare hands on inanimate objects or used to clean the skin, and is used to prevent degradation of the other ingredients.

The detergent composition may be in any convenient dry form, for example, a bar, a tablet, a scoop, a granule or a paste. It may also be a liquid detergent.

The digestive enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, for example, a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid or a boric acid derivative, for example, an aromatic borate ester or a phenylboronic acid derivative such as 4-formylphenylboronic acid, and the composition may be formulated as described in, for example, WO 92/19709 and WO 92/19708.

Antiseptics

Various embodiments of compositions comprising one or more digestive enzymes can also be used as antiseptic agents, for example, to reduce, eradicate or attenuate S. aureus on the skin or other living tissue. Antiseptics are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis or putrefaction. They should generally be distinguished from antibiotics, which destroy bacteria within the body, and disinfectants, which destroy microorganisms found on nonliving objects. Some antiseptics are true germicides, capable of killing microbes (bacteriocidal), while others are bacteriostatic and only prevent or inhibit their growth.

The antiseptics described here find particular use in hospital or health care settings, for example, in hand, face, or body wash formulations; as antiseptics for use before and after surgical treatment; and as antiseptics for use in cleaning and treating wounds, such as traumatic or surgical wounds. In the community setting, antiseptics are useful in any setting where

52/75 Community-acquired infections are of concern, e.g., day care settings, large institutions, schools, etc. Antiseptics may also be useful in the home environment in hand, face or body wash formulations, or for wound care.

An antiseptic may include one or more digestive enzymes, in various embodiments as described above, and may optionally include one or more active ingredients; or inactive, such as other antiseptic agents known to those skilled in the art, stabilizers (e.g., enzyme stabilizers), colorants, perfumes, and other excipients. Examples of antiseptic agents to include with one or more digestive enzymes include alcohols (e.g., ethanol, 1- and 2-propanol, or mixtures thereof), quaternary ammonium compounds (benzalkonium chloride, cetyl trimethylammonium bromide, cetylpyridinium chloride, and berizetonium chloride), boric acid, chlorhexidine gluconate, peroxides (e.g. hydrogen peroxide, benzoyl peroxide); iodine and iodophor solutions (e.g. povidone-iodine), octenidine dihydrochloride, phenolic (carbolic acid) and phenolic derivatives, sodium chloride, sodium hypochlorite and calcium hypochlorite.

An antiseptic as described herein may be bacteriocidal and/or bacteriostatic for S. aureus and in some embodiments may be bacteriocidal and/or bacteriostatic for MRSA or VRSA or both,

In one embodiment, an antiseptic composition comprises one or more digestive enzymes and optionally one or more of an anti-inflammatory agent, an analgesic, or an anesthetic.

An anti-inflammatory agent may include steroidal and non-steroidal anti-inflammatory compounds. In one embodiment, the antiseptic composition includes one or more steroids. In one embodiment, the antiseptic compositions may comprise an anti-inflammatory agent that is a non-steroidal anti-inflammatory drug. Non-limiting examples of 30 suitable non-steroidal anti-inflammatory drugs include aspirin (Anacin, Ascriptin, Bayer, Bufferin, Ecotrin, Excedrin), choline and magnesium salicylates (CMT, Trichosal, Trilisate), choline salicylate (Artliropan), celecoxib (Celebrex), didofenac potassium (Cataflam), diclofenac sodium (Voltaren, Voltaren XR), diclofenac sodium with misoprostol (Arthrotec), diflunisal (Dpipbid), etodolac (Lodina, Logins XL), fenoprofen calcium (Nalfene), flurbiprofen (Ansãid), íbuprpfenõ (Advíl, Motrin, Motrin IB, Nuprin), 5 indomethacin (Indõcin, Indocín SR), cétoprofenó (Action, Orudis, Orudis KT, Oruvail), magnesia salicylate (Arthritab, Bayer Select, Doan's Pills, Megan , Mobidin, Mobogesic), mectefenamate sodium (Meclomen), mefenamic acid (Ppnstei), méloxiçam (MObíc), nabumetone (Reiafen), naprpxen (Naprosyn, Naprêlan), nãpróxenô sodium (Ateve, Anàprox), óxaprózín 10 (Daypro) , piroxicam (Feldene),_: rofecoxib (Vioxx), salsalate (Amigesic, Anaflex 750, Dísalcid, Marthritlc, Mono-Gesíc, Salflex, Salsitab), sodium salicylate, sulindace (Clinoril), tolmetin sodium (Toiectin), valdecpxib (Béxtra) or a combination of these.

Non-limiting examples of analgesics include acetylsalicylic acid, codeine, ibuprofen, acetaminophen, or tea tree oil. Non-limiting examples of anesthetics include xylocaine, prilocaine, or benzocaine.

Exemplary methods of testing candidate antiseptic compositions are provided below. One skilled in the art will appreciate that other methods of testing antiseptic compositions are known in the art and are also suitable for testing candidate antiseptic compositions.

In vitro methods of determining the ability of candidate antiseptic compositions to kill or inhibit the growth of mycobacterial cells such as Staphylococcus aureus are well known in the art. In general, these methods involve contacting a culture of the cells of interest with various concentrations of candidate antiseptic compositions and monitoring the growth of the cell culture relative to an untreated control culture. A second control culture comprising cells contacted with a known antimicrobial agent may also be included in such tests, if desired.

For example, the ability of a candidate antiseptic composition to inhibit the growth of microbial cells can be readily assessed.

54/75 determined by measuring the minimum inhibiting concentration (MIC) for the antiseptic composition. The MIC is defined as the lowest concentration that inhibits the growth of the organism to a predetermined degree. For example, an MIC value of wo is defined as the lowest concentration that completely inhibits the growth of the organism, while an MIC value of 5 is defined as the lowest concentration that completely inhibits the growth of the organism.₃q is defined as the lowest concentration that inhibits growth by 90% is a MlC value_Sg is defined as the lowest concentration that inhibits growth by 50%. MIC values ​​are sometimes expressed as ranges, e.g. the MICW for an antiseptic composition may be expressed as the concentration at which no growth is observed or as a range between the concentration at which no growth is observed and the concentration of the dilution that immediately follows.

Antibacterial MICs for candidate antiseptic compositions can be measured using a 15-fold broth macro- or microdilution assay (see Amsterdam, D, (1996) “Susceptibility testing Of antimicrobials in liquid media”, pp. 52-111. In Loman, V., ed. Ant/biOtics in Laboratory Mathematics, 4^the ed. Williams and Wilkins, Baltimore, MD). A standardized antibacterial susceptibility test is provided by the National Committee for Clinical Laboratory Standards (NCCLS) as NCCLS, 2000; document M720 A58.

In the classical broth microdilution method, the candidate antiseptic composition is diluted in culture medium in a sterile, covered 96-well microtiter plate. An overnight culture of a single bacterial colony is diluted in sterile medium so that, 25 minutes after inoculation, each well in the microtiter plate contains an appropriate number of colony forming units (CFU)Anl (typically approximately 5 x 10⁵ CFU/ml). Culture medium only (no bacteria): This is also included as a negative control for each plate and known antibiotics are often included as positive controls. The inoculated microtiter plate is subsequently incubated at an appropriate temperature (e.g. 35°C-37°C for 16-48 hours). The turbidity of each well is then determined

55/75 mined by visual inspection and/or by measuring absorbance or optical density (OD) at 595 nm or 600 nm using a microplate reader and is used as an indication of the degree of bacterial growth,

Antimicrobial effects can also be expressed as the percent inhibition (%) of growth of a given microorganism over a predetermined period of time by treatment with a single concentration of a candidate antiseptic composition. This method provides a rapid method of evaluating the ability of an antiseptic composition to inhibit microbial growth, for example, prior to conducting more in-depth tests such as MIC determinations or in-wo testing.

The ability of any of the present disinfectant, detergent, sanitizing, and antiseptic compositions to kill or inhibit the growth of S. aureus bacteria can be tested using methods well known in the art, including the various methods described above. Methods and protocols for testing compositions against bacteria can be found, for example, in the Official Methods of Analysis of The CMC, 15^theEd., Arlington Virginia, 2291, USA (Assoc/ation of Official Analytical Chemists 20 (AOAC), InCi 1990), Designation* E 1054-91 Tractices for Evaluation Inactivators of Antimicrobial Agents Used in Disinfectant Sanitizer, Antiseptic or Preserved Products' ( American Society for Testing End Material (ASTM), 1991). As is well known in the art, in vitro Time-Kill assessments can be performed using a modification of the methods described in the Draft European Standard, prEH 12054, “Chemical Disinfectants and Antiseptics - Products for Hygienic and Surgical Handrub and Handwash Bactericidal Activity - Test Method and Requirements (1995)”. Additional methods that may be used include the log reduction test, the proliferation test, the AOAG usage dilution test, or the 30 inhibition zone test. Other methods are described in the Examples below.

Kits

Kits are also provided here. Typically, a kit includes a

56/75 or: more compositions as described herein. In certain embodiments, a kit may include one or more delivery systems, e.g., for administering a composition as provided above; and/or directions for use of the kit (e.g., instructions for treating a patient; instructions for disinfecting a surface). In another embodiment, the kit may include a composition as described herein and a label, e.g., a label indicating the amounts to be administered to a patient with an S. aureus or a label indicating how to use the composition as a disinfectant, sanitizer, detergent or antiseptic.

Methods of Use

The pharmaceutical compositions (e.g., antibiotic compositions) described above can be used to treat or prevent Staphylococcus aureus infection in animals, e.g., mammals and birds. In particular, the pharmaceutical compositions can be used to ameliorate one or more symptoms and side effects of such infections and/or reduce or eradicate the Staphylococcus aureus bacteria causing the infection. The pharmaceutical compositions may be in any suitable dosage form as previously described. In certain embodiments, the antibiotic compositions described herein are used to treat wounds or injuries that have become infected, e.g., wounds resulting from trauma or surgery. Such use may reduce healing and promote wound healing in patients having infected wounds. The compositions formulated for pharmaceutical use may also be used prophylactically, for example, as antiseptics. Such compositions find particular use in the prophylactic treatment of surgical incisions and other wounds to prevent infection by Staphylococcus aureus.

The digestive enzymes provided herein can be used to treat a variety of diseases and disorders associated with infection by SA bacteria or where SA bacteria are implicated. Certain embodiments include SA infections associated with medical devices and prostheses, e.g., catheters, grafts, prosthetic heart valves, artificial joints, etc. One to five percent of internal prostheses become infected, which

57/75 genalmeritis requires removal or replacement of the prosthesis. In some embodiments, a composition comprising one or more digestive enzymes may be coated onto the medical device at the time of manufacture of the device or after manufacture but prior to insertion of the device. Infection during hemodialysis is another source of infection. Infection is the second leading cause of death in patients on chronic hemodialysis. Approximately 23% of bacteremias are due to access site infections. Most graft infections are caused by coagulation-positive (SA) and coagulation-negative Sfepriy/ococc. To combat infection, digestive enzymes alone or in combination with an antibiotic may be applied as an ointment or cream to the dialysis site before each hemodialysis procedure.

In another embodiment, the compositions provided herein can be used to treat or prevent nasal and extranasal carriage of SA. Infection with this organism can result in impetigo lesions or infected wounds. SA is also associated with higher rates of infection following cardiac surgery, hemodialysis, orthopedic surgery, and neutropenia, both disease-induced and iatrogenic. Nasal and extranasal carriage of Stephy/ococcus may result in nosocomial outbreaks of the same strain of Sfapbyfococc/ that is colonizing the nasal passage or extranasal site of a patient or hospital employee. Close attention should be paid to eradication of nasal colonization, but treatment results have generally been unsatisfactory. The use of topical antimicrobial substances, such as Bacitracin, Tetradclin or Chlorhexidine, results in the suppression of nasal colonization, as opposed to its eradication.

Digestive enzymes alone or in combination with antibiotics are preferably applied intranasally, formulated for nasal application, or as an ointment, cream, or solution. Application may occur at multiple times until colonization by S. aphy/ococci is reduced or eliminated.

In some embodiments, the compositions provided herein can be used to treat or prevent burn wound infections. Although the occurrence of invasive burn wound infections has been significantly reduced, ongoing infection being the most common cause of morbidity and mortality in extensively burned patients, infection is the predominant determinant of wound closure, incidence of complications and outcome in burn patients. One of the main organisms responsible is SA. Frequent debridement and establishment of an epidermis, or a substitute, such as a graft or skin substitute, are essential for prevention of infection.

Digestive enzymes alone or in combination with other antibiotics and/or anesthetics or anti-inflammatories may be applied to burn wounds as an ointment or cream and/or administered systemically. Topical application may prevent systemic infection following superficial colonization or eradicate superficial infection. Application to the skin may be done once daily or as often as dressing changes. Systemic administration could be through intravenous, intramuscular or subcutaneous injections or infusions. Other routes of administration could also be used.

Surgical wounds, especially those associated with foreign material, e.g., sutures, can also be treated with the 20 compositions provided here. 71% of all nosocomial infections occur in surgical patients, 40% of which are operative site infections. Despite efforts to prevent infection, it is estimated that between 500,000 and 920,000 surgical wound infections complicate the approximately 23 million surgical procedures performed annually in the United States. The infecting organisms are varied, but Staphylococci are important organisms in these infections.

Digestive enzymes alone or with an antibiotic, anesthetic or anti-inflammatory may be applied as an ointment, cream or liquid to the wound site or as a liquid to the wound prior to and during wound closure. Following closure, a composition comprising one or more digestive enzymes could be applied at dressing changes. For wounds that are infected, the composition could be

59/75 applied topically and/or systemically.

In some cases, nosocomial pneumonia can be treated or prevented using the digestive enzymes provided here. Nosocomial pneumonia accounts for nearly 20% of all nosocomial infections; Patients at greatest risk of developing nosocomial pneumonia are those in intensive care units, patients with altered levels of consciousness, elderly patients, patients with chronic lung disease, ventilated patients, smokers, and postoperative patients. In a severely compromised patient, multiantibiotic-resistant nosocomial pathogens are likely the cause of pneumonia,

One of the main organisms responsible for this infection is SA. Digestive enzymes alone or in combination with other antibiotics could be administered orally, by aerosolization, or systemically to treat pneumonia. Administration could be once daily or multiple administrations per day. In some embodiments, compositions could be administered directly to the lung by inhalation or through placement of an endotracheal tube.

Cystic fibrosis (CF) (Cysfic F/bros/s) is the most common genetic disorder in the Caucasian population. Pulmonary disease is the 20th most common cause of premature death in patients with cystic fibrosis. Optimal antimicrobial therapy for CF is not known, and it is generally believed that the introduction of improved antipseudomonal antibiotics has been the major contributing factor to the increased life expectancy for patients with CF. One of the most common organisms associated with pulmonary disease25 is CF.

The digestive enzyme alone or in combination with other antibiotics may be given orally, systemically, or by aerosol to treat cystic fibrosis. Preferably, treatment is given for up to 3 weeks during acute lung disease and/or for up to 2 to 30 weeks every 2–6 months to prevent acute exacerbations.

Infective endocarditis results from infection of the cusps of the cardiac valve, although any part of the endocardium or any protective material

60775 tic inserted in the heart may be involved. It is usually fatal if left untreated. Most infections are of nosocomial origin, caused by pathogens that are highly resistant to available drugs. One of the main organisms responsible is SA.

Digestive enzymes alone or in combination with other antibiotics may be given orally or systemically to treat endocardiitis, although systemic administration is preferred. Treatment is preferably 2–6 weeks in duration and may be given as a continuous infusion or multiple administrations during the day.

In early acute onset of osteomyelitis the vascular supply to the bone is compromised by infection only extending into its surrounding tissue. Within this neurotic and ischemic tissue, bacteria may be difficult to eradicate even after an intense host response, surgery, and/or antibiotic therapy. The principal organisms responsible are SA and E. coli.

Digestive enzymes could be administered systemically alone or in combination with other antibiotics. Treatment could be 2-6 weeks in duration. The antibiotic could be given as a continuous infusion or multiple administrations throughout the day. A composition comprising one or more digestive enzymes could be used as an antibiotic-impregnated cement or as antibiotic-coated beads for joint replacement procedures.

Treatment or prevention of sepsis in immunocompromised guests is also provided. Treatment of infections in patients who are immunocompromised due to chemotherapy-induced granulocytopenia and immunosuppression related to organ or bone marrow transplantation represents a significant challenge. The neutropenic patient is especially susceptible to bacterial infection, so antibiotic therapy should be initiated promptly to cover likely pathogens if infection is suspected. Organisms likely to cause infections in granulocytopenic patients are: SA and E. cofe

The composition of digestive enzyme alone or with an antibotic

61/75 co is preferably administered either systemically or pharmatically for 2-6 weeks duration. Digestive enzymes could be given as a continuous infusion or by multiple administrations during the day.

Disinfectant, sanitizing and detergent compositions containing the compounds described herein may be applied to non-living surfaces in appropriate amounts and in a manner to reduce or eradicate S. aureus on such surfaces, and may thereby reduce or prevent transmission and/or infection of S. aureus. Concentrations, timing and frequency of treatment are parameters that can be determined by one of ordinary skill in the art.

Any surface can be disinfected with the compositions described, including a variety of medical devices used in the hospital and healthcare environment. As used herein, “medical device” refers to any device for use in or on a patient, such as an implant or prosthesis. Such devices include, without limitation, synthetic vascular grafts, blood monitoring devices, artificial heart valves, scalpel, knife, scissors, spatula, dilator, clamp, forceps, speculum, retractor, suture, valve, surgical mesh, chisel, drill, level, file, saw, splint, caliper, clamp, forceps, hook, lancet, needle, cannula, curette, depressor, dilator, elevator, articulator, extractor, probe, clamp, catheter, stent, tubing, bowl, tray, sponge, cord, spoon, syringe, passport, screw, plate and pin.

Other community, hospital, and healthcare surfaces suspected of carrying S. atirens can be disinfected, including large and small surfaces (floors, tables, changing tables, beds, ventilation systems, pipes, door handles, counters, food service surfaces, etc.). The compositions may also find use in hand or body washes, for example, at entry points to community settings, hospital rooms, or restrooms.

The compositions provided herein can be used as common disinfectants or in any situation where microorganisms are undesirable. For example, they can be used as surface disinfectants, in coatings for medical devices, in coatings for clothing, such as to inhibit the growth of bacteria or repel mosquitoes, in filters for air purification, such as in aircraft or in community or hospital environments, in water purification systems.

as constituents of shampoos and soups, as food preservatives, cosmetic preservatives, media preservatives, in herbicides or insecticides, as constituents of building materials, such as in silicone sealant, and in animal product processing, such as in the curing of animal hides or in slaughterhouses.

For these purposes, digestive enzymes are typically included in the compositions alone or in conjunction with disinfectants or detergents and applied with a suitable applicator. They may also be incorporated or impregnated into the material during manufacture, such as for an air filter, or applied to the material or object.

For example, in some embodiments, a composition described herein may be mixed with the material, for example, during the manufacture of the material or at a subsequent time. Further, a composition may be applied to the surface of a material, either during manufacture or at a subsequent time. As used herein, the term “suitable material** means any material on, in or into which digestive enzymes can be applied or incorporated, thereby incorporating an antimicrobial activity into/on the material. For example, a gauze pad or bandage may be manufactured with a composition comprising one or more digestive enzymes in or on the gauze and/or an ointment comprising one or more digestive enzymes may be applied to the gauze in this manner incorporating antimicrobial activity into the gauze. Examples of suitable materials where digestive enzymes may be used include, but are not limited to: food, liquid, a medical device (e.g., surgical instruments), a bead, a film, a monofilament, a nonwoven fabric, a sponge, clothing, a knitted fabric, a short fiber, a tube, a hollow fiber, an artificial organ, a catheter, a suture, a membrane, a bandage, and gauze. Enzymes

63/75 digestive but can be applied or mixed into various other types of materials that are suitable for use in medical, health, food safety or environmental cleaning activities.

Veterinary App/reactions

The compositions described herein, in pharmaceutical or disinfectant/sanitizer, detergent or antiseptic formats, may also be used in a variety of veterinary applications. For example, many mammals, including dogs, cats and cows, may be infected with S. aureus or act as carriers of the bacteria. Mastitis in cows is often caused by S. aureus infections. Thus, the present compositions can be used to treat animals infected with or suspected of carrying S. aumus in order to treat the infection or prevent transmission to other animals, including humans. Disinfectant and detergent compositions can be used to treat areas where animals live and equipment that comes into contact with animals, while antiseptic and antibiotic formulations can be used to treat animals to prevent or treat infection.

For example; the digestive enzymes provided herein may be used for the prevention and treatment of mastitis, particularly mastitis in dairy cattle, although any mastitis may be treated using the digestive enzymes provided herein. Mastitis in dairy cattle is an inflammation of the mammary gland in response to intramammary bacterial infection, mechanical trauma or chemical trauma. It is believed that contagious mastitis is primarily caused by Streptococcal aga/actiae. Ambient mastitis can be caused by a variety of different bacteria including, but not limited to, K. pneumoniae, E. coli, K. ephedra, Ent. aeragenes, Streptococcal uberis, Streptococcal bow's, and Streptococcal dysgalactia.

In some embodiments, prevention of bovine mastitis may include dripping onto the teats daily a solution comprising one or more digestive enzymes. In some embodiments, the solution comprising one or more digestive enzymes may include adding one or more

64/75 Additional antibiotics. When infection does occur, intramammary infusion of the urn or more digestive enzymes may be implemented. As above, additional antibiotics may also be administered in conjunction with the digestive enzymes. Typically, digestive enzymes are administered via intramammary injection; however, effective dosages may be administered parenterally, percutaneously, via implant, and also via imbibition. In some embodiments, bovine mastitis can be treated by administering an effective amount of one or more digestive enzymes to a cow. The administration can be prophylactic administration, where all cattle in the herd are treated with a digestive enzyme composition, or administration can occur when infection occurs in individual cows.

Introduction of SA bacteria and SA bacteria may occur during the preparation of beef, poultry, fish, and pork products. Thus, in some embodiments, infection reduction may be provided by administering one or more digestive enzymes to an animal (e.g., cow, chicken, turkey, fish, or pork) to reduce the presence of SA or SA bacteria in the intestines of the animal. Administration may occur by any suitable method including injection and by introducing one or more digestive enzymes into the feed.

In some embodiments, administration of one or more digestive enzymes to an animal can be used to prevent or reduce transmission of SÀ or E. coli bacteria from the animal to other animals or humans. Administration of the digestive enzymes can be accomplished by any available method known in the art. For example, prevention or reduction of transmission of SA bacteria from pigs to humans can be accomplished by administering one or more digestive enzymes to a pig to reduce the number of SA bacteria present in the pig, thereby reducing the transmission of SA bacteria to a human.

Applications in Affment

Also provided herein is a method of preventing infection by SA or £ coif of beef, poultry, fish and pork. Meat processing

65/75 of beef is a hot spot: during the slaughtering process, intestines or fecal matter on the skin could mix with the meat, allowing bacteria to grow in the warm, moist conditions. If the infected parts are then ground, the bacteria will move from the cut surface into the ground meat. Furthermore,_: In ground beef production, meat from multiple cattle is often ground together, allowing contamination from a single animal to infect an entire batch of ground beef. Thus, in some embodiments, infection reduction can be provided by administering one or more digestive enzymes to a cow to reduce the presence of bacteria in the cow's intestines. Administration may occur by any available method including injection and by introducing one or more digestive enzymes into the feed. In another embodiment, reduction of meat metabolization during slaughter and grinding may be provided through the use of sprays containing one or more digestive enzymes as provided herein.

Such sprays can be used, for example, for disinfecting slaughtering and grinding instruments or for disinfecting ground meat itself. The methods described above can also be used during the slaughtering and preparation of poultry, fish and pork products (for example, by administering one or more digestive enzymes to poultry, fish and/or pigs prior to slaughter).

E. coli and SA bacteria can also be spread through unwashed fruits and vegetables. Accordingly, also provided herein is a method of washing fresh fruits and vegetables using a solution, wash, spray, mist, gel or powder comprising one or more digestive enzymes as provided herein. A produce wash is a solution used to bathe the surface of the produce and is typically in contact with the produce for from about 30 s to about 5 min. A product rinse is a solution in which product items are immersed for a time of from about 30 s to about 30 min. However, the times and solutions may be used interchangeably unless otherwise noted. It is understood that the temperature at which the product is rinsed

66/75 The amount of time a duct is washed or rinsed will influence the length of time required to reduce or inactivate the bacteria in it, with higher temperatures leading to a shorter time required for treatment.

The washing or rinsing solutions described herein may be used to reduce bacterial numbers, especially bacterial pathogen numbers, on the surface of fruits, vegetables, cut meat products, fish, shellfish, in the consumer environment (at home), in a commercial food preparation environment, on fruits and/or vegetables prior to juice production, by wholesale and retail operators and/or at the harvest site, meat packing plant or slaughterhouse, fishing boat, etc. without limitation. The present methods are particularly useful for inactivating E. coli on the surfaces of fresh fruits and vegetables.

Methods for Ariwdade Assessment

The compositions described herein can be evaluated for a variety of activities by methods known to those skilled in the art. For example, enzymatic activities can be evaluated using standard enzyme assays. Minimum Inhibitory Concentrations (MIC) of the compositions can also be evaluated by methods known to those skilled in the art, as described above. Another 20 tests, including the Phenol coefficient, are also well known to those skilled in the art. See also the Examples below.

EXAMPLES

Exemplary Lfqofofás Compositions

A dry pancreatic enzyme composition containing about 25 to 200 USP units/mg protease, about 40 USP units/mg lipase, and about 250 USP units/mg amylase may be diluted with various diluents (water, saline, phosphate buffered solutions, pH stabilized solutions) and optionally with other active or inactive additives (enzyme stabilization systems, buffers, dyes, sanitizers, detergents, disinfectants, antiseptics) to form exemplary liquid compositions for the uses described herein. In some embodiments, the dry enzyme composition may be diluted in a ratio of mg of the

67/75 dry enzyme composition to ml of total diluent in the range from 1 mg of enzyme composition: ml of total diluent to 1 mg of enzyme composition: 10,000 ml of total diluent, or any value between them, for example, 1:2, 1'3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9» 1:10, 1:20» 1:50, 1:100, 1:200, , 5 1:500,1:1000,1:5000 or 1:10,000.

Exemplary Solid Compositions

A dry pancreatic enzyme composition containing about 200 USP units/mg of protease, about 40 USP units/mg of lipase, and about 250 USP units/mg of amylase may be mixed with 10 various active or inactive ingredients and additives (e.g., dry detergents, disinfectants, antiseptics, and sanitizers, such as alkyl sulfate ethoxylate, SDS, sodium lauryl sulfate, dodecylbenzene, sodium dodecylbenzenesulfonate, enzyme stabilization systems, excipients, colorants) to form exemplary solid compositions. In some embodiments, the dry enzyme composition can be admixed with total mg of the additives in the range of 1 mg enzyme composition : 1 mg total additives to 1 mg enzyme composition : 10,000 mg total additives or any value therebetween, for example, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:50, 1:100, 1:200, 1:500, 1:1000, 1:5000 or 1:10,000.

Exemplary Topical Formulations

A dry pancreatic enzyme composition containing about 200 USP units/mg protease, about 40 USP units/mg lipase, and about 250 USP units/mg amylase may be admixed with various carriers suitable for topical pharmaceutical formulations in ratios ranging from about 1 mg of the enzyme composition to 1 mg of the carrier to about 1 mg of the enzyme composition to about 200 mg of the carrier, or any ratio therebetween (e.g., 1:2, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:75, 1:100, 1:150). For example, in one embodiment a ratio of 1:25 is employed, and the carrier trans* 30 is petroiate,

Bacterial Limit Test-Assessment of Bacteriocidal Properties and Ethyl Ethers

68/75

The present inventors surprisingly discovered during the course of performing the bacterial limit test on a dried porcine pancreatic enzyme composition comprising about 200 USP units/mg of protease activity, about 40 USP units/mg of lipase activity and 250 USP units/mg of amylase activity that S. aureus does not grow in the presence of various dilutions of this material. Recovery of S. aureus CPUs from positive controls spiked with S. aureus was consistently low to absent. Bacterial dilution limit testing of the dry composition in both unencapsulated and lipid-encapsulated forms (soybean oil 20% by weight of the composition) demonstrates minimal to no recovery of the bacteria from the positive controls to which a known number of S. aureus CPUs were added. aureus had been added. A lack of recovery during such recovery methods suggests a bacteriostatic and/or bacteriocidal nature of the composition.

Methods and Materials

Sample Materials- Unencapsulated Porcine Pancreatic Enzyme Concentrate (uPEC) isolated from pig (Sus scrofa) was manufactured by a commercial supplier (Scientific Protein Labs) to contain approximately 200 U/mg protease activity, 40 U lipase activity/mg, and 25 U amylase activity/mg. A lipid encapsulated version of this material (éPÉC) was obtained using a modified fluidized bed process and a highly purified fully hydrogenated organic oil (fully hydrogenated soybean oil) which was then used to coat the enzyme particles at a weight percentage of about 20% of the final particles,

Methods: Both ePEC and uPEC were subjected to standard microbial analysis for the detection of microbes. Both compositions failed to show any significant contamination using Microbial Limits Testing 30 by USP methods. Furthermore, both samples were negative for 10 g of Sa/mone//ae £ co/f species. In summary, the procedure for examining test compositions for microbial suitability (i.e., estimation of

69/75 (total number of viable microorganisms free of specific organism) is given in USP Chapter 61, “Microbial Limit Tests”. USP 61 shows preparatory testing, during which test parameters are determined where the test composition itself no longer exhibits multiplication of 5 viable organisms. USP 1227 (“Validation”) also provides guidance for validating recovery methods. Methods for evaluating Total Aerobic Microbial Count, E. species. Salmonella collè, were performed.

Total Aerobic Microbial Count Preparation of test dilutions - Crude dilutions of ePEC and uPEC were prepared at 1:10, 1:50, 1:100, 10, and 1:200 in Tryptic Soybean broth containing 4% Polysorbate 20 and 0.5% Lecithin. The crude test sample dilution was then separated into separate 10 ml aliquots, where it was then inoculated with a low number of colony forming units (CFUs &lt;100/mL) of the appropriate microorganisms (S; aureus, P. aeruginosa, E. coli, S, enterics). One milliliter of the 15 inoculated aliquots was plated in duplicate using the appropriate solid medium (agar). Positive controls were prepared, inoculated and plated in a manner similar to the test compounds. Negative controls were prepared, inoculated with sterile saline and plated in a manner similar to the test samples. The plates were incubated at 30°C for 20–35°C for two days. At the end of the period the recovery was calculated. Recovery of inoculated organisms should be at least 70% of the positive control in order to show no inhibition of growth by the test composition. Triphenyltetrazolium chloride was used to count the plaques.

SafmOnéOã species test - Dilutions prepared with 25% lactose broth containing 204% polysorbate and 0.5% lecithin were inoculated with &lt;= 100 CFU of Salmonella enterica. The inoculated dilutions were incubated at 30°C for 35^QC for 24 hours before 1 ml was transferred to both Selenite Cystine and Tetrathionate broths. The selective broths were incubated for 18 h at 30°C to 35°C before being layered onto brilliant green bismuth sulfite and xylose lysine deoxycholate agars. The selective agar plates were incubated at 30°C to 35°C for 24 hours. The plates were observed for colony characteristics.

7QZ75 Salmonella species. Where observed, a representative colony was confirmed to be Salmonella species using an API 2000 biochemical identification test.

E. coli Test - Dilutions prepared with 5% lactose broth containing 40% polysaccharide 20 and 0.5% lectin were inoculated with &lt;1000 CFU of E. coli. The inoculated dilutions were incubated at 0°C for 24 h before being layered onto MaoKonkey agar. Plates were observed for characteristic E. coli colonies. Where observed, a representative colony was confirmed to be E. coli using an API 20e biochemical identification test.

Results

Total Aerobic Count The results of the test using uncoated PEC (uPEC) are shown in Table 1, below. As shown, for dilutions of 1;50, 1100, and 200, the percent recovery of S. aureus was 0%, demonstrating the bacteriocidal and/or bacteriostatic action of uPEC on S. aureus.

Table 1

<td colspan="6">Microorganism Recovery Following uPEC Incubation</td>

<td>Body</td><td>Contfoíe Negative</td><td>Hire Positive</td><td>Dilution</td><td>Medium of Test</td><td>% of Recovery</td>

<td>S. aureus</td><td>0 GFU</td><td>S7GFU</td><td>1:50 1:100 1?2Óp</td><td>0 CFU Q ÇFU OÇFU</td><td> 0% 0% 0%</td>

<td>E. coli</td><td>0CFU</td><td>63ÇFU</td><td> 1:50 1:100 1:200</td><td>ÕCFÜ 1 CFU 1GFU</td><td> 0% 2% 2%</td>

<td>S. eniériça</td><td>OÇRJ</td><td>66 CPU</td><td> 1:50 1:100 1:200</td><td>46ÇFU 62 CPU 56 CFU</td><td> 73% 94% 85%</td>

The percent recovery of uPEC for £. e&amp;lí is also given in Table 1. For a dilution of 1:50 the recovery was 0%, for a

71/75 dilution of 1:100; recovery was 2% and for a dilution of 1:200 the recovery was 2%.

The percent recovery of uPEC for S. enterica is also given in Table 1. For a 1:50 dilution the recovery was 7.3%, for a 1:100 dilution the recovery was 94% and for a 1:2 dilution the recovery was 85%.

The results using lipid-encapsulated PEC (PECj) are given in Table 2 below. As shown, for 1:50 dilutions the recovery of S. aureus was 29%, for a 1:100 dilution the recovery was 0% and for a 1:20 dilution the percent recovery of S. aureus was also 0%.

Table 2

<td colspan="6">Microorganism Recovery Following Incubation with ePEC</td>

<td>Body</td><td>Hire Negative</td><td>Confrofe Positive</td><td>Dilution</td><td>Average of Test</td><td>%of Recovery</td>

<td>S. swei/s</td><td>QCFU</td><td>34 CPU</td><td> 1:50</td><td>10 CPU</td><td> 29%</td>

<td></td><td></td><td></td><td> 1:100 1:200</td><td>0CPU 0CFU</td><td>THE% 0%</td>

<td>E. co li</td><td>0CFO</td><td>34 CPU</td><td> 1:50 1:100 1 200</td><td>12 CPU 9 CPU 28 CPU</td><td> 32% 17% 78%</td>

<td>S. eoteriçâ</td><td>0 CPU</td><td>61 CPU</td><td> 1:5$</td><td>53 CFU</td><td> 87%</td>

<td></td><td></td><td></td><td>1:100 1:200_______ i</td><td>N/P N/P</td><td>N/P N/P</td>

The percentage recovery of ePEC for E. cofi is also shown in Table 1. For a dilution of 1:50 the recovery was 32%. For a dilution of 1:100 the recovery was 17% and for a dilution of 1:200 the recovery was 78%.

The percentage recovery of ePÊC for S. entenca is also given in Table 2. For a dilution of 1:50 the recovery was 87%, for a dilution of 1:100 and one of 1:200 the tests were not performed.

Positive controls as reported in Tables 1 and 2 clearly demonstrate that the growth media for all microbiota cultures were functioning effectively. From Table 1, it can be seen that the uPEC composition was highly effective on both S. aureus and E. coli. This is substantiated by the fact that recovery of 5 positively spiked controls failed to meet USP criteria for positive recovery (at least 70% of spiked sample CPUs were recovered);

It is also important to note that the bacteriostatic/bactericidal activity showed species specificity. S enteroã samples showed excellent recovery using both coated and uncoated PEG. Because bacteria share common cell wall and membrane structures such as lipid and peptide bonds, it is possible that these results point to the sensitivity of a more specific cellular component such as a protein. If lipase or amylase action alone were sufficient to induce bacterial death or growth suppression, then it would be unlikely to see recovery of en/erica to such robust levels. It is possible that species-specific protein(s) in S. aureus and E. coty share similar peptide sequences and the appropriate local tertiary structure to allow enzymatic attack by one or more of the 20 proteases present in PEG, leading to subsequent destruction of the bacteria. These results, however, would not rule out a multistage event where action by amylase lipases within the bacterial cell wall and membrane first exposes extracellular or transmembrane proteins that have the appropriate primary and tertiary structures to lend themselves to enzymatic degradation by one or more of the PEC proteases.

These results demonstrate a clear bacteriostatic effect for both lipid-coated and uncoated PEG. Because this experiment is based on an endpoint reading of bacterial colony growth while the subject bacteria remain in the presence of 30 PEG, it is not possible on the basis of these results to eliminate the possibility of a bacteriostatic effect alone. Thus, failure to recover a sufficient number of viable colony-forming units could be the

73775 result of the continued suppressive presence of the PEG material, as opposed to the induction of bacterial killing. Consequently, additional experimental testing was performed in order to more conclusively evaluate the bacterial capabilities of PEG.

„ 5 Materials &amp; Methods: The uncoated PEC formulation previously described above was used to evaluate the bacteriocidal activity of the formulations.

Preparation of test samples - Crude dilutions of uPEC were prepared at 1:100 and 1:200 in Tryptic Soy broth containing 4% Polysorbate 20 and 0.5% lecithin. The crude dilution was then separated into separate 10 ml aliquots, which were then inoculated with small numbers of colony forming units (CPUs &lt; 100) of the appropriate microorganisms. One milliliter of the inoculated aliquots was plated in duplicate using an appropriate solid medium (agar). Positive controls were prepared, inoculated and plated in a manner similar to the test samples. Negative controls were prepared, inoculated with sterile reagent and plated in a manner similar to the test samples. The plates were incubated at 30^theC to 35°C for two days. Following this initial incubation, the cultured material was then collected, washed in 20% Phosphate Buffered Saline (PBS) and filtered to remove PEG. The material was resuspended in Tryptic Soy Broth as above and replated onto fresh solid medium (agar) and incubated for a further 2 days. At the end of this period, colonies were enumerated and the percentage recovery calculated. At the end of the period, the recovery was calculated. The recovery of inoculated organisms must be at least 70% in order to show no inhibition of growth. Triphenyltetrazolium chloride was used to count the plants.

Tests for E. coli - Dilutions prepared with lactose broth containing 40% polysorbate 20 and 0.5% lecithin were inoculated with &lt;= 100 30 CFU of E. coli. The inoculated dilutions were incubated at 30°C to 35°C for 24 hours before being layered on MacKonkey agar. The plates were observed for characteristic colonies of E. coli.

74/75 observed, a representative colony was confirmed to be E. co/z using an API 20e biochemical identification test.

The tests were also repeated for both S. aureus and E cafi at dilutions of 1:20, 1:40 and 1:80.

cfo Test Results

The results of the test using uncoated ePEC are given in Tables 3 and 4 below, demonstrating recovery of bacteria following replating of bacteria alone, post-incubation with uPEC and washing. As shown in Table 3, for dilutions of 1:100 and 1:20, the percent recovery of S. aureus was 13% and 48%, respectively, clearly demonstrating the bacteriocidal action of uPEC on S. aureus. As shown in Table 4, the percent recovery of S; aureus at a 1'20 dilution is 2%.₍ for a dilution of 1:40 it is 4% and for a dilution of 1:80 it is 23%

Table 3

<td colspan="6">Bactericidal Action After Washing/Recovery of Wero-organisms</td>

<td>Body</td><td>Control Negative</td><td>Control Positive</td><td>Dilution</td><td>Average of Test</td><td>%d© Recovery</td>

<td>S. aureus</td><td>OCFU</td><td>48 CFU</td><td></td><td></td><td></td>

<td>£ call</td><td>OCFÜ</td><td>46 CFU</td><td></td><td></td><td></td>

Table 4

<td>Bacter Action</td><td colspan="5">elda Post-Wash/Microorganism Recovery</td>

<td>Body</td><td>Control Negative</td><td>Control Positive</td><td>Dilution</td><td>Test Average</td><td>%de Recoveries</td>

<td>S. aur&amp;us</td><td>0 CFU</td><td>47 CFU</td><td>1:20 1-40 T.80</td><td>1 CFU2 2 CFU 11 CFU</td><td> 2% 4% 23%</td>

<td>E. ç&amp;lí</td><td>0 CFU</td><td>43 CFU</td><td> 1:20 1:40 1:80</td><td>0CFU 0 CFU 5 CFU</td><td> 0% 0% : 12%</td>

The recovery of E cofi is also given in Tables 3 and 4.

75/75

As shown in Table 3, for a dilution of 1:100 the recovery was 2% and for a dilution of 1:200 the recovery was 28%. As shown in Table 4, the percentage recovery of E. co/f in a dilution of 1:20 is 0%, for a dilution of 1:40 it is 0% and for a dilution of 1:80.5 it is 12%.

Positive controls demonstrate that the growth media for all microbial cultures were functioning effectively. This demonstrates a true bactericidal effect when the selected organisms are exposed to PEC. If the action of PEC were solely bacterial, removal of PEC from the coculture with the test bacteria would result in a release of any suppressive action by PEC with a recovery of active growth. However, removal of PEC did not result in any such recovery, even if the cultures were allowed to grow for a full 24 h. This strongly supports the notion that the reason the 15 selected organisms failed to show recovery of CPUs upon exposure to PEC was because of a bactericidal action of PEC. This action could include mechanisms such as physical and irreversible damage to cell surface lipids, membrane proteins or bacterial capsule through enzymatic degradation, leading to electrolytic and ionic imbalances between extracellular and intracellular cells, changes in acidity and damage to genetic material.

Various embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the claims which follow.