HK1225961B - Compositions and methods for the treatment or prevention of staphylococcus aureus infections and for the eradication or reduction of staphylococcus aureus on surfaces - Google Patents

Description

Compositions and methods for treating or preventing Staphylococcus aureus infection and for eradicating or reducing Staphylococcus aureus on surfaces

This application is a divisional application of an invention patent application filed on January 6, 2010, with application number 201080004045.6 and invention name “Compositions and methods for treating or preventing Staphylococcus aureus infection and for eradicating or reducing Staphylococcus aureus on surfaces”.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application Serial No. 61/142,714, filed on January 6, 2009, U.S. Provisional Application Serial No. 61/153,274, filed on February 17, 2009, and U.S. Provisional Application Serial No. 61/170,915, filed on April 20, 2009, all of which are incorporated herein by reference in their entirety.

Technical Field

The present disclosure relates to compositions, including pharmaceutical compositions (e.g., antibiotic compositions), and methods of using the compositions to treat or prevent Staphylococcus aureus (SA) infections in humans and other animals. The present disclosure also relates to compositions (e.g., sterilants, disinfectants, antiseptics, and detergents), and methods of using the compositions to eradicate or reduce the presence of S. aureus on surfaces (including inanimate/non-living and biotic surfaces (e.g., skin, wounds)), and/or methods of using the compositions to reduce the infectivity of S. aureus to prevent and/or reduce the spread of S. aureus infections.

Background Art

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

Staphylococci are Gram-positive, spherical bacteria that exist in microscopic clusters resembling grapes. In 1884, Rosenbach described two types of colored colonies of Staphylococci and recommended appropriate nomenclature: Staphylococcus aureus (yellow) and Staphylococcus albus (white). The latter is currently known as Staphylococcus epidermidis. Staphylococci are facultative anaerobic microorganisms that grow by aerobic respiration or by fermentation, primarily producing lactic acid. The bacteria are catalase-positive and oxidase-negative. Staphylococcus aureus can grow at temperatures ranging from 15 to 45 degrees Celsius and in NaCl concentrations up to 15%.

Some Staphylococci are resistant to antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) is a type of Staphylococcus that is resistant to a class of antibiotics called beta-lactams. Beta-lactam antibiotics include methicillin and other more commonly used antibiotics, such as oxacillin, penicillin, and amoxicillin. Most MRSA infections occur among patients in hospitals or other healthcare facilities (called hospital-acquired MRSA or HA-MRSA infections); however, Staphylococci and MRSA can also cause illness in people outside of hospitals and healthcare facilities. MRSA infections acquired by people who have not recently (within the past year) visited a doctor or had a medical procedure (e.g., dialysis, surgery, catheterization) are considered community-acquired MRSA (CA-MRSA) infections. Approximately 75% of CA-MRSA infections are located in the skin and soft tissues and can usually be effectively treated. However, CA-MRSA strains exhibit increased virulence, spread more rapidly, and cause more severe illness than traditional HA-MRSA infections and can affect vital organs, leading to widespread infection (sepsis), toxic shock syndrome, and pneumonia.

The reason why some healthy people develop treatable CA-MRSA skin infections while others infected with the same strain develop severe, fatal infections is unknown. Studies have shown that CA-MRSA infection rates are increasing. In 1999, four children in Minnesota and North Dakota were reported to have died from an outbreak of CA-MRSA infections. A study of children in South Texas found a 14-fold increase in CA-MRSA cases between 1999 and 2001. In 2007, CA-MRSA was the most common cause of skin and soft tissue infections seen in emergency rooms in the United States.

Hospital strains of Staphylococcus aureus are often resistant to a variety of different antibiotics. A few strains are resistant to all clinically useful antibiotics except vancomycin, and vancomycin-resistant strains (VRSA) are being reported more and more. Methicillin resistance is widespread (MRSA), and most methicillin-resistant strains are also resistant to multiple drugs. In addition, Staphylococcus aureus shows resistance to preservatives and biocides, such as quaternary ammonium compounds, which can help it survive in hospital environments.

In the United States, MRSA infections now kill more people than AIDS. According to a CDC report published in the October 17, 2007, issue of the Journal of the American Medical Association, MRSA caused an estimated 94,000 life-threatening infections and 18,650 deaths in 2005. This national estimate is more than double the rate of invasive MRSA reported five years earlier. That same year, approximately 16,000 Americans died from AIDS, according to the CDC.

Summary of the Invention

The present disclosure relates to the use of pharmaceutical compositions to prevent and/or treat Staphylococcus aureus infections, including MRSA and VRSA infections, comprising one or more digestive enzymes that decompose food components, such as pancreatic or other digestive tract enzymes (e.g., porcine pancreatic enzymes) or enzymes derived from plants, fungi, or microorganisms. As used herein, pharmaceutical compositions can be used for human or veterinary indications. Thus, the pharmaceutical compositions can be used for preventive and/or therapeutic treatment of humans or other mammalian groups (e.g., pigs, horses, cows, sheep, goats, monkeys, rats, mice, cats, dogs) or poultry groups (e.g., ducks, geese, chickens, turkeys).

The pharmaceutical composition can be used alone and/or in combination with other antibacterial or antibiotic (eg, anti-S. aureus) therapies and/or in combination with other post-infection therapeutic agents or antibiotic agents to treat S. aureus infections.

The present disclosure also provides bactericidal and/or bacteriostatic compositions containing one or more digestive enzymes for use as bactericides, disinfectants, detergents, and preservatives, for example, in hospitals, nursing homes, nurseries, daycare centers, schools, workplaces, food service facilities, public transportation, and restroom facilities to reduce, attenuate, and/or eliminate Staphylococcus aureus present in these facilities. The surfaces treated with the compositions can be large (e.g., operating room tables, doors, switchboards, ventilation systems) or small (e.g., medical equipment, door handles); inanimate or non-living (tables) or living tissue (hands, e.g., detergents for hand washing; wounds, e.g., surgical wounds or wounds caused by accidents/trauma). Thus, the compositions can be used to treat surfaces to reduce or eradicate Staphylococcus aureus thereon, or to attenuate or reduce the infectivity of Staphylococcus aureus, thereby preventing or reducing the spread of Staphylococcus aureus.

Therefore, an object of the present disclosure is to provide a method for treating or preventing Staphylococcus aureus infection in poultry or mammals, comprising administering to the poultry 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, cellulases, sucrases, maltase, papain, and lipases. In some embodiments, the one or more 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 porcine pancreas.

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

In some embodiments, the pharmaceutical composition is in a dosage form selected from the group consisting of a pill, tablet, capsule, caplet, powder, cream, lotion, aerosol, emulsion, powder, liquid, gel, and any combination thereof.

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

The present disclosure also provides methods for treating a mammal or bird displaying 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.

The present disclosure also provides a method for promoting wound healing and/or reducing scar formation in an individual with a wound, comprising administering a pharmaceutical composition comprising one or more digestive enzymes to the individual. The wound may be, for example, a surgical wound or a traumatic wound.

The present disclosure also provides a method for disinfecting or sterilizing a surface to reduce the number of or eradicate Staphylococcus aureus thereon, comprising applying a composition comprising one or more digestive enzymes to the surface. The surface can be a living surface (e.g., skin, a wound) or an inanimate or non-living surface (e.g., a medical device such as a scalpel, knife, scissors, spatula, extender, clamp, tweezers, metal mirror, retractor, suture, valve, surgical mesh, chisel, drill, level, rasp, saw, splint, caliper, clamp, forceps, hook, lancet, needle, cannula, curette, depressor, dilator, elevator, articulator, extractor, probe, staple, catheter, stent, tube, bowl, tray, sponge, snare, spoon, syringe, pacemaker, screw, plate, and nail).

The present disclosure also provides methods for reducing the number of Staphylococcus aureus present on a skin area, tissue, or wound of a mammal or bird comprising applying a composition comprising one or more digestive enzymes to the skin area, tissue, or wound.

The present disclosure also provides a bactericide containing one or more digestive enzymes, wherein the bactericide has a phenol coefficient of >1 to about 20 against Staphylococcus aureus or Escherichia coli.

The present disclosure also provides an antibiotic comprising one or more digestive enzymes, wherein the antibiotic is bactericidal and/or bacteriostatic with respect to Staphylococcus aureus.

Similarly, the present disclosure provides detergents containing one or more digestive enzymes, wherein the detergents are bactericidal and/or bacteriostatic with respect to Staphylococcus aureus.

The present disclosure also provides a preservative comprising one or more digestive enzymes, wherein the preservative is bactericidal and/or bacteriostatic with respect to Staphylococcus aureus.

Similarly, the present disclosure also provides a bactericidal agent comprising one or more digestive enzymes, wherein the bactericidal agent is bacteriostatic and/or bacteriostatic against Staphylococcus aureus.

The details of one or more embodiments of the present invention are set forth in the following drawings and the specification. Any references disclosed herein, such as patents, patent applications, specifications, books, papers, or scientific publications, are incorporated by reference in their entirety. Other features, objects, and advantages of the present invention will become apparent from the specification, drawings, and claims.

DETAILED DESCRIPTION

The term "administering" refers to a method of administering a composition or a dose of a pharmaceutical composition to a vertebrate or invertebrate (including mammals, birds, fish or amphibians) 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 various factors, for example, the components of the pharmaceutical composition, the location of the disease, the disease involved, and the severity of the disease.

The term "mammal" is used in its ordinary biological sense. Thus, it specifically includes humans, cows, horses, dogs, and cats, but also includes many other species.

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, isotonic agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. In addition, any media or agents that are incompatible with the active ingredient are contemplated for use in therapeutic compositions. Supplementary active ingredients, such as antibiotics, antifungals, and antimicrobials, may also be added to the composition. 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 including various components in pharmaceutical compositions are described, for example, in Gilman et al. (eds.) (2006); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 11th ed., The McGraw-Hill Companies.

As used herein, "subject" or "patient" or "individual" refers to a human or a non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate, or bird, such as a chicken, as well as any other vertebrate or invertebrate.

A "therapeutically effective amount" or "pharmaceutically effective amount" generally refers to an amount sufficient to achieve the desired effect, which may vary depending on the nature and severity of the disease condition, the nature of the subject, and the efficacy of the composition. It will be understood that different concentrations may be used for the prevention of active disease than for the treatment of active disease. The dosage may also depend on the patient's height, weight, sex, age, and medical history.

A therapeutic effect alleviates one or more symptoms of a disease to some extent and includes curing the disease. "Cure" refers to the elimination of symptoms of an active disease. However, certain long-term or lasting effects of a disease may exist even after a cure is achieved (e.g., tissue damage).

As used herein, "treat" refers to the administration of a pharmaceutical composition for therapeutic purposes. The term "therapeutic treatment" refers to treating a patient already suffering from a disease, thereby causing a therapeutically beneficial effect, such as ameliorating existing symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic cause of symptoms, delaying or preventing further development of the disease, and/or reducing the severity of symptoms that will develop or are expected to develop.

The present disclosure provides compositions containing one or more digestive enzymes and methods of using the same to treat and/or prevent infections caused by Staphylococcus aureus (including antibiotic-resistant forms of Staphylococcus aureus, such as MRSA and VRSA). The present disclosure also provides compositions containing one or more digestive enzymes and methods of using the same as antiseptics, detergents, antiseptics, and disinfectants (e.g., as bactericidal and/or bacteriostatic compositions) to eradicate or weaken Staphylococcus aureus and/or reduce its infectivity. The compositions described herein contain one or more digestive enzymes, and it is hypothesized that the one or more digestive enzymes assist in reducing, weakening, or eradicating Staphylococcus aureus, thereby preventing Staphylococcus aureus infection or treating Staphylococcus aureus infection (e.g., ameliorating or improving symptoms or shortening the course of infection).

Composition

The compositions described herein may include one or more digestive enzymes. Without wishing to be bound by theory, it is believed that the digestive enzymes in the composition can degrade the cell wall, cell membrane, and/or protein structure of S. aureus, resulting in bacteriostatic and/or bactericidal activity. The compositions exhibit species-specific bactericidal/bacteriostatic activity against S. aureus and E. coli, but not against Salmonella enterica (S. enterica), possibly indicating that the vulnerability of the two organisms arises from proteolytic degradation of similar protein sequences present in both organisms.

The digestive enzymes described herein are enzymes that can break down one or more food components (e.g., proteins, fats, carbohydrates). The digestive enzymes can be of animal origin (e.g., pancreatic or other digestive tract enzymes), or of plant, fungal, or microbial origin, or can be synthetically prepared. Many digestive enzymes are commercially available or can be isolated or purified from other sources by methods known to those skilled in the art. The enzymatic activity of the enzymes can also be assessed using standard assays.

The digestive enzymes can be used in combination with any type of enzyme and any source of enzyme. In some embodiments, the one or more digestive enzymes comprise one or more enzymes selected from the group consisting of proteases, amylases, cellulases, sucrases, maltase, 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 contains 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 compositions described herein consist essentially of or consist of one or more digestive enzymes.

In certain embodiments, the composition comprises at least one amylase, at least two proteases, and at least one lipase. In certain embodiments, the composition may further comprise one or more hydrolases, papain, bromelain, papaya, cellulase, pancreatin, sucrase, and maltase.

As noted, the one or more digestive enzymes can be derived from an animal source. In some embodiments, the animal source is porcine, such as porcine pancreas. Porcine 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, pancreatic enzyme compositions can be purchased from Scientific Protein Laboratories (referred to as PEC). For example, the pancreatic enzyme composition or any composition herein can be adjusted by production and/or processing methods or by selectively adding exogenous enzymes, activators or inhibitors to the composition to change the amount of one or more digestive enzymes contained herein, such as lipase, amylase or protease content.

In some cases, it may be desirable that protease activity is relatively higher than lipase.Therefore, in some embodiments, compositions contains at least one protease and at least one lipase, and wherein the ratio scope of total protease and total lipase (in USP units) is 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, 15:1, 16:1, 17:1, 18:1, 19:1 and 20:1, and all numerical values in the scope.In some embodiments, the ratio scope of protease and lipase is about 4:1 to about 10:1, including 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 and 10:1, and all numerical values in the scope.

In some cases, it may be useful to vary the amount of a particular enzyme activity in a given composition. The activity of one or more digestive enzymes can be regulated by various methods known to those skilled in the art, such as by increasing the amount of a particular enzyme or by adjusting the components of the composition, for example, by using stabilizers, inhibitors, and activators. In some embodiments, the compositions described herein comprise one or more proteases having an activity of 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, the compositions described herein comprise one or more lipases having an activity of 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, 9, 10, 12, 14, 16, 18, 20, 22, 25, 28, 30, 35, 38, 40, 45 USP units per mg). In some embodiments, the compositions described herein comprise one or more amylases having an activity of about 0.05 to about 400 USP units per mg of 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, the compositions described herein comprise one or more proteases having an activity in the above ranges, one or more lipases having an activity in the above ranges, and one or more amylases having an activity in the above ranges. 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.

In some embodiments, the composition is formulated to stabilize one or more digestive enzymes, for example, to protect the enzymatic activity of the enzyme. Stabilization technology can limit or inhibit the automatic degradation of one or more enzymes in the composition and help maintain enzymatic activity, increase shelf life and contribute to the tolerance of the composition activity to changes in temperature, humidity and storage conditions. For example, in some embodiments, one or more enzymes in the composition are encapsulated, for example, lipid encapsulated. In other applications, changes in excipients, pH, enzyme inhibitors, etc. can be utilized to help enzyme stabilization. Suitable stabilization technology depends on the intended application (for example, antibiotics relative to detergents), route of administration, composition form, intended location/intended activity and other factors of the composition, 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 calcium salts; alkyl or branched alcohols, such as ethanol and isopropanol; alkanolamines, such as triethanolamine; acids, such as organic acids; and mixtures of petroleum fractions.

In certain embodiments, the enzyme activity stabilizer can be a composition selected from the following groups: (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 boron anion at an appropriate concentration. In some cases, the activity stabilizer is solvated in a polyol and can be combined with an enzyme stabilizing synergist or an adjuvant that forms an enzyme stabilizing system. Preferred "micelle inhibitors" include substances known to alter and inhibit micelle formation, which can be selected from water-miscible solvents, such as C1-C6 alkanols, C1-C6 diols, C2-C24 alkylene glycol ethers, alkylene glycol alkyl ethers, and mixtures thereof. Particularly preferred micelle inhibitors are di(propylene glycol) methyl ether ("DPM") and its analogs that alter micelle formation.

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

The active stabilizer can be selected to substantially minimize the minimum inhibitory concentration ("MIC") of the digestive enzymes in the formulation. The MIC is a measure of the lowest concentration of a biocide that successfully inhibits bacterial growth in a culture medium over a given period of time (e.g., 24 hours). Details of the MIC test are found on page 177 of "Bailey & Scott'Diagnostic Microbiology," 8th edition, 1990.

In some embodiments, the compositions described herein can be coated with various natural or synthetic coatings, for example, to provide timed release of the enzyme, to provide flavor or taste masking, or to stabilize the enzyme. Enzyme preparations containing one or more coatings for use in the methods and compositions described herein, including lipid-coated or lipid-encapsulated enzyme compositions, are disclosed in U.S. Serial No. 12/386,051, filed April 13, 2009, which is incorporated herein by reference in its entirety. Such coating formulations can provide desirable characteristics, including increased storage stability, reduced aerosolization of powders or solid formulations, odor and taste masking, enzyme stabilization, and delayed or timed release of the enzyme.

Other additives that may be included in the compositions described herein can be determined by one skilled in the art and are based on a number of characteristics, including the intended application, such as human and veterinary applications; the desired release profile; the desired pharmacokinetics; safety; stability and physical properties (odor, color, taste, pourability, aerosolization). Appropriate formulation ingredients, excipients, binders, fillers, flavorants, colorants, etc. can be determined and evaluated by one skilled in the art using known methods.

Pharmaceutical compositions and antibiotics for human or veterinary use

The compositions described herein can be formulated into pharmaceutical compositions, for example, compositions comprising the aforementioned compositions formulated together with one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical compositions are used to treat or prevent Staphylococcus aureus infections in humans and other animals, such as mammals (e.g., cows, horses, pigs, sheep, goats, monkeys, cats, dogs, mice, rats) and poultry (chickens, turkeys, ducks, geese). In the present invention, pharmaceutical compositions for treating Staphylococcus aureus infections may also be referred to as antibiotics or antibiotic compositions.

The sensitivity of Staphylococcus aureus (including MRSA and VRSA) to the antibiotic compositions described herein can be determined by methods known to those skilled in the art. A rapid method uses commercially available filter paper discs impregnated with a specific amount of the antibiotic composition. These filter paper discs are placed on the surface of an agar plate streaked with a culture medium containing the Staphylococcus aureus to be tested, and the plate is observed for growth inhibition zones. The liquid culture dilution sensitivity test involves preparing test tubes containing serially diluted compositions in liquid culture medium, and then inoculating the organism to be tested into the test tubes. After an appropriate incubation period, the lowest concentration that inhibits bacterial growth is reported as the minimum inhibitory concentration (MIC).

The resistance or sensitivity of S. aureus to the antibiotics described herein can be determined based on clinical outcomes, i.e., whether administration of the antibiotic to a subject infected with the organism successfully cures the subject. Alternatively, to facilitate the use of in vitro test results to identify antibiotic resistance or sensitivity, the National Committee for Clinical Laboratory Standards (NCCLS) has formulated standards for antibiotic susceptibility that correlate clinical outcomes with in vitro determinations of the minimum inhibitory concentration of an antibiotic.

The pharmaceutical compositions of the present invention can be administered by any acceptable mode of administration for agents of similar use, including, but not limited to, oral, subcutaneous, intravenous, intranasal, topical, transdermal, transmucosal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, or intraocular. For example, oral, transmucosal, topical, and parenteral administration are commonly used to treat Staphylococcus aureus infection indications.

In the pharmaceutical composition, effective concentrations of one or more digestive enzymes are mixed with suitable pharmaceutical excipients or carriers. The concentration of the digestive enzymes in the composition is such that upon administration, an amount of the composition is delivered that is effective for reducing or eradicating Staphylococcus aureus and/or treating or ameliorating one or more symptoms associated with Staphylococcus aureus infection.

The antibiotic composition can be formulated for single-dose administration. To formulate the composition, a weight fraction of the digestive enzyme is dissolved, suspended, dispersed in or mixed with a selected carrier at an effective concentration that reduces or eradicates bacteria, alleviates the condition being treated, or improves one or more symptoms.

The digestive enzyme is included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically effective effect without causing adverse side effects in the patient to be treated. The therapeutically effective concentration can be determined empirically by testing the digestive enzyme in vitro and in vivo, and then extrapolated to a human dose.

The concentration of the digestive enzyme in the pharmaceutical composition depends on the absorption, inactivation and excretion rates of the enzyme, the physicochemical properties of the enzyme, the dosage form, the dosage form and the amount of administration, as well as other factors known to those skilled in the art.

The pharmaceutical composition can be administered immediately or divided into many small doses administered at intervals. It should be understood that the precise dosage and duration of treatment vary with the disease to be treated and can be determined empirically from in vivo or in vitro test data using known test methods or by extrapolation. It should be noted that concentrations and dosage values can also vary with the severity of the condition to be alleviated. It should also be understood that for a particular subject, a specific dosage regimen should be adjusted over a period of time according to individual needs and the professional judgment of the person administering the composition or directing the administration of the composition, and it should be understood that the concentration ranges listed herein are merely exemplary and not intended to limit the scope or practice of the claimed composition.

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

The composition for pharmaceutical application can be used as a crystalline or amorphous product. Pharmaceutically acceptable compositions include solid, semisolid, liquid, gel, powder and aerosol dosage forms, for example, tablets, capsules, caplets, powders, powders, liquids, suspensions, emulsions, gels, suppositories, aerosols, etc. For example, they can be obtained as a film by methods such as precipitation, crystallization, freeze drying, spray drying or evaporative drying. Microwave or radio frequency drying can be used for this purpose. The composition can also be used in a sustained release or controlled release dosage form, including long-acting injections, osmotic pumps, pills, special coatings (such as enteric coatings) on oral dosage forms, transdermal effects (including electromigration) patches, etc., for extending and/or timing, pulsed administration at a predetermined rate. In some embodiments, the composition is provided in a unit dosage form suitable for a single administration of an exact dose.

The composition can be administered alone or, more generally, in combination with conventional pharmaceutical carriers, excipients, and the like. The term "excipient" as used herein is used to describe any ingredient other than the compound (enzyme) used in the composition. Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin; self-emulsifying drug delivery systems (SEDDS), such as succinic acid d-α-tocopheryl polyethylene glycol 1000 ester; surfactants used in pharmaceutical dosage forms, such as Tween or other similar polymer delivery matrices; serum proteins, such as human serum albumin; buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and lanolin. Cyclodextrins such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-β-cyclodextrin, or other dissolved derivatives may also be advantageously used to increase the delivery of the compositions described herein. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art, for example, see Remington: The Science and Practice of Pharmacy, 21st edition (Lippincott Williams & Wilkins. 2005).

In a preferred embodiment, the composition will take the form of a unit dosage form such as a pill or tablet, so in addition to the active ingredient, the composition may contain a diluent such as lactose, sucrose, dicalcium phosphate, etc.; a lubricant such as magnesium stearate, etc.; and a binder such as starch, gum arabic, polyvinyl pyrrolidone, gelatin, cellulose, cellulose derivatives, etc. In another solid dosage form, the powder, solution or suspension (for example, in propylene carbonate, vegetable oil or triglyceride) is encapsulated in a gelatin capsule. Unit dosage forms in which two or more components are physically separated are also contemplated, for example, a capsule containing enzyme granules and granules of other ingredients; a double-layer tablet; a double-chamber capsule, etc.

Liquid pharmaceutical compositions can be prepared, for example, by dissolving or dispersing one or more digestive enzymes and optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, ethylene glycol, ethanol, etc.) to form a solution or suspension. If necessary, the pharmaceutical composition may also contain a small amount of non-toxic auxiliary substances, such as wetting agents, emulsifiers, solubilizers, pH buffers, etc. (e.g., sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, etc.). Injectables can be prepared in conventional forms, as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolution or suspension in a liquid prior to injection.

Solid compositions can be provided in a variety of different types of dosage forms, depending on the physicochemical properties of the enzyme, the desired dissolution rate, cost considerations, and other criteria. In one embodiment, the solid composition is a single unit. This means that one unit dose of the drug is contained in a single, physically solid form or article. In other words, the solid composition is cohesive, in contrast to a multiple unit dosage form in which the units are not cohesive.

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

On the other hand, for certain applications, solid compositions can also be formed as multi-unit dosage forms. Examples of multi-units are powders, granules, microgranules, microcapsules, pills, beads, lyophilized powders, and the like. In one embodiment, the solid composition is a lyophilized powder. This dispersed lyophilized system comprises a large number of powder particles. Because the powder is formed using a lyophilization process, each particle has an irregular, porous microstructure through which the powder can absorb water very quickly, resulting in rapid dissolution. In another embodiment, the solid composition is a powder.

Another type of multiparticulate system capable of achieving rapid drug dissolution is a system derived from a powder, granule, or pellet coated with a water-soluble excipient of a composition component (e.g., an enzyme) such that the enzyme is located on the exterior surface of the individual particles. In this type of system, a water-soluble, low molecular weight excipient is used to prepare the core of the coated microparticles, which can then be coated with a coating composition containing the enzyme and preferably one or more other excipients, such as a binder, porogen, sugar, sugar alcohol, film-forming polymer, plasticizer, or other excipient for drug coating compositions.

The appropriate dosage for treating or preventing S. aureus infection depends on the patient (species, age, weight, health status), 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 skilled 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 also be understood that for a particular subject, the specific dosage regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition.

In some embodiments, the pharmaceutical composition comprises, per dose: an amylase having an activity of about 10,000 to about 60,000 U.S.P., including 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, and 60,000 U.S.P. and all values therebetween; an amylase having an activity of about 10,000 to about 70,000 U.S.P., including 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, and 60,000 U.S.P.; and all values therebetween, and lipases having an activity of about 4,000 to about 30,000 U.S.P., including 4,000, 5,000, 10,000, 15,000, 20,000, 25,000, and 30,000 U.S.P. and all values therebetween. The pharmaceutical composition may include one or more of: about 2 to about 5 mg, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg, and all values therebetween, of chymotrypsin; about 60 to about 100 mg, including 50, 65, 70, 75, 80, 85, 90, 95, and 100 mg, and all values therebetween, of trypsin; 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, and all values therebetween, of papain; and about 30 to about 60 mg, including 30, 35, 40, 45, 50, 55, and 60 mg, and all values therebetween, of papain.

Additional formulations of specific dosage forms of the compositions are provided below.

1. Composition for oral administration

Oral pharmaceutical dosage forms are solids, gels, or liquids. Solid dosage forms include tablets, capsules, granules, and bulk powders. Oral tablet types include compressed tablets, chewable lozenges, and tablets that can be enteric-coated, sugar-coated, or film-coated. Capsules can be hard or soft gelatin capsules, while granules and powders can be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art.

a. Solid compositions for oral administration

In certain embodiments, the preparation is a solid dosage form. In one embodiment, the preparation is a capsule or tablet. Tablets, pills, capsules, lozenges, etc. may contain one or more of the following ingredients or compounds of similar properties: binder; lubricant; diluent; glidant; disintegrant; colorant; sweetener; flavoring; wetting agent; enteric coating; and film coating. Examples of binders include microcrystalline cellulose, tragacanth, glucose solution, acacia mucilage, gelatin solution, syrup, polyvinyl pyrrolidone, povidone, crospovidone, sucrose, and starch paste. Lubricants include talc, starch, magnesium stearate or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Flow aids include, but are not limited to, colloidal silicon dioxide. Disintegrants include cross-linked sodium carboxymethyl cellulose, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar, and carboxymethyl cellulose. Colorants include, for example, any approved water-soluble FD and C dyes and mixtures thereof; and water-insoluble FD and C dyes suspended in hydrated alumina. Sweeteners include sucrose, lactose, mannitol, and artificial sweeteners such as saccharin, and many spray-dried spices. Fragrances include natural flavors extracted from plants such as fruits, and synthetic mixtures of pleasant-smelling compounds such as, but not limited to, mint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Enteric coatings include fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalate. Film coatings include hydroxyethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

The digestive enzymes can be provided in a composition that protects them from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains the integrity of the composition in the stomach and releases the digestive enzymes in the intestines. The composition can also be formulated with an antacid or other such ingredients.

When the dosage unit form is a capsule, it may contain, in addition to materials of the types described above, a liquid carrier, such as a fatty oil. Furthermore, the dosage unit form may contain a variety of other materials that modify the physical form of the dosage unit, such as sugar coatings and other enteric solvents. Digestive enzymes may also be administered as a component of elixirs, suspensions, syrups, tablets, powders, chewing gums, and the like. In addition to containing active digestive enzymes, syrups may contain sucrose as a sweetener and certain preservatives, dyes, colorants, and flavorings.

The digestive enzymes may also be mixed with 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 of digestive enzymes, up to about 98% by weight, may be included.

In all embodiments, tablet and capsule formulations may be coated by techniques known to those skilled in the art to modify or maintain dissolution of the digestive enzymes. Thus, for example, they may be coated with conventional enteric digestible coatings such as phenyl salicylates, waxes, and cellulose acetate phthalate.

b. Liquid compositions for oral administration

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

Elixirs are clear, sweetened hydroalcoholic preparations. Pharmaceutically acceptable carriers for elixirs include solvents. Syrups are concentrated aqueous solutions of sugars, such as sucrose, and may contain preservatives. Emulsions are two-phase systems in which one liquid is dispersed throughout another liquid in the form of droplets. Pharmaceutically acceptable carriers for emulsions are non-aqueous liquids, emulsifiers, and preservatives. Suspensions utilize pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances for non-effervescent granules for reconstitution into liquid oral dosage forms include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable substances for effervescent granules for reconstitution into liquid oral dosage forms include organic acids and a source of carbon dioxide. Colorants and flavorings are used in all of the above dosage forms.

Solvents include glycerin, sorbitol, ethanol, and syrup. Examples of preservatives include glycerin, methyl and propyl parabens, benzoic acid, sodium benzoate, and alcohol. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifiers include gelatin, gum arabic, gum tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, gum tragacanth, Veegum, and gum arabic. Sweeteners include sucrose, syrup, glycerin, and artificial sweeteners such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Organic acids include citric acid and tartaric acid. Carbon dioxide sources include sodium bicarbonate and sodium carbonate. Colorants include any approved water-soluble FD and C dyes and mixtures thereof. Fragrances include natural flavors extracted from plants, such as this fruit species, and synthetic mixtures of compounds that produce a pleasant taste.

In one embodiment, for solid dosage forms, a solution or suspension of, for example, propylene carbonate, vegetable oil, or triglyceride is encapsulated in a gelatin capsule. Such solutions and their preparation and encapsulation are disclosed in U.S. Patents 4,328,245, 4,409,239, and 4,410,545. For liquid dosage forms, for example, a solution of polyethylene glycol can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, such as water, to allow for easy measurement for administration.

Alternatively, liquid or semisolid oral formulations can be prepared by dissolving or dispersing the digestive enzymes in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate), and other such carriers and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those described in U.S. Patents RE28,819 and 4,358,603. Briefly, such formulations include, but are not limited to, formulations containing the digestive enzymes provided herein, dialkylated mono- or poly-alkylene glycols (including, but not limited to, 1,2-dimethoxymethane, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether, tetraethylene glycol dimethyl ether, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol), and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholic solutions containing pharmaceutically acceptable acetals. The alcohol used in these formulations is any pharmaceutically acceptable water-miscible solvent 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. Injections, solutions and emulsions

In one embodiment, the present invention also contemplates parenteral administration characterized by subcutaneous, intramuscular, or intravenous injection. Injections can be prepared in conventional forms: liquid solutions or suspensions, emulsions, or solid forms suitable for forming liquid solutions or suspensions before injection. The injections, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, glucose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic adjuvant substances, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubilizers, and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

The present invention also contemplates the implantation of slow-release or extended-release systems so that a constant level of dosage is maintained (e.g., see U.S. Patent No. 3,710,795). Briefly, the digestive enzymes provided herein are dispersed in a solid internal 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 rubber, polydimethylsiloxane, silicone carbonate copolymers, or the like. The parenteral composition comprises a polymeric outer membrane, such as polyethylene, polypropylene, ethylene/propylene copolymer, ethylene/ethyl acrylate copolymer, ethylene/vinyl acetate copolymer, silicone rubber, polydimethylsiloxane, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomers polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, which is insoluble in body fluids. The digestive enzyme diffuses through the outer polymeric membrane at a controlled release rate. The percentage of digestive enzyme contained in such parenteral compositions is highly dependent on their characteristics, the activity of the digestive enzyme or mixture thereof, and the needs of the subject.

Parenteral administration of the composition includes intravenous, subcutaneous and intramuscular administration. Preparations for parenteral administration include sterile solutions for injection, sterile dried soluble products, such as lyophilized powders that are mixed with a solvent just before use, including subcutaneous injection tablets, sterile suspensions for injection, sterile dried insoluble products that are mixed with a medium just before use, and sterile emulsions. The solution may be 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 for parenteral formulations include aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, chelating agents and other pharmaceutically acceptable substances.

Examples of aqueous media include sodium chloride injection, Ringers injection, isotonic dextrose injection, sterile water injection, dextrose, and emulsified Ringers injection. Non-aqueous parenteral media include fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers, which include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, sodium thiomercurate, benzyl chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (80). Chelating agents for metal ions include EDTA. For aqueous media, pharmaceutical carriers also include ethanol, polyethylene glycol, and propylene glycol; for pH adjustment, pharmaceutical carriers also include sodium hydroxide, hydrochloric acid, citric acid, or lactic acid.

Unit dose parenteral preparations are packaged in ampoules, vials or syringes with needles.All preparations for parenteral administration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing the digestive enzymes is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing the digestive enzymes, which can be injected as needed to produce the desired pharmacological effect.

Injections are designed for local and systemic administration. In one embodiment, a therapeutically effective dose is formulated to contain at least about 0.1% w/w up to about 90% w/w or more, and in certain embodiments, greater than 1% w/w of the digestive enzyme for the tissue to be treated.

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

3. Freeze-dried powder

Contemplated herein are also lyophilized powders that can be reconstituted into solutions, emulsions, and other mixtures for administration. They can also be reconstituted and formulated into solids or gels.

Sterile lyophilized powders are prepared by dissolving the digestive enzymes provided herein in a suitable solvent. The solvent may contain excipients that improve the stability of the powder or the reconstituted solution prepared from the powder or other pharmacological components. Useful excipients include, but are not limited to, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, in one embodiment, at a pH of approximately neutral. The solution is then sterile filtered, followed by lyophilization under standard conditions known to those skilled in the art to provide the desired formulation. In one embodiment, the resulting solution is divided into vials for lyophilization. Each vial contains a single dose or multiple doses of the digestive enzyme. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other appropriate carrier. The exact amount depends on the digestive enzyme selected. This amount can be determined empirically.

4. Topical application

Topical mixtures can be prepared as described for local and systemic administration. The resulting mixture can be a solution, suspension, emulsion, or the like, and can be formulated into a cream, gel, ointment, emulsion, powder, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, rinse, spray, suppository, bandage, dermal patch, or any other formulation suitable for topical administration.

Digestive enzymes can be formulated as aerosols for topical application, such as by inhalation (see, for example, U.S. Patents 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivering steroids for treating inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a finely divided powder for insufflation, alone or in combination with an inert carrier (e.g., lactose). In this case, in one embodiment, the diameter of the formulation particles is less than 50 microns, and in one embodiment, the diameter of the formulation particles is less than 10 microns.

The digestive enzymes can be formulated for topical application, such as in the form of gels, creams, and lotions for topical application to the skin and mucous membranes (e.g., eyes), as well as for application to the eye or for intracisternal or intraspinal administration. Topical administration is contemplated for transdermal delivery, as well as for application to the eye or mucous membranes, or for inhalation therapy. Nasal solutions of the digestive enzymes can also be administered alone or in combination with other pharmaceutically acceptable excipients.

These solutions, particularly those used in ophthalmology, can be formulated with appropriate salts to provide 0.01% to 10% isotonic solutions, with a pH of about 5-7.

Powders can be formed with the aid of a suitable powder base, such as talc, lactose, starch, and the like. Solutions can be formulated with aqueous or non-aqueous bases and may also contain one or more dispersants, suspending agents, solubilizing agents, and the like. Topical gels are prepared using polymers having a molecular weight and concentration level effective to form viscous solutions or colloidal gels of aqueous or non-aqueous solutions or suspensions of digestive enzymes. Polymers that can be used to prepare topical gels include polyphosphates, polyethylene glycols, high molecular weight polylactic acids, hydroxypropyl celluloses, chitosan, polystyrene sulfonates, and the like.

For example, in aqueous or oily bases, add suitable thickener, gelling agent, stabilizer, emulsifier, dispersant, suspending agent or compatibility regulator etc. to prepare ointment, cream and lotion.Matrix comprises water, alcohol or oil, for example liquid paraffin, mineral oil or vegetable oil, for example peanut oil or castor oil.The thickener that can be used according to the character of matrix comprises soft paraffin, aluminum stearate, cetearyl alcohol, propylene glycol, polyethylene glycols, polyphosphates, polylactic acid, hydroxyethyl cellulose, hydroxypropyl cellulose, cellulose gum, acrylate polymers, hydrophilic gelling agent, chitosan, polystyrene sulfonate, petrolatum, lanolin, hydrogenated lanolin, beeswax etc.

Ointments, pastes, creams, gels and lotions may also include excipients, such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, zinc oxide and mixtures thereof. Powders and sprays may also include excipients, such as silicic acid, aluminum hydroxide, calcium silicate and polyamide powder or mixtures thereof. Solutions, suspensions or dispersants may be converted into aerosols or sprays by any known method commonly used to prepare topical aerosols. Typically, this method comprises compressing or providing a means of compressing a solution, suspension or dispersion container, usually with an inert carrier gas, and transmitting compressed gas through an aperture. Sprays and aerosols may also contain conventional propellants, such as chlorofluorocarbons or volatile unsubstituted hydrocarbons, such as butane and propane.

Excipients include compounds that promote skin absorption, such as dimethyl sulfoxide (DMSO), fatty acid partial glycerides, and the like, present at levels up to about 10% by weight of the total weight of the formulation. Examples of fatty acid partial 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 contain inert ingredients to improve cosmetic acceptability, including, but not limited to, humectants, surfactants, fragrances, colorants, emollients, fillers, and the like.

The topical composition may also include other antibiotic agents, examples of which include bacitracin, neomycin, polymyxins, beta-lactams including penicillins, methicillin, latamoxef, and cephalosporins such as cefaclor, cefdroxil, cefmandole sodium, cefazolin, cefixime, cefmetazole, cefonicid, cefoperazone, cefoperazone, cefotanme, cefotaxime, cefotetan, cefoxitin, cefpodoxime proxetil, ceftazidime, ceftizoxime, ceftriaxone, ceftriaxone, cefuroxime, cephalexin, cephalosporin C, cephalosporin C sodium salt, cephalothin, cephalothin sodium salt, cephalothin dihydrate, cefpirin, cephradine, cefuroxime axetil, loracarbef, etc. Essentially any anti-infective/antibiotic agent that is effective when applied topically can be used. Thus, the methods of the present invention for treating active infections and eliminating pathogen populations on the skin include methods using digestive enzymes alone or in combination, without or in combination with at least one other anti-infective agent.

Topical compositions can be applied directly to the desired area of the skin by dusting, spraying, or applying the ointment, cream, lotion, solution, or gel film to the desired area of the skin with the patient's or health care provider's fingertips or other conventional dressings, such as swabs or wipes. The product can be applied to the skin first and then spread with the fingertips or applicator, or applied to the fingertips and then spread on the skin. The composition can also be optionally first applied to the surface of a topical applicator (e.g., a bandage, swab, moist woven or non-woven wipe, etc.), which is then applied to the portion of the skin to receive the composition.

The topical compositions of the present invention can be prepared from basic formulations whose components, amounts, and preparation methods are generally known to those skilled in the art and require no further description. The topical compositions of the present invention can also be prepared as creams or lotions based on emulsion formulations, which, in addition to having good skin compatibility and wound healing properties, are particularly suitable for formulations containing digestive enzymes and possess hitherto unknown bactericidal activity.

As noted above, the present invention is not limited to topical creams or lotions. Topical formulations based on conventional sprays, mists, aerosols, lotions, creams, aqueous and non-aqueous solutions or liquids, oils, gels, creams, pastes, ointments, emulsions, and suspensions may contain an amount of digestive enzymes and, optionally, one or more other anti-infective agents in a total concentration of about 0.125% by weight to about 10% by weight or more, again recognizing that the optimal dosage may vary by only 0.05% by weight, such that typical cream and lotion embodiments contain each 0.05% by weight concentration increment within this range.

The topical compositions of the present invention are used to treat skin infections and wound infections, such as superficial and penetrating wounds. Suitable wounds 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 remove Staphylococcus aureus bacteria to prevent secondary infections, including pretreatment prior to surgical procedures or catheterization.

Mucosal delivery formulations can include digestive enzymes as described herein, combined with or co-administered with suitable carriers or media for mucosal delivery. As used herein, the term "carrier" refers to a pharmaceutically acceptable solid or liquid filler, diluent, or encapsulating material. Aqueous liquid carriers can include pharmaceutically acceptable additives, such as acidulants, alkalizers, antimicrobial preservatives, antioxidants, buffers, chelating agents, complexing agents, solubilizers, wetting agents, solvents, suspending agents, and/or tackifiers, tonicity agents, wetting agents, or other biocompatible materials. A list of the aforementioned components can be found in U.S. Pharmacopeia National Formulary, 1900, pp. 1857-1859. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffers, and other nontoxic, compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives, and antioxidants may also be present in the composition as desired by the formulator. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. The amount of digestive enzyme that can be combined with the carrier material to prepare a single dosage form will vary depending on the particular mode of administration.

Mucosal preparations are generally sterile, particulate-free, and stable for pharmaceutical use. As used herein, the term "particulate-free" refers to a preparation that meets the requirements of the USP regulations for small-volume parenteral solutions. The term "stable" refers to a preparation that meets all chemical and physical requirements for identity, strength, quality, and purity as established by good pharmaceutical manufacturing practices as set forth by the appropriate governmental regulatory agencies.

In mucosal delivery compositions, a variety of delivery facilitators can be used, which increase the delivery of digestive enzymes into or through mucosal surfaces. As used herein, "mucosal delivery facilitator" includes (for example, from a formulation delivery medium) release or solubility, diffusion rate, permeability and opportunity, intake, residence time, stability, effective half-life, peak or sustained concentration level, clearance and other required mucosal delivery features (for example, measured at the delivery site, or measured at selected active target sites such as blood flow or central nervous system). Therefore, the increase of mucosal delivery can occur by any of a variety of mechanisms, such as by increasing diffusion, transport, the persistence or stability of digestive enzymes, increasing membrane fluidity, regulating the effectiveness or effect of calcium and other ions (the ion regulation intracellular or intercellular penetration), dissolving mucosal components (for example, lipids), changing non-protein and protein sulfhydryl levels in mucosal tissue, increasing the water flow by mucosal surface, regulating epithelial junctional physiology, reducing the viscosity of the mucus covering mucosal epithelium, reducing mucociliary clearance and other mechanisms.

In some embodiments, the nasal delivery facilitator of the present invention is used to deliver the drug to the patient in a manner that is suitable for the patient's nasal delivery of the drug. Although the absorption promoting mechanism can be changed according to the different intranasal delivery facilitators of the present invention, about this point, useful reagent will not adversely affect mucosal tissue basically, and is selected according to the physicochemical properties of specific digestive enzymes or other active or delivery facilitators. About this point, the delivery facilitator that increases the permeability of mucosal tissue or the permeability usually causes some changes in the protective permeability barrier layer of mucosa. For this valuable delivery facilitator in the present invention, it is usually required that any significant change in the permeability of mucosa be reversible in the time frame suitable for the drug delivery required duration. Moreover, there should be no real accumulation toxicity, and the barrier properties of the mucosa of long-term use do not have any inductive lasting harmful changes.

In some embodiments, the absorption enhancers used in conjunction with the digestive enzymes described herein or in combination formulations are selected from small hydrophilic molecules, including but not limited to dimethyl sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and 2-pyrrolidone. Alternatively, long-chain hydrophilic lipid molecules, such as dodecyl methyl sulfoxide, azone, sodium lauryl sulfate, oleic acid, and bile salts, can be used to improve the mucosal permeability of the digestive enzymes. In other aspects, surfactants (e.g., polysorbates) are used as adjuvant compounds, treatment agents, or formulation additives to improve the intranasal delivery of digestive enzymes. These penetration enhancers typically interact with the polar head groups or hydrophilic tail regions of molecules comprising the lipid bilayers of the epithelial cells lining the nasal mucosa (Barry, Pharmacology of the Skin, Vol. 1, pp. 121-137, Shroot et al., eds., Karger, Basel, 1987; and Barry, J. controlled Release 6: 85-97, 1987). The interaction at these sites may have the effect of destroying lipid molecule assembly, increasing the mobility of bilayer and promoting digestive enzyme transport through mucosal barrier. The interaction of these penetration enhancers and polar head groups may also cause or allow the hydrophilic regions of adjacent bilayers to absorb more water and separate from each other, thus opening the cell paralateral channel to transport digestive enzymes. In addition to these effects, some promoters may have a direct effect on the volume properties of the nasal mucosa aqueous region. If present in a delivery environment (for example, by pre-administration or in combination with therapeutic preparations) with sufficiently high concentrations, promoters such as DMSO, polyethylene glycol and ethanol can enter the mucosal aqueous phase and change its solubility, thus promoting digestive enzymes to separate from the medium and enter the mucosa.

Other mucosal delivery enhancers for use in the coordinated administration and treatment methods and combined formulations of the present invention include, but are not limited to, mixed micelles; enamines; nitric oxide donors (e.g., S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4, preferably co-administered with NO scavengers such as carboxy-PITO or diclofenac sodium); sodium salicylate; acetoacetin (e.g., glyceryl-1,3-diacetoacetate or 1,2-isopropylglycerol-3-acetoacetate); and other release-diffusion agents or intraepithelial or transepithelial penetration enhancers that are physiologically compatible with mucosal delivery. Other absorption enhancers are selected from a variety of carriers, matrices, and excipients that promote mucosal delivery, stability, activity, or transepithelial permeability of digestive enzymes. These include, inter alia, cyclodextrins and β-cyclodextrin derivatives (e.g., 2-hydroxypropyl-β-cyclodextrin and heptakis (2,6-di-O-methyl-β-cyclodextrin). These compounds, optionally combined with one or more active ingredients and further optionally formulated in an oleaginous base, enhance the bioavailability of the mucosal formulations of the present invention. However, other suitable absorption enhancers for mucosal delivery include medium-chain fatty acids, including mono- and diglycerides (e.g., sodium caprate-coconut oil extract, Capmul) and triglycerides (e.g., starch dextrins, Estaram 299, Miglyol 810).

Mucosal therapeutic and preventive compositions can be supplemented with any suitable permeation enhancer that promotes the absorption, diffusion or penetration of digestive enzymes through the mucosal barrier. The permeation enhancer can be any pharmaceutically acceptable enhancer. Therefore, in a more detailed aspect of the present invention, the composition comprises one or more permeation enhancers selected from sodium salicylate and salicylic acid derivatives (e.g., acetylsalicylate, choline salicylate, salicylalamine); amino acids and their salts (e.g., monoaminocarboxylic acids, such as glycine, alanine, phenylalanine, proline, hydroxyproline; hydroxyamino acids, such as serine; acidic amino acids, such as aspartic acid, glutamic acid; and basic amino acids, such as lysine, including their alkali metal salts 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 salts (alkali metal salts and alkaline earth metal salts). The methods and compositions of the present invention also provide penetration enhancers, which are substances commonly used as emulsifiers (e.g., sodium oleyl phosphate, sodium lauryl phosphate, sodium lauryl sulfate, sodium myristyl sulfate, polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters, etc.), caproic acid, lactic acid, malic acid and citric acid and alkali metal salts thereof, pyrrolidone carboxylic acid, alkylpyrrolidone carboxylic acid esters, N-alkylpyrrolidone, proline acyl esters, etc.

5. Compositions for other routes of administration

Other routes of administration, such as transdermal patches, including iontophoretic and electrophoretic devices, and rectal administration are also contemplated by the present invention.

Transdermal patches including iontophoresis and electrophoresis devices are well known to those skilled in the art. For example, such patches are disclosed in U.S. Patents 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433 and 5,860,957.

For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules, and tablets for systemic effects. Rectal suppositories as used herein refer to solids inserted into the rectum that melt or soften at body temperature, releasing one or more pharmacologically active or therapeutically active ingredients. Pharmaceutically acceptable substances for rectal suppositories are bases or media and agents that increase the melting point. Examples of bases include cocoa butter (cocoa bean oil), glycerol-gelatin, polyethylene glycol products (polyethylene glycol), and suitable mixtures of monoglycerides, diglycerides, and triglycerides of fatty acids. A mixture of multiple bases can be used. Agents that increase the melting point of suppositories include spermaceti and wax. Rectal suppositories can be prepared by compression or molding. In one embodiment, the weight of the rectal suppository is about 2 to 3 grams.

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

6. Sustained-release preparations

The present invention also provides sustained-release formulations for delivering digestive enzymes to a desired target. It will be appreciated that the level of digestive enzymes can be maintained as desired over a period of time, and such levels can be readily determined by those skilled in the art. Such sustained- and/or timed-release formulations can be prepared by sustained-release means using delivery devices well known to those skilled in the art, as described in U.S. Patents 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 4,710,384, 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, each of which is 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, hydropropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, and 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, the present invention contemplates single unit dosage forms suitable for oral administration that are suitable for sustained release, such as, but not limited to, tablets, capsules, gelcaps, caplets, powders, and the like.

In one embodiment, the sustained formulation comprises an active compound, such as, but not limited to, microcrystalline cellulose, maltodextrin, ethyl cellulose, and magnesium stearate. As described above, the present invention contemplates all known methods for encapsulation, as long as it is compatible with the properties of the disclosed digestive enzymes. The sustained release formulation is encapsulated by coating the microparticles or particles of the pharmaceutical composition provided herein with a slowly soluble polymer coating of varying thicknesses, or by microencapsulation. In one embodiment, the sustained release formulation is encapsulated with a coating material of varying thicknesses (e.g., about 1 micron to 200 microns), which allows the pharmaceutical composition to dissolve within about 48 hours to about 72 hours after being administered 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, which is prepared by compressing the drug and a slowly soluble polymer carrier into a tablet. In one embodiment, the size of the coated particles ranges from about 0.1 to about 300 microns, as disclosed in U.S. Patents 4,710,384 and 5,354,556, which are incorporated herein by reference in their entirety. Each particle is in the form of a micromatrix in which the active ingredient is uniformly distributed throughout the polymer.

The digestive enzymes provided herein can be formulated as sustained- and/or timed-release formulations. All sustained-release pharmaceutical products share the common goal of improving the therapeutic efficacy of a drug over that achieved by its non-sustained counterpart. Ideally, the use of a medically optimally designed sustained-release formulation is characterized by the minimal use of digestive enzyme to cure or control a condition. Advantages of sustained-release formulations may include: 1) prolonged activity of the composition, 2) reduced dosing frequency, and 3) increased patient compliance. Additionally, sustained-release formulations can be used to influence the time of onset of action or other characteristics, such as blood levels of the composition, and thus, the occurrence of side effects.

The sustained-release formulations provided herein are designed to initially release a certain amount of the therapeutic composition to rapidly produce the desired therapeutic effect, and then gradually and continuously release the remaining amount 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 must be released from the dosage form at a rate that replaces the composition that is metabolized and excreted from the body.

Sustained release of the active ingredient can be stimulated by various inducers, such as pH, temperature, enzymes, water or other physiological conditions or compounds.

Formulations for oral administration can be suitably formulated to achieve controlled release of the digestive enzymes. In one embodiment, the digestive enzymes are formulated as controlled-release powders of discrete microparticles that can be readily formulated into liquid forms. Sustained-release powders comprise microparticles containing the active ingredient and, optionally, an excipient with at least one nontoxic polymer.

Powders can be dispersed or suspended in a liquid medium and maintain their sustained release characteristics over an effective period of time. These dispersions or suspensions have chemical stability and dissolution rate stability. Powders can include excipients containing polymers, which can be soluble, insoluble, permeable, non-permeable or biodegradable. The polymer can be a polymer or a copolymer. The polymer can be a natural or synthetic polymer. Natural polymers include polypeptides (e.g., zein), polysaccharides (e.g., cellulose) and alginic acid. Typical synthetic polymers include but are not limited to those described in columns 3, rows 33-45 of U.S. Patent No. 5,354,556, which is incorporated by reference in its entirety. Particularly suitable polymers include but are not limited to those described in columns 3, row 46 to column 4, row 8 of U.S. Patent No. 5,354,556, which is incorporated by reference in its entirety.

Sustained release compositions provided by the invention can be formulated for parenteral administration, for example, by intramuscular injection or for implants and transdermal devices for subcutaneous tissue and various body cavities. In one embodiment, intramuscular injection is mixed with water or oil suspension. In aqueous suspension, the sustained release effect is partially due to the solubility reduction or dissolution rate reduction of the digestive enzyme under complexation. Oil suspension and solution take a similar approach, wherein the rate of release of the digestive enzyme is determined by the digestive enzyme and oil separation and entering the surrounding aqueous medium. Only oil-soluble and digestive enzymes with required separation characteristics are suitable. Oils that can be used for intramuscular injection include but are not limited to sesame oil, olive oil, peanut oil, corn oil, almond oil, soybean oil, cottonseed oil and castor oil.

Co-administration with other pharmaceutical compositions

The pharmaceutical composition can be used alone and/or in combination with other treatments or antibacterial (e.g., anti-Staphylococcus aureus) therapies. For example, other therapeutic agents, such as anti-inflammatory drugs or anesthetics, can be administered to the patient to address other aspects of Staphylococcus aureus infection (e.g., pain, tissue damage) or other diseases that the patient may face. In other embodiments, the pharmaceutical composition described herein and one or more other antibiotics can be administered to the patient. The one or more other antibiotics can be effective against Staphylococcus aureus or other bacteria, or against both (e.g., if the patient suffers from multiple infections), and can be in the same or different form as the pharmaceutical composition of the present invention (e.g., one can be a liquid and one can be a topical antibiotic). The major classes of antibiotics are (1) β-lactams, including penicillins, cephalosporins, and monoamides; (2) aminoglycosides, for example, gentamicin, tobramycin, ethoxycycline, and amikacin; (3) tetracyclines; (4) sulfonamides and trimethoprim; (5) fluoroquinolones, for example, ciprofloxacin, norfloxacin, and ofloxacin; (6) vancomycin; (7) macrolides, which include, for example, erythromycin, azithromycin, and clarithromycin; and (8) other antibiotics, for example, polymyxins, chloramphenicol, and lincosamides.

In some embodiments, the other antibiotic can be a β-lactam antibiotic (e.g., penicillin or penicillin derivative, cephalosporin, monobactam, penicillin, penicillin-like, oxopenicillanic acid, carbapenem or carbapenam, cephem, carbacephem, oxacephem, monobactam). In some embodiments, the other antibiotic can be a β-lactamase inhibitor. In some embodiments, the other antibiotic can be an aminoglycoside antibiotic. In some embodiments, other antibiotics are selected from penicillin or penicillin derivatives, oxacillin, amoxicillin, nafcillin, cloxacillin, methicillin, temocillin, ampicillin, amoxicillin/clavulanic acid (co-amoxiclav), azlocillin, carbenicillin, carbenicillin, mezlocillin, piperacillin, cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefmandole, cefotetan, cefoxitin, ceftriaxone, cefotaxime, cefpodoxime, ceftazidime, cefepime, cefpirome, vancomycin, teicoplanin, telavancin, bleomycin, ramoplanin, decaplanin, oritavancin, dalbavancin. Antibiotic compositions described herein and other antibiotics can be administered alone or in a single dosage form. If administered alone, they can be administered in any order and relative frequency.

biocides and disinfectants

Compositions containing one or more digestive enzymes described herein can also be used as antiseptics and disinfectants, for example, to disinfect inanimate objects and surfaces in (but not limited to) hospitals, health centers, homes, and community settings by eradicating, attenuating, or reducing Staphylococcus aureus in such settings. Antiseptics are antimicrobial agents that are applied to inanimate objects to destroy microorganisms. Antiseptics should generally be distinguished from antibiotics that destroy microorganisms in the body, and from antiseptics that destroy microorganisms on living tissue. Disinfectants are antiseptics that reduce the number of microorganisms to a safe level. One definition of a disinfectant describes a disinfectant as one that must be able to kill 99.999% (referred to as a 5-log reduction) of a specific bacterial test population, and do so within 30 seconds. The main difference between a disinfectant and a sterilizer is that at a specific use dilution, a sterilizer must have a higher pathogen-killing ability than a disinfectant.

In various embodiments as described above, the disinfectants or sanitizers described herein can include one or more digestive enzymes and can optionally include other active and inactive ingredients, including stabilizers (e.g., enzyme stabilizers), other disinfectants known to those skilled in the art, formulation excipients, colorants, flavorings, etc. Those skilled in the art can select other active or inactive ingredients to include in the disinfectant. Examples of other antiseptics include: active chlorine sources (i.e., hypochlorites, chloramines, dichloroisocyanurates, trichloroisocyanurates, wet chlorine, chlorine dioxide, etc.); active oxygen sources (peroxides, such as peracetic acid, potassium persulfate, sodium perborate, sodium percarbonate, and urea peroxide); iodine and iodophor solutions (iodine povidone (povidone-iodine, Betadine), Lugol's solution, iodine tincture, iodinated nonionic surfactants); concentrated alcohols (primarily ethanol, 1-propanol (also known as n-propanol), and 2-propanol (known as isopropanol), and mixtures thereof; and 2-phenoxyethanol and 1- and 2-phenoxypropanols); phenolic substances (e.g., phenol (also known as "carbolic acid"), cresols (combined with liquid potassium soap, known as "lysole"), halogenated (chlorinated, brominated) phenols, such as hexachlorophene, [0014] Examples of the present invention include triclosan, trichlorophenol, tribromophenol, pentachlorophenol, dibromol, and its salts); cationic surfactants, such as some quaternary ammonium cations (e.g., benzalkonium chloride, cetyltrimethylammonium bromide or cetyltrimethylammonium chloride, dodecyldimethylammonium chloride, cetylpyridinium chloride, benzethonium chloride), and others; non-quaternary compounds, such as chlorhexidine, glucoprotamine, octenidine dihydrochloride; strong oxidants, such as ozone and permanganate solution; 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 acid, nitric acid, sulfuric acid, aminosulfuric acid, toluenesulfonic acid) and bases (sodium hydroxide, potassium hydroxide, calcium hydroxide), such as at pH <1 or >13, especially at elevated temperatures (above 60°C). In some cases, the antiseptics described herein consist essentially of one or more digestive enzymes. In some cases, the antiseptics consist essentially of one or more digestive enzymes and do not contain other antiseptics.

Antiseptic compositions containing one or more digestive enzymes described herein can be combined with other ingredients to form a variety of antiseptic products, including but not limited to hand cleansers, mouthwashes, surgical brushes, body sprays, hand sanitizer gels and foams, antiseptic wipes, and similar personal care products. Other types of products include antiseptic foams, creams, mousses, and the like, as well as compositions containing organic or inorganic filler materials, such as emulsions, lotions, creams, pastes, and the like. The compositions can also be used as antibacterial cleansers for hard surfaces, such as sinks and countertops in hospitals, food service areas, and meat processing plants. The antiseptic compositions can also be used as antiseptic aerosols and antiseptic mists. The digestive enzyme compositions of the present invention can be prepared as dilute, ready-to-use compositions or as concentrates that are diluted prior to use. Depending on the nature of the product, various products employing antiseptics can also include fragrances. For example, rosin or lemon fragrances can be used in kitchen wipes because their combination with cleanliness appeals to many consumers. Additionally, gels or aerosols can be aromatized for similar or other reasons.

In one embodiment, the antiseptic composition can be used to prepare an antiseptic wipe. Antiseptic wipes can be used to clean a variety of hard and other surfaces, including, for example, human hands and skin, medical devices and equipment, countertops, floors, walls and windows. The wipes can be made from a variety of fabrics. Fabric is defined as including cloth and paper, as well as woven and non-woven materials. Woven or non-woven fabrics can be made from suitable materials such as rayon, nylon or cotton, and combinations thereof. Examples of non-woven fabrics are described in U.S. Patents 3,786,615, 4,395,454 and 4,199,322, which are incorporated herein by reference. The fabric or paper can be impregnated with the antiseptic solution by any method known in the art. The wipes can be packaged in any manner known in the art, including individual bubble packs or packaged or stacked multi-packs.

In another embodiment, a disinfectant composition containing one or more digestive enzymes can be formulated as a gel or gelatin disinfectant composition. In addition to the disinfectant composition, the gel disinfectant can include a thickener or gelling agent, wherein "thickener" and "gelling agent" are used interchangeably. As used herein, the term "gel" or "gelatin" disinfectant composition refers to a disinfectant liquid material having a viscosity of about 1,000 centipoise to about 100,000 centipoise, or 2,000 centipoise to 50,000 centipoise. In another embodiment, these ranges are not limiting. For example, the viscosity of a hand gel can be much lower than that of a gel used for industrial cleaning or disinfection purposes. Examples of gelling agents or thickening agents include, but are not limited to, natural gums such as guar gum and guar gum derivatives, synthetic polymers, clays, oils, waxes, aloe vera gel, acrylate homopolymers, acrylate copolymers, carbomers, cellulose, cellulose derivatives, algin, algin derivatives, water-insoluble C8-C20 alcohols, carrageenan, fumed silica, and mixtures thereof. The gelling agent may be present in the gelatin disinfectant composition in an amount of about 0.1% to 50% by weight of the gelatin composition. In another embodiment, the gelling agent is present in an amount of 0.25% to 10% by weight of the gelatin composition. The amount of the gelling agent may depend on a variety of factors, including the type of gelling agent and the desired viscosity of the gel. Gelatin disinfectants can be used in a variety of applications, including human skin disinfection, for example, gel hand disinfectants and hard surface disinfectants. In one embodiment, the antiseptic composition can be mixed with natural aloe vera gel to form an antiseptic aloe vera formulation. Such formulations can be applied to burns, skin infections, and other irritations. Aloe vera may serve as a thickening agent, or, depending on the desired viscosity of the antiseptic gel, another of the above-mentioned thickening or gelling agents may also be included.

In another embodiment, the antiseptic composition containing one or more digestive enzymes can be formulated into an antiseptic foam or foam composition. The antiseptic foam or foam composition comprises the antiseptic composition and a foaming agent. Any foaming agent known in the art can be used depending on the desired application and characteristics of the resulting antiseptic foam. Like the antiseptic composition, the antiseptic foam of the present invention can be used for both human (e.g., hand washing) and industrial applications.

In another embodiment, a biocide composition containing one or more digestive enzymes can be formulated as a biocide aerosol or mist. Nebulization, also known as thermal aerosolization, is the process of dispersing a biocide into an aerosolized form. Aerosolized microparticles of biocide remain suspended in the air for a period of time, disinfecting the air itself and surfaces, including hard-to-reach areas of buildings, such as air vents. The aerosolized microparticles of biocide can have a particle size of about 5 μm to about 200 μm. In another embodiment, the aerosolized microparticles can have a particle size of about 20 μm to about 150 μm.

Methods for evaluating the bactericidal ability of a particular composition are known to those skilled in the art. Typically, the relative effectiveness of a bactericide can be determined by comparing its bactericidal ability to that of known bactericides. Phenol is a known bactericide standard, and the corresponding rating system is called the "phenol coefficient." The bactericide to be tested is compared to phenol on a standard microorganism, such as Escherichia coli or Staphylococcus aureus. Bactericides that are more effective than phenol have a coefficient > 1. Bactericides that are less effective than phenol have a coefficient < 1. To calculate the phenol coefficient, the concentration of the test compound that 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 or absence of a standard amount of added organic matter. One specific phenol coefficient test uses the Rideal-Walker method. The United States Department of Agriculture also has a method for generating USDA coefficients. Other methods are known to those skilled in the art.

The antiseptics described herein can have a phenol coefficient greater than 1 for S. aureus, 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 from about 2 to about 20, e.g., from about 4 to about 10, from about 2 to about 6, from about 6 to about 12, or from about 10 to about 15.

The bactericides described herein can have a phenol coefficient for E. coli greater than 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. In some cases, the phenol coefficient is from about 2 to about 20, e.g., from about 4 to about 10, from about 2 to about 6, from about 6 to about 12, or from about 10 to about 15.

The antiseptics or disinfectants described herein can be antiseptics and/or bacteriostatics against Staphylococcus aureus. In some embodiments, the antiseptics or disinfectants described herein can be antiseptics and/or bacteriostatics against MRSA or VRSA, or both.

detergent

The antiseptic compositions described herein containing one or more digestive enzymes can also be formulated as detergents. Detergents are materials used to aid in cleaning. Detergents can include one or more of the above-mentioned digestive enzymes in a formulation suitable for maintaining their antibacterial properties, and can include optional active or inactive ingredients, such as enzyme stabilizers, other antiseptics, bleaching agents, soaps, surfactants, colorants and fragrances, abrasives, pH adjusters, acids, bases, or corrosive compounds, water softeners, oxidants, suspending agents, fabric softeners, foaming or defoaming agents, viscosity modifiers, corrosion inhibitors, and fluorescent brighteners. The detergents described herein can be antibacterial and/or antiseptic for Staphylococcus aureus, and in some embodiments, can be antibacterial and/or antiseptic for MRSA or VRSA, or both.

Detergent compositions containing one or more digestive enzymes, particularly those for use with water, may include other ingredients such as surfactants to "cut" (dissolve) grease and wet surfaces, abrasives for rinsing, substances to adjust pH or affect performance or stability, acids for descaling or caustics for breaking down organic compounds, water softeners to counteract the effects of "hard" ions, oxidizing agents (oxidants) for bleaching, disinfecting and breaking down organic compounds, non-surfactant materials to keep soils in suspension, and detergents for digesting proteins, fats or carbohydrates in dyes or for altering fabrics. Sensory enzymes, components that modify the foaming properties of cleaning surfactants or serve to stabilize or counteract foaming, components that increase or decrease the viscosity of a solution, or components that maintain other ingredients in a detergent used as an aqueous solution or gel, components that affect the aesthetics of the items to be cleaned or the detergent itself before or during use, such as fluorescent brighteners, fabric softeners, colorants and fragrances, corrosion inhibitors to counteract damage to equipment in which the detergent is used, ingredients that reduce skin damage or provide benefits to the skin when the detergent is used with bare hands on inanimate objects or for cleaning the skin, and preservatives to prevent deterioration of other ingredients.

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

The digestive enzymes of the detergent compositions of the present invention may be stabilized using conventional stabilizers, for example polyols, such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives, such as aromatic borate esters or phenylboronic acid derivatives, such as 4-formylphenylboronic acid, and the compositions may be formulated as described, for example, in WO 92/19709 and WO 92/19708.

preservative

Various embodiments of compositions containing one or more digestive enzymes can also be used as antiseptics, for example, to reduce, eradicate, or attenuate Staphylococcus aureus on the skin or other living tissue. Antiseptics are antimicrobial substances that are applied to living tissue/skin to reduce the likelihood of infection, sepsis, or corruption. They are generally distinguished from antibiotics, which destroy bacteria in the body, and from bactericides, which destroy microorganisms found on non-living objects. Some antiseptics are true bactericides that destroy microorganisms (bactericidal), while other antiseptics are bacteriostatics that only prevent or inhibit their growth.

The antiseptics described herein can be particularly useful in hospitals or healthcare settings, for example, in preparations for washing hands, face, or body; as antiseptics for use before and after surgical procedures; and as antiseptics for cleaning and treating wounds, such as trauma or surgical wounds. In community settings, the antiseptics are used in any setting where community-acquired infections are a concern, such as childcare facilities, large research institutes, schools, and the like. The antiseptics can also be used in home settings, in preparations for washing hands, face, or body, or for treating wounds.

In various embodiments described above, the preservative may include one or more digestive enzymes and may optionally include one or more active or inactive ingredients, such as other preservatives, stabilizers (e.g., enzyme stabilizers), colorants, flavorings, and other excipients known to those skilled in the art. Examples of preservatives that include one or more digestive enzymes include alcohols (e.g., ethanol, 1- and 2-propanol, or mixtures thereof), quaternary ammonium compounds (benzalkonium chloride, cetyltrimethylammonium bromide, cetylpyridinium chloride, and benzethonium chloride), boric acid, chlorhexidine gluconate, peroxides (e.g., hydrogen peroxide, benzoyl peroxide); iodine and iodophor solutions (e.g., povidone-iodine), octenidine dihydrochloride, phenol (carbolic acid) and phenol derivative compounds, sodium chloride, sodium hypochlorite, and calcium hypochlorite.

The antiseptics described herein can be bactericidal and/or bacteriostatic against S. aureus, and in some embodiments, can be bactericidal and/or bacteriostatic against MRSA or VRSA, or both.

In one embodiment, the antiseptic composition contains one or more digestive enzymes and optionally one or more anti-inflammatory agents, analgesics, or anesthetics.

Anti-inflammatory agents may include steroidal or nonsteroidal anti-inflammatory compounds. In one embodiment, the antiseptic composition includes one or more steroidal compounds. In one embodiment, the antiseptic composition may include a nonsteroidal anti-inflammatory drug anti-inflammatory agent. Non-limiting examples of suitable nonsteroidal anti-inflammatory drugs include aspirin (Anacin, Ascriptin, Bayer, Bufferin, Ecotrin, Excedrin), choline and magnesium salicylate (CMT, Tricosal, Trilisate), choline salicylate (Artliropan), celecoxib (Celebrex), diclofenac potassium (Cataflam), diclofenac sodium (Voltaren, Voltaren XR), diclofenac and misoprostol (Arthrotec), diflunisal (Dolobid), etodolac (Lodine, Lodine XL), fenoprofen calcium (Nalfon), flurbiprofen (Ansaid), ibuprofen (Advil, Motrin, Motrin IB, Nuprin), indomethacin (Indocin, Indocin SR), ketoprofen (Actron, Orudis, Orudis KT, Oruvail), magnesium salicylate (Arthritab, Bayer Select, Doan's Pills, Magan, Mobidin, Mobogesic), meclofenamic acid sodium (Meclomen), mefenamic acid (Ponstel), meloxicam (Mobic), nabumetone (Relafen), naproxen (Naprosyn, Naprelan), naproxen sodium (Aleve, Anaprox), oxaprozin (Daypro), piroxicam (Feldene), rofecoxib (Vioxx), salsalate (Amigesic, Anaflex 750, Disalcid, Marthritic, Mono-Gesic, Salflex, Salsitab), sodium salicylate, sulindac (Clinoril), tolmetin sodium (Tolectin), valdecoxib (Bextra), or a combination thereof.

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

Typical methods for testing candidate preservative compositions are provided below. It will be appreciated by those skilled in the art that other methods for testing preservative compositions are known in the art and are also suitable for testing candidate preservative compositions.

In vitro methods for determining the ability of a candidate antiseptic composition to kill or inhibit microbial cells (e.g., Staphylococcus aureus) are known in the art. Typically, these methods involve contacting a culture medium of cells of interest with a candidate antiseptic composition at various concentrations and monitoring the growth of the cell culture medium relative to an untreated control culture medium. If desired, such tests may also include a second control culture medium containing cells contacted with a known antimicrobial agent.

For example, the ability of a candidate antiseptic composition to inhibit the growth of microbial cells can be readily determined by measuring the minimum inhibitory concentration (MIC) of the antiseptic composition. The MIC is defined as the lowest concentration that inhibits the growth of an organism within a predetermined range. For example, the MIC ₁₀₀ value is defined as the lowest concentration that completely inhibits the growth of an organism, while the MIC ₉₀ value is defined as the lowest concentration that inhibits 90% of growth, and the MIC ₅₀ value is defined as the lowest concentration that inhibits 50% of growth. MIC values are sometimes expressed as a range, for example, the MIC ₁₀₀ of an antiseptic composition can be expressed as the concentration at which no growth is observed, or as the range between the concentration at which no growth is observed and the immediately following dilution concentration.

The antibacterial MIC of a candidate antiseptic composition can be determined using a liquid media macro- or microdilution assay (see Amsterdam, D. (1996) "Susceptibility testing of antimicrobials in liquid media," pp. 52-111. In Loman, V., ed., Antibiotics in Laboratory Medicine, 4th ed. Williams and Wilkins, Baltimore, MD). Standardized antibacterial susceptibility testing is provided by the National Committee for Clinical Laboratory Standards (NCCLS), NCCLS, 2000; Document M7-A58.

In a typical liquid culture microdilution method, the candidate antiseptic composition is diluted in culture medium in a sterile, covered 96-well microtiter plate. A single colony cultured overnight is diluted in sterile culture medium so that after inoculation, each well in the microtiter plate contains an appropriate number of colony-forming units (CFU) AnI (usually about 5x10 ⁵ CFU/ml). Culture medium (without bacteria) is also included as a negative control for each plate, and a known antibiotic is usually included as a positive control. The inoculated microtiter plate is then incubated at an appropriate temperature (e.g., 35°C-37°C for 16-48 hours). The turbidity of each well is then measured by visual inspection and/or absorbance or optical density (OD) at 595nm or 600nm using a microplate reader and used as an indicator of the extent of bacterial growth.

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

The ability of any bactericide, detergent, disinfectant and antiseptic composition to kill or inhibit the growth of Staphylococcus aureus can be tested using methods known in the art, including the various methods described above. Methods and protocols for testing compositions against specific bacteria can be found, for example, in Official Methods of Analysis of the AOAC, 15th Edition, Arlington Virginia 22201, USA (Association of Official Analytical Chemists (AOAC), Inc. 1990), Designation: E 1054-91 "Practices for Evaluation Inactivators of Antimicrobial Agents Used in Disinfectant, Sanitizer, Antiseptic, or Preserved Products" (American Society for Testing and Materials (ASTM), 1991). As is known in the art, in vitro time-kill assessments can be performed using a modification of the method described in Draft European Standard, prEN 12054, "Chemical Disinfectants and Antiseptics - Products for Hygienic and Surgical Handrub and Handwash - Bactericidal Activity - Test Method and Requirements (1995)". Other methods that can be used include log reduction assays, proliferation assays, AOAC use dilution assays, or zone of inhibition assays. Other methods are described in the Examples below.

Reagent test kit

The present invention also provides kits. Typically, the kits comprise one or more compositions described herein. In certain embodiments, the kits may comprise one or more delivery or administration systems, e.g., for delivering or administering the compositions described above, and/or instructions for use of the kit (e.g., instructions for treating a patient; instructions for disinfecting a surface). In another embodiment, the kit may comprise a composition described herein and a label, e.g., a label indicating the amount to be administered to a patient suffering from a Staphylococcus aureus infection, or a label indicating how to use the composition as a sterilant, disinfectant, detergent, or preservative.

How to use

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

The digestive enzymes provided herein can be used to treat diseases and disorders associated with SA bacterial infection or infection involving SA bacteria. Certain embodiments include SA infections associated with medical devices or implants, such as catheters, grafts, prosthetic heart valves, artificial joints, and the like. 1-5% of intrinsic prosthetics become infected, which typically requires removal or replacement of the prosthesis. In some embodiments, a composition containing one or more digestive enzymes can be applied to the medical device before or after the device is prepared but before insertion. Infection during hemodialysis is another source of infection. Infection is the second leading cause of death in chronic hemodialysis patients. Approximately 23% of bacteremias are attributed to access site infection. Most transplant infections are caused by coagulation-positive (SA) and coagulation-negative Staphylococci. To combat infection, digestive enzymes alone or in combination with antibiotics can be applied as an ointment or cream to the dialysis site before each hemodialysis session.

In another embodiment, the compositions provided herein can be used to treat or prevent nasal and extranasal carriage of Staphylococcus aureus. Infection with this organism can result in pustular lesions or infected wounds. It is also associated with increased infection rates following cardiac surgery, hemodialysis, orthopedic surgery, and neutropenia, both disease-induced and due to physician-administered treatment. Nasal and extranasal carriage of Staphylococci can lead to hospital outbreaks of the same Staphylococcus aureus strain that colonizes the nasal passages or extranasal sites of patients or hospital workers. Much attention has been paid to eradicating nasal colonization, but treatment results are generally unsatisfactory. Topical application of antimicrobial substances, such as bacitracin, tetracycline, or chlorhexidine, results in inhibition of nasal colonization, rather than eradication.

The digestive enzymes alone or in combination with antibiotics are preferably administered intranasally as an ointment, cream or solution formulated for nasal application. This can be applied once or repeatedly until Staphylococcus colonization is reduced or eliminated.

In some embodiments, the compositions provided herein can be used to treat or prevent burn wound infections. Although the incidence of invasive burn wound infections has decreased significantly, infection remains the most common cause of morbidity and mortality in patients with extensive burns. Infection is a major determinant of wound healing, complication rates, and prognosis in burn patients. A major cause of infection is SA. Frequent debridement and the establishment of an epidermis, or a replacement, such as a graft or skin replacement, is essential for infection prevention.

Digestive enzymes, alone or in combination with other antibiotics and/or anesthetics or anti-inflammatory drugs, can be applied to burn wounds and/or systemically as ointments or creams. Topical application can prevent systemic infection or eradicate epidermal infection after epidermal grafting. Application to the skin can be once daily or at any time when dressings are changed. Systemic administration can be by intravenous, intramuscular, or subcutaneous injection or infusion. Other routes of administration are also possible.

Surgical wounds, particularly wounds associated with foreign matter, such as sutures, can also be treated with the compositions provided by the present invention. Up to 71% of all nosocomial infections occur in surgical patients, with 40% of infections occurring at the surgical site. Despite efforts to prevent infection, it is estimated that between 500,000 and 920,000 surgical wound infections complicate approximately 23 million surgical procedures performed in the United States each year. The infecting organisms vary, but Staphylococcus aureus is an important organism in these infections.

Digestive enzymes, alone or in combination with antibiotics, anesthetics, or anti-inflammatory drugs, can be applied to the wound site as an ointment, cream, or liquid, or as a liquid before and during wound closure. After closure, a composition containing one or more digestive enzymes can be applied during dressing changes. For infected wounds, the composition can be applied topically and/or systemically.

In some embodiments, the digestive enzymes provided herein can be used to treat or prevent nosocomial pneumonia. Nosocomial pneumonia accounts for nearly 20% of all hospital infections. Patients at greatest risk for developing nosocomial pneumonia are those in intensive care units, those with altered levels of consciousness, elderly patients, those with chronic lung disease, those receiving oxygen, smokers, and postoperative patients. In severely affected patients, nosocomial pathogens that are resistant to multiple antibiotics may cause pneumonia.

One of the main organisms causing this infection is SA. Digestive enzymes, alone or in combination with other antibiotics, can be administered orally, by aerosol, or systemically to treat pneumonia. Administration can be once a day or multiple times a day. In some embodiments, the composition can be administered directly into the lungs via inhalation or via an endotracheal tube device.

Cystic fibrosis (CF) is the most common genetic disease in the Caucasian population. Lung disease is the most common cause of premature death in patients with CF. While the optimal antimicrobial therapy for CF is unknown, the introduction of better antipseudomonal antibiotics is generally considered to be the major factor in extending the life expectancy of CF patients. The most common organism associated with lung disease in CF is SA.

Digestive enzymes alone or in combination with other antibiotics can be administered orally or systemically or via aerosol to treat cystic fibrosis. Preferably, treatment is administered for 3 weeks for acute lung disease and/or 2 weeks every 2-6 months to prevent acute exacerbations.

Although it can involve any part of the endocardium or any prosthetic material inserted into the heart, infective endocarditis results from infection of the heart valve cusps. If left untreated, it is often fatal. Most infections are hospital-acquired and are caused by pathogens that are increasingly resistant to available drugs. A major cause of infection is SA.

Although systemic administration is preferred, digestive enzymes alone or in combination with other antibiotics can be administered orally or systemically to treat endocarditis. Treatment preferably lasts 2-6 weeks and can be administered as a continuous infusion or multiple times a day.

During the initial acute phase of osteomyelitis, the vascular connections of the bone are compromised by infection that extends into the surrounding tissues. Within the necrotic and ischemic tissue, bacteria are difficult to eradicate, even after a vigorous host response, surgery, and/or antibiotic therapy. The main infecting organisms are SA and Escherichia coli.

Digestive enzymes can be administered systemically, alone or in combination with other antibiotics. Treatment can last 2-6 weeks. Antibiotics can be administered as a continuous infusion or multiple times daily. Compositions containing one or more digestive enzymes can be used as antibiotic-impregnated cements or as antibiotic-coated beads for joint replacement procedures.

The present invention also provides for the treatment and prevention of sepsis in immunocompromised hosts. Treatment of infections in immunocompromised patients due to chemotherapy-induced neutropenia and immunosuppression associated with organ or bone marrow transplantation is particularly challenging. Neutropenic patients are particularly susceptible to bacterial infections, and if infection is suspected, antibiotic therapy should be initiated promptly to cover potential pathogens. Organisms that may cause infections in neutropenic patients include SA and E. coli.

The digestive enzyme composition alone or in combination with antibiotics is preferably administered orally or systemically for 2-6 weeks. The digestive enzyme can be administered by continuous instillation or multiple times a day.

The antiseptic, disinfectant, and detergent compositions described herein can be applied to inanimate surfaces in appropriate amounts and manners to reduce or eradicate Staphylococcus aureus on such surfaces and thereby reduce or prevent Staphylococcus aureus transmission and/or infectivity. The concentration, timing, and frequency of treatment are parameters determined by one skilled in the art.

Any surface can be disinfected with the composition, including various medical devices used in hospitals or health care institutions. As used herein, "medical device" refers to any device used for a patient, such as an implant or prosthesis. Such devices include, but are not limited to, synthetic vascular grafts, blood monitoring devices, artificial heart valves, scalpels, knives, scissors, spatulas, expanders, clamps, tweezers, metal mirrors, retractors, sutures, surgical mesh, chisels, drill bits, levels, rasps, saws, splints, calipers, clamps, pliers, hooks, lancets, needles, cannulas, curettes, depressors, dilators, openers, articulators, extractors, probes, staples, catheters, stents, tubes, bowls, trays, sponges, snares, spoons, syringes, pacemakers, screws, plates, and nails.

Other community and hospital or healthcare surfaces suspected of harboring S. aureus can be disinfected, including large or small surfaces (floors, tables, switching tables, beds, ventilation systems, tubs, door handles, counters, food service surfaces, etc.). The composition can also be used to wash hands or bodies, for example, at points of entry to community institutions, hospital settings, or bathrooms.

The compositions provided herein can be used in the same manner as conventional antiseptics or in any location where microorganisms are not desired. For example, they can be used as surface antiseptics in medical device coatings, fabric coatings to inhibit bacterial growth or repel mosquitoes, for example, on aircraft or in community or hospital settings, as air purification filters in water purification systems, as shampoo and soap components, as food preservatives, cosmetic preservatives, as media preservatives, in herbicides or pesticides, for example, in silicone sealants, as building material components, and in animal product processing, for example, curing animal hides or in slaughterhouses.

For these purposes, the digestive enzymes, used alone or in combination with other antiseptics or detergents, are typically included in the composition and applied with a suitable applicator. They can also be added or impregnated into the material, such as an air filter, during preparation, or applied to the material or object.

For example, in some embodiments, the compositions described herein can be mixed with the material during or subsequently during the preparation of the material. In addition, the compositions can be applied to the surface of the material during or subsequently during the preparation of the material. As used herein, the term "appropriate material" refers to that the digestive enzyme can be applied or incorporated into any material thereon or therein, thereby incorporating antimicrobial activity in/on the material. For example, a gauze pad on a bandage can be prepared from a composition containing one or more digestive enzymes in or on the gauze, and/or an ointment containing one or more digestive enzymes can be used for gauze, thereby incorporating antimicrobial activity on the gauze. Examples of suitable materials that can use digestive enzymes include, but are not limited to, food, liquid, medical devices (such as surgical instruments), beads, films, monofilaments, non-woven fabrics, sponges, cloth, knitted fabrics, staple fibers, tubes, hollow fibers, artificial organs, catheters, sutures, membranes, bandages, and gauze. Digestive enzymes can be used for or incorporated into many other types of materials suitable for medical treatment, health, food safety, or environmental cleaning activities.

Veterinary applications

The compositions described herein in the form of pharmaceuticals or antiseptics/disinfectants, detergents, or antiseptics can also be used in various veterinary applications. For example, many mammals, including dogs, cats, and cows, can be infected with Staphylococcus aureus or serve as carriers of the bacteria. Mastitis in cows is commonly caused by infection with Staphylococcus aureus. Therefore, the compositions of the present invention can be used to treat animals infected with or suspected of carrying Staphylococcus aureus to treat the infection or prevent transmission to other animals, including humans. Antiseptic and detergent compositions can be used to treat animal housing and equipment that come 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 can be used to prevent and treat mastitis, particularly dairy cow mastitis, although any mastitis can be treated using the digestive enzymes provided herein. Dairy cow mastitis is an inflammation of the breast caused by bacterial infection, mechanical trauma or chemical trauma in the udder. It is believed that contagious mastitis is mainly caused by SA and Streptococcus agalactiae. Environmental mastitis can be caused by a variety of different bacteria, including but not limited to Klebsiella pneumoniae (K.pneumoniae), Escherichia coli (E.coli), Klebsiella oxytoca, Enterobacter aerogenes (Enterobacter aerogenes), Streptococcus uberis (Streptococcal uberis), Streptococcus bovis (Streptococcal bovis) and Streptococcus dysgalactiae (Streptococcal dysgalactia).

In some embodiments, the prevention of bovine mastitis can include daily dipping of the teats with a solution containing one or more digestive enzymes. In some embodiments, the solution containing one or more digestive enzymes can also include one or more other antibiotics. When an infection does occur, intramammary instillation of one or more digestive enzymes can be performed. As above, other antibiotics can also be administered in combination with the digestive enzymes. Typically, the digestive enzymes are administered via intramammary injection; however, effective doses can be administered parenterally, transdermally, by implantation, and by dipping. In some embodiments, bovine mastitis can be treated by administering an effective amount of one or more digestive enzymes to the cow. Administration can be prophylactic, i.e., all cows in the herd are treated with the digestive enzyme composition, or administration can be performed when an infection occurs in a single cow.

The introduction of SA bacteria and E. coli can occur during the preparation of beef, poultry, fish, and pork products. Therefore, in some embodiments, one or more digestive enzymes can be administered to animals (e.g., cows, chickens, turkeys, fish, or pigs) to reduce the presence of E. coli or SA bacteria in the animal's intestines, thereby reducing infection. Administration can be performed by any available method, including injection and by introducing one or more digestive enzymes into feed.

In some embodiments, administering one or more digestive enzymes to an animal can be used to prevent or reduce the spread of SA bacteria or E. coli 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, preventing or reducing the spread of SA bacteria from pigs to humans can be achieved by administering one or more digestive enzymes to pigs to reduce the number of SA bacteria present in the pigs, thereby reducing the spread of SA bacteria to humans.

Food applications

The present invention also provides methods for preventing SA or E. coli infection in beef, poultry, fish, and pork. The beef processing process is a common point of contamination: during the slaughter process, the contents of the intestines or fecal material on the skin can mix with the meat, thereby allowing bacteria to multiply under warm, humid conditions. If the infected portion is subsequently ground, the bacteria leave the cut surface and enter the ground block. In addition, in the preparation of ground beef, meat from multiple cows is often ground together, allowing contamination from a single animal to infect the entire batch of ground beef. Therefore, in some embodiments, one or more digestive enzymes can be administered to cows to reduce the presence of bacteria in the cow's intestines, thereby reducing infection. Administration can be performed by any available method, including injection and by introducing one or more digestive enzymes into the feed. In another embodiment, reducing contamination of meat during slaughter and grinding can be provided by utilizing a spray containing one or more digestive enzymes provided herein. For example, such a spray can be used to sterilize slaughtering and grinding equipment, or to sterilize the ground meat itself. The methods described above can also be used during the slaughtering and preparation of poultry, fish, and pork products (e.g., by administering one or more digestive enzymes to poultry, fish, and/or pigs before slaughter).

Escherichia coli and SA bacteria can also be spread by unwashed fruits and vegetables. Therefore, the present invention also provides a method for washing fresh fruits and vegetables using a solution, detergent, aerosol, smoke, gel or powder containing one or more digestive enzymes provided herein. A product detergent is a solution for rinsing the surface of the product, typically in contact with the product for about 30 seconds to about 5 minutes. A product soaker is a solution in which the product is immersed for about 30 seconds to about 30 minutes. However, unless otherwise distinguished, these terms and solutions are used interchangeably. It should be understood that the temperature at which the product is washed or soaked will affect the duration required to reduce or inactivate the bacteria thereon, and warmer temperatures result in a shorter processing time.

The washing or soaking solutions described herein can be used to reduce bacteria counts, particularly bacterial pathogens, on fruits, vegetables, raw cut meat products, fish, shellfish, at the consumer level (home), in commercial food preparation environments, on fruits and/or vegetables prior to juicing, in wholesale or retail operations, and/or at the harvest, in slaughterhouses or abattoirs, on fishing vessels, etc. The methods of the present invention are particularly useful for inactivating E. coli on the surfaces of fresh fruits and vegetables.

Methods for assessing activity

The various activities of the compositions described herein can be assessed by methods known to those skilled in the art. For example, enzyme activity can be assessed using standard enzyme activity assays. The minimum inhibitory concentration (MIC) of the compositions can also be assessed by methods known to those skilled in the art as described above. Other tests, including the phenol coefficient, are also well known to those skilled in the art. See also the following examples.

Example

Exemplary liquid 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 can be diluted with various diluents (water, saline, phosphate buffer solutions, pH stabilizing solutions) and optionally other active or inactive additives (enzyme stabilization systems, buffers, colorants, disinfectants, detergents, biocides, preservatives) to form exemplary liquid compositions for the uses described herein. In some embodiments, the dry enzyme composition can be diluted at a ratio of mg of dry enzyme composition to ml of total diluent ranging from 1 mg enzyme composition: 1 ml total diluent to 1 mg enzyme composition: 10,000 ml total diluent, or at any value within this range, 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 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 can be mixed with various active or inactive dry ingredients and additives (e.g., dry detergents, bactericides, preservatives, and disinfectants, such as ethoxylated alkyl sulfates, SDS, sodium lauryl sulfate, dodecylbenzene, sodium 4-dodecylbenzene sulfonate, enzyme stabilization systems, excipients, colorants) to form an exemplary solid composition. In some embodiments, the dry enzyme composition can be mixed with the additive in a ratio ranging from 1 mg enzyme composition: 1 mg total additive to 1 mg enzyme composition: 10,000 mg total additive, or any value within this range, 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 of protease, about 40 USP units/mg of lipase, and about 250 USP units/mg of amylase can be mixed with a variety of carriers suitable for topical pharmaceutical formulations at a ratio of about 1 mg of enzyme composition: 1 mg of carrier to 1 mg of enzyme composition: about 200 mg of carrier, or any ratio within this range (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 used and the carrier is petrolatum.

Bacterial Limit Test - Evaluating Bactericidal and Bacteriostatic Properties

During bacterial limit testing of a dried porcine pancreatic enzyme composition containing approximately 200 USP units/mg of protease activity, approximately 40 USP units/mg of lipase activity, and 250 USP units/mg of amylase activity, the inventors unexpectedly discovered that Staphylococcus aureus did not grow in the presence of various dilutions of this material. Correspondingly, very low to non-existent CFUs of S. aureus were recovered from a positive control spiked with S. aureus. Dilution bacterial limit testing of the dried composition in both unencapsulated and lipid-encapsulated (20% soybean oil by weight of the composition) forms demonstrated very low to no recovery of bacteria from a positive control spiked with a known number of S. aureus CFUs. The lack of recovery during this recovery process demonstrates the bacteriostatic and/or bactericidal properties of the composition.

Materials and methods

Sample material: Unencapsulated porcine pancreatic enzyme concentrate (uPEC) isolated from pigs (wild boars) was produced by a commercial supplier (Scientific Protein Labs) and contained approximately 200 U/mg of protease activity, 40 U of lipase activity/mg, and 250 U of amylase activity/mg. A lipid-encapsulated form (ePEC) of this material was obtained by coating enzyme microparticles with organic oil at approximately 20% by weight of the final microparticles using a modified fluidized bed process and fully hydrogenated, highly purified organic oil (fully hydrogenated soybean oil).

Methods: Both ePEC and uPEC were subjected to standard microbiological analysis for the detection of microorganisms. Using the microbial limit test according to the USP method, neither composition showed any obvious contamination. In addition, both samples were negative/10g for Salmonella species and E. coli. Briefly, the procedures for verifying the microbial suitability of the test compositions (i.e., estimating the total number of viable microorganisms and exempting specific organisms) are outlined in USP, Chapter 61, "Microbial Limit Tests." USP61 outlines preliminary testing, during which the test parameters are determined, wherein the test composition itself no longer inhibits the reproduction of viable organisms. USP1227 ("Validation") also provides guidance for validating the recovery method. Methods for evaluating the total aerobic microbial count, E. coli, and Salmonella species were performed.

Total Aerobic Microbial Count: Preparation of Test Dilutions: Prepare batch dilutions of ePEC and uPEC at 1:10, 1:50, 1:100, and 1:200 in tryptic soy broth containing 4% polysorbate 20 and 0.5% lecithin. The batch test sample dilutions are then divided into individual 10 ml aliquots and inoculated with a low number of colony-forming units (CFU<100/ml) of the appropriate microorganism (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica). Duplicate 1 mm inoculum aliquots are smeared on appropriate solid medium (agar). Positive controls are prepared, inoculated, and smeared in a manner similar to the test samples. Negative controls are prepared, inoculated, and smeared in a manner similar to the test samples using sterile reagents. Incubate the plates at 30°C to 35°C for two days. Calculate the recovery rate at the end of the period. Recovery of the inoculum organisms must be at least 70% of that of the positive control to demonstrate that the test composition does not inhibit growth. Triphenyltetrazolium chloride was used to count the plates.

For testing of Salmonella species: a dilution prepared with a lactose liquid medium containing 4% polysorbate 20 and 0.5% lecithin is inoculated with <=100 CFU of enteric Salmonella. The inoculated dilution is incubated at 30°C to 35°C for 24 hours, and then 1 ml is transferred to a selenite cystine and tetrathionate liquid medium (Tetrahionate broth). The selected liquid medium is incubated at 30°C to 35°C for 18 hours, and then streaked onto brilliant green, bismuth sulfite, xylose, lysine and deoxycholate agar. The selected agar plate is incubated at 30°C to 35°C for 24 hours. The plate is observed for characteristic colonies of the Salmonella species. Once observed, the API 20e biochemical identification test is used to confirm that the typical colony is a Salmonella species.

For E. coli testing: Inoculate a dilution prepared in lactose broth containing 4% polysorbate 20 and 0.5% lecithin with <100 CFU of E. coli. Incubate the inoculated dilution at 30°C to 35°C for 24 hours, then streak onto MacKonkey agar. Observe the plate for characteristic E. coli colonies. Once observed, confirm that the representative colonies are E. coli using the API 20e biochemical identification test.

result

Total Aerobic Count: The results of the test using uncoated PEC (uPEC) are shown below in Table 1. As shown, the percent recovery of S. aureus was 0% for dilutions of 1:50, 1:100, and 1:200, demonstrating the bactericidal and/or bacteriostatic effect of uPEC on S. aureus.

Table 1

The recovery of uPECs from E. coli is also shown in Table 1. For a dilution of 1:50, the recovery was 0%, for a dilution of 1:100, the recovery was 2%, and for a dilution of 1:200, the recovery was 2%.

The uPEC recovery of Salmonella enterica is also shown in Table 1. For a dilution of 1:50, the recovery was 73%, for a dilution of 1:100, the recovery was 94%, and for a dilution of 1:200, the recovery was 85%.

The results of the lipid encapsulated PEC (ePEC) assay are shown below in Table 2. As shown, for a 1:50 dilution, the recovery of S. aureus was 29%, for a 1:100 dilution, the recovery was 0%, and for a 1:200 dilution, the recovery of S. aureus was also 0%.

Table 2

The ePEC recovery of E. coli is also shown in Table 2. 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 ePEC recovery of Salmonella enterica is also shown in Table 2. For a dilution of 1:50, the recovery was 87%, and dilutions of 1:100 and 1:200 were not tested.

The positive controls reported in Tables 1 and 2 clearly demonstrate that the growth media for all microbial cultures was functionally effective. In Table 1, it can be seen that the uPEC composition was very effective against both S. aureus and E. coli. This is confirmed by the fact that the positive spiked controls did not meet the USP standard for positive recovery (recovery of at least 70% of the spiked sample CFU).

It is also important to note that the antibacterial/bactericidal activity exhibited species specificity. Samples of Salmonella enterica exhibited excellent recoveries using both coated and uncoated PECs. Because bacteria share common cell wall and membrane structures, such as lipids and peptidoglycans, it is possible that these results point to sensitivity to more species-specific cellular components, such as proteins. If lipase or amylase action alone were sufficient to induce bacterial death or growth inhibition, it would be unlikely that Salmonella enterica recoveries would be observed at such robust levels. It is possible that species-specific proteins of Staphylococcus aureus and Escherichia coli share similar peptide sequences and appropriate local tertiary structures, allowing enzymatic attack by one or more proteases present in PECs, leading to subsequent bacterial destruction. However, these results do not rule out a multi-stage event in which the action of lipases and amylases on the bacterial cell wall and membrane first contacts extracellular or transmembrane proteins that possess appropriate primary and tertiary structures to support their enzymatic degradation by one or more PEC proteases.

These results demonstrate a clear antibacterial effect for both lipid-coated and uncoated PECs. Because this assay relies on an endpoint readout of colony growth, and the test bacteria remain present within the PECs, it is not possible to rule out the possibility of an antibacterial effect based solely on these results. Therefore, the inability to recover sufficient numbers of viable colony-forming units could be the result of the persistent inhibitory presence of the PEC material, as opposed to inducing bacterial death. Therefore, additional experiments were performed to more definitively assess the bactericidal capacity of the PECs.

Materials and Methods: The uncoated PEC formulations described above were used to evaluate the bactericidal activity of the formulations.

Preparation of Test Dilutions: Prepare batch dilutions of uPECs at 1:100 and 1:200 in Tryptic Soy Broth containing 4% polysorbate 20 and 0.5% lecithin. The batch dilutions are then divided into individual 10 ml aliquots and inoculated with a low number of colony-forming units (CFU<100/ml) of the appropriate microorganism. Duplicate 1 mm inoculated aliquots are smeared onto appropriate solid medium (agar). Positive controls are prepared, inoculated, and smeared similarly to the test samples. Negative controls are prepared, inoculated, and smeared similarly to the test samples using sterile reagents. Incubate the plates at 30°C to 35°C for two days. After the initial incubation, the culture material is collected, washed in phosphate-buffered saline (PBS), and filtered to remove PECs. The material is resuspended in the diluted Tryptic Soy Broth described above and replated onto fresh solid (agar) medium before incubation for an additional two days. At the end of this period, colonies are counted and the percent recovery is calculated. At the end of this period, the recovery rate is calculated. Recovery of the inoculum organism must be at least 70% to show no growth inhibition. Triphenyltetrazolium chloride is used to count the plates.

For E. coli testing: Inoculate a dilution prepared in lactose broth containing 4% polysorbate 20 and 0.5% lecithin with <100 CFU of E. coli. Incubate the inoculated dilution at 30°C to 35°C for 24 hours, then streak onto MacKonkey agar. Observe the plate for characteristic E. coli colonies. Once observed, confirm that the representative colonies are E. coli using the API 20e biochemical identification test.

The S. aureus and E. coli tests were repeated at dilutions of 1:20, 1:40 and 1:80.

Test results

The results of the experiments using uncoated uPECs are shown in Tables 3 and 4 below, indicating bacterial recovery rates after re-smearing individual bacteria, incubation with uPECs, and washing. As shown in Table 3, the recovery rates of Staphylococcus aureus were 13% and 48% for dilutions of 1:100 and 1:200, respectively, clearly demonstrating the bactericidal effect of uPECs against S. aureus. As shown in Table 4, the recovery rates of S. aureus were 2% for a dilution of 1:20, 4% for a dilution of 1:40, and 23% for a dilution of 1:80.

Table 3

Table 4

The recovery rate of E. coli is also shown in Tables 3 and 4. As shown in Table 3, the recovery rate was 2% for a dilution of 1:100, and 28% for a dilution of 1:200. As shown in Table 4, the recovery rate of E. coli was 0% for a dilution of 1:20, 0% for a dilution of 1:40, and 12% for a dilution of 1:80.

The positive control showed that the growth medium used for all microbial cultures was functionally effective. This demonstrated a true bactericidal effect when the selected organisms were contacted with PECs. If the effect of PECs was merely bacteriostatic, removing PECs from the co-culture medium containing the test bacteria would result in a removal of any inhibitory effect caused by the PECs, manifested as an active growth recovery. However, removing PECs did not result in any such recovery, even when the culture medium was allowed to grow over a full 24-hour period. This strongly supports the view that the failure of the selected organisms to show CFU recovery when contacted with PECs was due to the bactericidal effect of the PECs. This effect could include mechanisms such as physical and irreversible damage to cell surface lipids, membrane proteins, or bacterial capsules through enzymatic degradation, leading to extracellular and intercellular ion-electrolyte imbalances, acidic changes, and damage to genetic material.

A number of embodiments of the present invention have been described. However, it will be appreciated that various modifications may be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims.

Claims (22)

1. Use of a pharmaceutical composition in the preparation of a medicament for the treatment or prevention of Staphylococcus aureus infection in birds or mammals, said pharmaceutical composition comprising a therapeutically effective amount of a digestive enzyme, said digestive enzyme comprising protease, amylase and lipase, said digestive enzyme being derived from an animal source, said animal source being porcine pancreas, and said pharmaceutical composition being bactericidal or bacteriostatic against Staphylococcus aureus. 2. The use according to claim 1, wherein the pharmaceutical composition further comprises a β-lactam antibiotic. 3. The use according to claim 1, wherein the pharmaceutical composition further comprises one or more enzymes selected from the group consisting of cellulase, sucrase, and maltase. 4. The use according to claim 1, wherein the ratio of total protease to total lipase in the pharmaceutical composition is in the range of 1:1 to 20:1 in United States Pharmacopeia (U.S.P.) units. 5. The use according to claim 1, wherein the ratio of total protease to total lipase in the pharmaceutical composition is in the range of 4:1 to 10:1 in U.S.P. units. 6. The use according to claim 1, wherein the ratio of total protease to total lipase in the pharmaceutical composition is 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1 or 12:1 in U.S.P. units. 7. The use according to claim 1, wherein the pharmaceutical composition comprises a dosage form selected from the group consisting of pills, tablets, capsules, pouches, powders, creams, aerosols, emulsions, powders, liquids, gels, and any combination thereof. 8. The use according to claim 1, wherein the digestive enzyme comprises a mixture of proteases, wherein the proteases comprise chymotrypsin and trypsin. 9. The use according to claim 1, wherein the mammal is a pig, horse, cow, dog, cat, monkey, rat, mouse, sheep, goat or human. 10. The use according to claim 1, wherein the poultry is a chicken, duck, turkey or goose. 11. The use according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. 12. The use according to claim 11, wherein the pharmaceutically acceptable carrier or excipient comprises a solid filler, a liquid filler, a diluent, an encapsulating material, or a combination thereof. 13. The use according to claim 11, wherein the pharmaceutically acceptable carrier or excipient is selected from the group consisting of: acidifiers, alkalizers, antimicrobial preservatives, antioxidants, buffers, chelating agents, complexing agents, solubilizers, solvents, suspending agents and/or thickeners, tensioning agents, wetting agents, and combinations thereof. 14. The use according to claim 11, wherein the pharmaceutically acceptable carrier or excipient is selected from the group consisting of: starch, cellulose, powdered astragalus gum, malt, gelatin, talc, cocoa butter, suppository wax, oil, polyol, ester, agar, alginic acid, pyrogen-free water, isotonic saline, ethanol solution, lubricant, colorant, release agent, coating agent, sweetener, flavoring agent, preservative, surfactant, emollient, dimethyl sulfoxide (DMSO), and combinations thereof. 15. The use according to any one of claims 1-14, wherein the pharmaceutical composition is formulated for topical application. 16. The use according to claim 15, wherein the composition is formulated for topical administration to the skin of birds or mammals. 17. The use according to any one of claims 1-14, wherein the pharmaceutical composition is formulated for administration via mucosal application. 18. The use according to any one of claims 1-14, wherein the pharmaceutical composition is formulated for oral administration. 19. The use according to any one of claims 1-14, wherein the pharmaceutical composition further comprises a coating. 20. The use according to claim 19, wherein the coating comprises lipids. 21. Use of the composition in the preparation of a bactericide for eradicating, attenuating or reducing Staphylococcus aureus, wherein the composition comprises a therapeutically effective amount of a digestive enzyme, the digestive enzyme comprising protease, amylase and lipase, wherein the digestive enzyme is derived from an animal source, and the animal source is porcine pancreas. 22. Use of the composition in the preparation of a detergent for eradicating, attenuating or reducing Staphylococcus aureus, wherein the composition comprises a therapeutically effective amount of a digestive enzyme, the digestive enzyme comprising protease, amylase and lipase, wherein the digestive enzyme is derived from an animal source, and the animal source is porcine pancreas.