Methods and Compositions for the Prevention and Treatment of Influenza

Abstract

This disclosure relates to the prevention and treatment of Influenza, and more particularly Influenza A Virus Subtype H1N1, with the use of a pharmaceutical composition comprising one or more digestive enzymes, such as pancreatic enzymes and porcine pancreatic enzymes. The disclosure further relates to the use of an individual's fecal chymotrypsin level as an indicator, e.g., biomarker of whether an individual may be more susceptible to Influenza, e.g., Influenza A Subtype H1N1, and/or whether an individual will benefit from administration of the described pharmaceutical compositions. Use of the compositions as sanitizers, antiseptics, disinfectants, and detergents, e.g., to reduce or eradicate influenza virus present on living or inanimate surfaces is also contemplated.

Claims

1. A method for the treatment or prevention of Influenza in a mammal or a bird, comprising administering to the mammal or bird a therapeutically effective amount of a pharmaceutical composition comprising one or more digestive enzymes.

2. The method of claim 1 wherein the Influenza is Influenza Type A.

3. The method of claim 2 where the Influenza Type is Subtype H1N1.

4. The method of claim 1 wherein the one or more digestive enzymes comprise one or more enzymes selected from the group consisting of proteases, amylases, celluloses, sucrases, maltases, papain, and lipases.

5. The method of claim 1 wherein the one or more digestive enzymes comprise one or more pancreatic enzymes.

6. The method of claim 1 wherein the one or more of the digestive enzymes comprise avian enzymes or pig enzymes.

7. The method of claim 4 wherein the proteases comprise chymotrypsin and trypsin.

8. The method of claim 1 wherein 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.

9. The method of claim 1 wherein the mammal is a pig, horse, cow, dog, cat, monkey, rat, mouse, sheep, goat or human.

10. The method of claim 8 where the animal source is a pig pancreas.

11. The method of claim 1 wherein the pharmaceutical composition comprises at least one amylase, a mixture of proteases comprising chymotrypsin and trypsin, and at least one lipase.

12. The method of claim 1, further comprising vaccinating the mammal or bird with a TIV or LAIV, or treating the mammal or bird with an anti-viral medication, or both.

13. The method of claim 1 wherein the pharmaceutical composition comprises: amylases from about 10,000 to about 60,000 U.S.P, proteases from about 10,000 to about 70,000 U.S.P, lipases from about 4,000 to about 30,000 U.S.P., chymotrypsin from about 2 to about 5 mg, trypsin from about 60 to about 100 mg, papain from about 3,000 to about 10,000 USP units, and papaya from about 30 to about 60 mg.

14. The method of claim 1 wherein the pharmaceutical composition comprises at least one protease and at least one lipase, and wherein the ratio of total proteases to total lipases (in USP units) ranges from about 1:1 to about 20:1.

15. The method of claim 11 wherein the ratio of proteases to lipases ranges from about 4:1 to about 10:1.

16. The method of claim 1 wherein the pharmaceutical composition is a dosage formulation selected from the group consisting of: pills, tablets, capsules, microcapsules, mini-capsules, time released capsules, mini-tabs, sprinkles, and a combination thereof.

17. A method of diagnosing a patient as immune-compromised comprising: a. obtaining a fecal sample from the patient; b. determining a level of chymotrypsin present in the fecal sample; and c. diagnosing the patient as having a compromised immune system if the determined fecal chymotrypsin level is less than a control level.

18. The method of claim 17 wherein the determined fecal chymotrypsin level is less than 8.4 U/gram.

19. The method of claim 17 wherein the determined fecal chymotrypsin level is less than 4.2 U/gram.

20. The method of claim 17 wherein the level of chymotrypsin present in the fecal sample is determined using an enzymatic photospectrometry method.

21. The method of claim 17 further comprising administering to the patient an effective amount of a pharmaceutical composition comprising one or more digestive enzymes if the patient is diagnosed as having a compromised immune system.

22. The method of claim 21 further comprising determining if the administration of the pharmaceutical composition reduces or ameliorates the propensity or frequency of contracting bacterial or viral illnesses or the severity of bacterial or viral illnesses.

23. The method of claim 22 further comprising comparing the post-administration measurement of one or more symptoms of the illness to a pre-administration measurement of the one or more symptoms of illness.

24. A method of identifying a patient likely to benefit from administration of a pharmaceutical composition comprising one or more digestive enzymes comprising: obtaining a fecal sample from the patient; determining a level of chymotrypsin present in the fecal sample; and identifying the patient as likely to benefit from administration of the pharmaceutical composition if the determined fecal chymotrypsin level is less than a control level and the patient is diagnosed with a compromised immune system or the patient is exhibiting one or more symptoms associated with a bacterial or viral illness.

25. The method of claim 24 further comprising determining if the patient exhibits one or more symptoms of a compromised immune system.

26. The method of claim 24 wherein the benefit comprises a reduction or amelioration of one or more symptoms associated with a bacterial or viral illness.

27. The method of claim 24 wherein the level of chymotrypsin present in the fecal sample is determined using an enzymatic photospectrometry method.

28. The method of claim 24 further comprising administering to the patient an effective amount of a pharmaceutical composition comprising one or more digestive enzymes.

29. A pharmaceutical composition comprising one or more digestive enzymes, wherein the one or more digestive enzymes comprise at least one lipase and at least one protease, and wherein the ratio of total proteases to total lipases (in USP units) ranges from about 1:1 to about 20:1.

30. The pharmaceutical composition of claim 29 wherein the ratio of total proteases to total lipases ranges from about 4:1 to about 10:1.

31. A pharmaceutical composition comprising at least one amylase, a mixture of proteases comprising chymotrypsin and trypsin, and at least one lipase.

32. The pharmaceutical composition of claim 31 wherein the pharmaceutical composition further comprises papain.

33. The pharmaceutical composition of claim 29 or 31 wherein the pharmaceutical composition is lipid encapsulated.

34. A method for treating a mammal or bird exhibiting one or more symptoms of Influenza comprising administering to the mammal or bird a therapeutically effective amount of a composition comprising one or more digestive enzymes.

35. The method of claim 34 where the symptoms of influenza are selected from the group consisting of: fever, headache, tiredness, cough, sore throat, runny or stuffy nose, body aches, diarrhea and vomiting, and a combination thereof.

36. The method of claim 34 wherein the mammal is a human or a pig.

37. The method of claim 34 wherein the preparation is administered orally via a dosage formulation selected from the group consisting of: emulsions, liquids, pills, tablets, capsules, microcapsules, mini-capsules, time released capsules, mini-tabs, powders, sprinkles, and a combination thereof.

38. A method of preventing infection of an individual with Influenza or of treating an individual diagnosed with Influenza comprising: measuring a level of fecal chymotrypsin in a stool sample of the individual; comparing the level of fecal chymotrypsin with a normal fecal chymotrypsin level; and administering a composition comprising one or more digestive enzymes to the individual if the level of fecal chymotrypsin in the individual is less than a normal fecal chymotrypsin level.

39. A method for sanitizing or disinfecting a surface to reduce the amount of influenza virus thereon or to eradicate the influenza virus thereon, comprising applying to the surface a composition comprising one or more digestive enzymes.

40. A method for reducing the amount of influenza virus present on a skin region, tissue, or wound of a mammal or bird comprising applying to the skin region, tissue, or wound a composition comprising one or more digestive enzymes.

Description

DETAILED DESCRIPTION

[0144] The present disclosure provides pharmaceutical compositions comprising one or more digestive enzymes and methods of using the same for the treatment and/or prevention of Influenza. The present disclosure also provides compositions comprising one or more digestive enzymes and methods of using the same as antiseptics, detergents, disinfectants, and sanitizers, e.g., as viricidal and/or viristatic compositions to kill or attenuate the influenza virus. The compositions described herein include one or more digestive enzymes, which are postulated to assist in the reduction, weakening, or eradication of Influenza virus, and thus to prevent contraction of Influenza; and/or to ameliorate gastrointestinal dysfunction or to enhance the normal gastrointestinal function, in order to prevent contraction of Influenza or to treat Influenza (e.g., improve or ameliorate the symptoms or reduce the time course of the infection). In addition, the pharmaceutical compositions can be utilized to enhance immune system response for individuals with compromised immune systems and/or to augment immune system functions is nonimmuno-compromised individuals, e.g., to assist in the prevention or treatment of Influenza.

[0145] In certain embodiments, the pharmaceutical compositions can include one or more digestive enzymes, wherein the one or more digestive enzymes comprise at least one lipase and at least one protease, and wherein the ratio of total proteases to total lipases (in USP units) ranges from about 1:1 to about 20:1, including 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 and 20:1 along with all values in-between. In some cases, the ratio of total proteases to total lipases ranges from about 4:1 to about 10:1, including 4;1, 4;1, 6:1, 7:1, 8:1, 9:1, and 10:1 along with all values in-between. In some embodiments, the pharmaceutical composition is encapsulated, e.g., lipid-encapsulated. Enzyme preparations comprising one or more digestive enzymes useful for the methods described herein are disclosed in U.S. Ser. No. 12/386,051, incorporated herein by reference.

[0146] In some cases, a pharmaceutical composition for use herein comprises at least one amylase, at least one protease, and at least one lipase. In certain embodiments, the composition can comprise at least one amylase, at least two proteases, and at least one lipase. In certain embodiments the pharmaceutical composition includes multiple proteases, including, without limitation, chymotrypsin and trypsin. In certain embodiments, the composition can further include one or more hydrolases, papain, bromelain, papaya, celluloses, pancreatin, sucrases, and maltases.

[0147] The one or more enzymes can be independently derived from animal, plant, fungal, microbial, or synthetic sources. In some embodiments, the one or more enzymes are derived from pig, e.g. pig pancreas or avian bird proventriculus or small intestine.

[0148] One exemplary formulation for the prevention of Influenza or treatment of symptoms of Influenza is as follows:

Amylase 10,000-60,000 U.S.P

Protease 10,000-70,000 U.S.P

Lipase 4,000-30,000 U.S.P

Chymotrypsin 2-5 mg

Trypsin 60-100 mg

[0149] Papain 3,000-10,000 USP units/mg

Papaya 30-60 mg

[0150] Additional formulations comprising one or more digestive enzymes may be advantageous including formulations in which the ratio of total proteases to total lipases (in USP units) is from about 1:1 to about 20:1, including 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 and 20:1 along with all values in-between. In some embodiments, the ratio of total proteases to total lipases is from about 4:1 to about 10:1 including 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1 along with all values in-between. Such formulations are useful for the prevention of Influenza or treating symptoms of Influenza, or the enhancement of immune system functions.

[0151] The pharmaceutical compositions can be formulated in dosage forms for any route of administration, including oral, parenteral, IV, inhalation, and buccal dosage formulations. In certain embodiments, a dosage formulation may be administered by an oral preparation including, but not limited to, an encapsulated tablet, mini-tabs, microcapsule, mini-capsule, time released capsule, sprinkle, powder or other methodology. In one embodiment, the oral preparation is encapsulated using one or more lipids. Alternatively, the oral preparation may be encapsulated using enteric coating or organic polymers. A formulation may also be prepared using Prosolv technology, direct compression, dry granulation, wet granulation, and/or a combination of these methods.

[0152] Fecal chymotrypsin level is a sensitive, specific measure of proteolytic activity, see, e.g. U.S. Pat. No. 6,660,831, incorporated by reference herein. Normal levels of chymotrypsin are typically considered to be greater than 8.4 U/gram. Decreased values (less than 4.2 U/gram) suggest diminished pancreatic output (pancreatic insufficiency), hypoacidity of the stomach or cystic fibrosis. Elevated chymotrypsin values suggest rapid transit time, or less likely, a large output of chymotrypsin from the pancreas.

[0153] For the fecal chymotrypsin test, a stool sample can be collected from each of the subjects. Each stool sample can be analyzed using an enzymatic photospectrometry analysis to determine the level of fecal chymotrypsin in the stool; in some cases the assay is performed at 30 C., see, e.g. U.S. Pat. No. 6,660,831, incorporated by reference herein. Alternatively, other methods, such as the colorimetric method, use of substrates, use of assays, and/or any other suitable method may be used to measure the fecal chymotrypsin levels. The levels of fecal chymotrypsin in the samples e.g., of individuals suspected of or diagnosed as having a compromised immune system, are compared to the levels of fecal chymotrypsin in normal or control individuals (e.g., individuals not suspected or diagnosed with a compromised immunes system), to determine if the individuals would benefit from the administration of a composition as described herein.

[0154] The nature of the human digestive tract creates challenges for the delivery of digestive enzymes to patients including the general population, those with compromised immune systems, or those with influenza. Multiple temperature and pH changes over the course of the digestive tract make specific delivery a necessity and a challenge. For instance, pH as low as 1 is encountered in the stomach, but rapidly increases to a more basic pH of 5-6 in the proximal small intestine. For example, generally the pH in the stomach is approximately 1.2, the pH in the duodenum is about 5.0 to 6.0; the pH in the jejunum is about 6.8, and the pH is about 7.2 in the proximal ileum and about 7.5 in the distal ileum. The low pH in the stomach which changes rapidly to a more basic pH of 5-6 in the proximal small intestines, calls for a specific delivery method depending upon where the enzyme is to be delivered. For veterinary applications, a specific delivery location may also be necessary, depending on the animal to be treated.

[0155] Delivery of digestive enzymes can also be challenging due to the rapid degradation and denaturing of enzymes at ambient room temperature, as well as the enhanced degradation and denaturing that can occur with high temperature, pressure, humidity and/or exposure to light. Moisture and heat together can quickly destabilize enzymes, reducing their effectiveness, and shortening shelf life, leading to inaccurate dosing. Denaturization or destabilization of the enzymes can reduce their effectiveness by reducing the dose of active enzymes to less than the amount needed for effective treatment. Alternatively, attempting to compensate for the denaturization or destabilization by increasing the dose to ensure an effective level of active enzyme, could risk an overdose or overfilling a capsule or other dosage form. To protect and stabilize the compositions from unfavorable conditions, the digestive enzymes may be coated or encapsulated in a continuous coating containing a crystallizable lipid.

[0156] Manufacturers of enzyme preparations have used enteric coatings to deliver lipases in individuals requiring administration of lipases, such as individuals with cystic fibrosis.

[0157] Coatings in the digestive/pancreatic enzyme preparations create a barrier to degradation and denaturation, and allow more accurate levels of active enzymes to reach the treated individuals.

[0158] For example, a lipid coating of this disclosure provides a significant barrier to moisture, heat, humidity and exposure to light by allowing for a physical barrier as well as one that prevents and or reduces hydrolysis. The coated enzyme preparations undergo less hydrolysis as a result of protection from moisture in the environment by the lipid coating. As a result of the present disclosure, pancreatic/digestive enzymes are provided which can tolerate storage conditions (e.g., moisture, heat, oxygen, etc.) for long periods of time thus enabling extended shelf life. The coating of the encapsulated enzyme preparation protects the enzyme from the environment and provides emulsification in a solvent without detracting from the abrasion resistance of the coating.

[0159] In some embodiments, the coatings on the digestive enzyme particle cores are preferably continuous coatings. By continuous, it is meant that the pancreatic/digestive enzyme is uniformly protected. The continuous coating of the fully surrounds or encapsulates the pancreatic/digestive enzymes. The encapsulation provides protection of the pancreatic/digestive enzyme from conditions such as moisture, temperature, and conditions encountered during storage.

[0160] As discussed, the encapsulation can provide controlled release of the digestive enzymes. The emulsification properties of the coating in a solvent allows for controlled release of the enzyme in the gastrointestinal system, preferably the region of the GI tract where the enzymes are to be utilized. In some embodiments, the dissolution profile may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 minutes. Dissolution profiles may be obtained using methods and conditions known to those of skill in the art. For example, dissolution profiles can be determined at various pH's, including pH. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0161] Encapsulate as used herein means that the coating completely surrounds the pancreatic/digestive enzyme. In a population of encapsulated particles, encapsulated enzyme preparations may include contaminating or small portion of particles with a substantially continuous coating as long as the release profiles of the encapsulated particles are not significantly altered. A coated or encapsulated particle may contain one or more digestive enzyme particles enveloped in one coating to form one coated or encapsulated digestive enzyme particle in the coated or encapsulated digestive enzyme preparation.

[0162] The crystallizable lipid is any lipid or wax, lipid or wax mixture, or blend of lipid and/or waxes, where the crystalliable lipid forms a solid coating via crystallization at typical storage temperatures. The crystallizable lipid can be a vegetable or animal derived-lipid. In some embodiments, the crystallizable lipid is emulsifiable upon contact with physiological conditions and consists essentially of, or comprises one or more monoglycerides, diglycerides or triglycerides, or other components including, for example, emulsifiers found in hydrogenated vegetable oils. In another embodiment the crystallizable lipid is a non-polar lipid, for example hydrogenated soybean oil

[0163] As used herein, animal and/or vegetable derived lipids can include fats and oils originating from plant or animal sources and/or tissues, and/or synthetically produced based on the structures of fats and oils originating from plant or animal sources. Lipid material may be refined, extracted or purified by known chemical or mechanical processes. Certain fatty acids present in lipids, termed essential fatty acids, must be present in the mammalian diet. The lipid may, in some embodiments, comprise a Type I USP-National Formulary vegetable oil.

[0164] The digestive enzyme used in the present disclosure can be, for example, any combination of digestive enzymes of a type produced by the pancreas, including, but not limited to digestive enzymes from a pancreatic source or other sources. The scope of the disclosure is not limited to pancreatic enzymes of porcine origin, but can be of other animal, microbial, or plant origin as well as those which are synthetically derived. The digestive enzyme may be derived from mammalian sources such as porcine-derived digestive enzymes. The enzyme may include one or more enzymes, and can also be plant derived, synthetically derived, recombinantly produced in microbial, yeast, fungal or mammalian cells, and can include a mixture of enzymes from one or more sources. Digestive enzymes, can include, for example, one or more enzymes from more or more sources mixed together. This includes, for example, the addition of single digestive enzymes to digestive enzymes derived from pancreatic sources in order to provide appropriate levels of specific enzymes that provide more effective treatment for a selected disease or condition. One source of digestive enzymes can be obtained, for example, from Scientific Protein Laboratories (see Table 6). The digestive enzyme may be, for example a pancreatin/pancrelipase composition. In one embodiment, the digestive enzymes will comprise or consist essentially of 25 USP units/mg protease, 2 USP Unit/mg, and 25 USP Units/mg amylase.

[0165] In some embodiments, the digestive enzyme particles used as cores in the present disclosure include digestive enzyme particles where about 90% of the particles are between about #40 and #140 USSS mesh in size, or between about 105 to 425 m, including 105, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, and 425 um or where at least about 75% of the particles are between about #40 and #80 mesh, or about 180 to 425 m in size. Particles between #40 and #140 mesh in size pass through #40 mesh but do not pass through #140 mesh. The coated or encapsulated digestive enzyme particles in one embodiment of this disclosure may comprise less than about 35, 30, 25, 20, 15 or 10% of the particles which can be sieved through #100 mesh (150 m). In some embodiments, the term non-aerosolizable refers to a coated or encapsulated enzyme preparation where less than about 20% or less than about 15% of the particles can be sieved through #100 mesh (150 m). The encapsulated digestive enzyme preparation can be an encapsulated digestive enzyme composite where the digestive enzyme particles contain two or more enzymes.

[0166] The minimum amount of pancreatic enzyme present in the core can vary, and in some embodiments is at least about 5% active enzymes by weight of the coated enzyme preparation, but in other embodiments may be at least about 30%, or at least about 50% by weight. The maximum amount of pancreatic/digestive enzyme present in the composite can vary, and in some cases is at most about 95% by weight, and in other embodiments at most about 90%, 85%, 80%, 75% or 70% of the coated enzyme preparation. In other embodiments, the amount of pancreatic enzyme present in the composite is about 10%, 15%, 20%, 25%, 35%, 40%, 45%, 55%, 60%, 65%, 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 87.5%, or 92.5% by weight or anywhere in between.

[0167] The composition which contains the encapsulated digestive enzyme preparation or composite can be delivered as a sprinkle, powder, capsule, tablet, pellet, caplet or other form. Packaging the encapsulated enzyme preparations in an enzyme delivery system that further comprises single dose sachet-housed sprinkle preparations allows for ease of delivery, and accurate dosing of the enzyme, by allowing a specific amount of enzyme to be delivered in each dosing. Allowing for specific unit dosing of an enzyme preparation which maintains the enzyme activity within specific stability parameters in an enhancement over other sprinkle formulations, which are housed, in a multi-unit dosing form that allows for air, moisture and heat to depredate and denature the enzyme preparation. In a preferred embodiment the powder or sachet is housed in a trilaminar foil pouch, or similar barrier to keep out moisture and to protect the enzyme preparation from adverse environmental factors.

[0168] Further, in some embodiments, the lipid encapsulation methodology reduces the aerosolization of the enzyme preparation that may be caustic to an individual if inhaled through the lungs or the nose. The lipid encapsulation reduces aerolization and the potential for caustic burn, aspiration, and/or aspiration pneumonias in receivers and administrators of the enzyme preparation, thereby reducing the potential for illness in those already compromised by influenza or reduced immune system functionality, and leading to safer administration.

[0169] As used herein, the term non-aerosolizable will be used to refer to a coated or encapsulated enzyme preparation where substantially all of the particles are large enough to eliminate or reduce aerosolization upon pouring of the coated enzyme preparation compared to uncoated enzyme particles. For example, the term non-aerosolizable may refer to a coated or encapsulated enzyme preparation where at least about 90% of the particles are between about #40 and #140 mesh in size, or between about 106 to 425 m, or where at least about 75% of the particles are between about #40 and #80 mesh, or about 180 to 425 m. The term non-aerosolizable may also refer to a coated or encapsulated enzyme preparation where less than about 35, 30, 25, 20, 15 or 10% of the particles can be sieved through #100 mesh (150 m). In some embodiments, the term non-aerosolizable refers to a coated or encapsulated enzyme preparation where less than about 20% or less than about 15% of the particles can be sieved through #100 mesh (150 m).

[0170] The choice of suitable enzymes and of suitable lipid coatings, including choice of the type or amount of enzymes or coating, are guided by the specific enzyme needs of the individual to be treated.

[0171] Additives can be blended with a crystallizable lipid. Selection of the lipid(s) and additives will control the rate of release of the bioactive substance. In the case of the digestive and or pancreatic enzymes, the lipid can be chosen to release the bioactive substance in the area of the digestive tract selected for release to optimize treatment.

[0172] The disclosure further relates to the administering of the coated and/or encapsulated enzyme preparation in a sachet or pouch preparation for ease of delivery to children and adults. In some embodiments, the disclosure specifically relates to the administration of a coated enzyme particle preparation, housed in a sachet or pouch. This facilitates administration, including but not limited to, administration in food or drink, direct administration into the oral cavity, or administration directly into the GI system through an NG-tube, G-tube or other GI entrances or deliveries.

[0173] In some embodiments, each dose contains about 100 to 1500 mg of coated or encapsulated enzyme preparation, and each dose may contain about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1500 mg of coated or encapsulated enzyme preparation. About can include 80 to 125% of the recited preparation. Each dose may also be plus or minus 10% of the recited weight. In one embodiment each does will have a protease activity of not less than about 156 USP units/mg plus or minus 10%. The protease activity may also be not less than about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 USP units/mg.

[0174] In other embodiments, the disclosure relates to methods of treatment comprising administering to a subject, at least two doses of a composition comprising a therapeutically effective amount of the coated digestive enzyme preparations for the prophylaxis or treatment or influenza. In certain embodiments, about 80% of the enzyme is released by about 30 minutes in a dissolution test performed at pH 6.0. In other embodiments, about 80% of the enzyme is released by about 30 minutes after the coated digestive enzyme preparations reach the small intestine.

[0175] In other embodiments, the disclosure relates to methods of treatment comprising administering to a subject, at least three doses of a composition comprising a therapeutically effective amount of the coated digestive enzyme preparations for the prophylaxis or treatment or influenza. In certain embodiments, about 80% of the enzyme is released by about 30 minutes in a dissolution test performed at pH 6.0. In other embodiments, about 80% of the enzyme is released by about 30 minutes after the coated digestive enzyme preparations reaches the small intestine.

[0176] Another embodiment of the disclosure relates to the improvement of delivery of enzymes to humans by reducing the use of excipients, extenders and solvents currently used in the preparations for delivery of digestive enzymes to humans. For example, the encapsulated digestive enzyme preparation may contain only one excipient, which increases the safety of administration by decreasing the chance of an allergic response. In one embodiment, the excipient is hydrogenated soybean oil.

[0177] The lipid coating surprisingly does not appear to be reduced or destroyed by HCl (hydrochloric acid) present in the stomach, thereby protecting the enzyme from degradation following administration until the enzyme preparation reaches its target region in the GI tract. Further the lipid coat reduces the exposure of the enzyme to attack by water, thereby reducing hydrolysis, and further protecting the digestive enzymes from degradation. In addition, an excipient containing only lipid can be used to coat or encapsulate digestive enzyme particles containing lipase.

[0178] The disclosure therefore relates to improvement of the delivery of digestive enzymes to humans or animals based specifically upon needed delivery times, and dissolution profiles. For example, in certain aspects of the disclosure, the rate of release and dissolution characteristics are unique to the lipid encapsulations of this disclosure

[0179] For prophylaxis of influenza or treatment of patients with influenza or immune compromised individuals who require delivery of protease enzymes for effective treatment, the lipid encapsulate can be modified to deliver the protease during an earlier transit time window, in the proximal small intestine, to optimize virion protein digestion. In another example, for elderly patients with slower GI transit times, still another release profile may be advantageous to deliver enzymes for effective treatment. The lipid and/or additive selection will be made to obtain enzyme release at later times after administration. In veterinary applications, still another release profile may be necessary.

[0180] The present disclosure also relates to methods of making the enzyme preparations by lipid coating and/or encapsulation of pancreatic and/or digestive enzymes. The methods comprise providing a crystallizable lipid, and coating pancreatic/digestive enzyme particles with the lipid, where the pancreatic/digestive enzymes comprise 5-90% including 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, and 90% along with all values in-between of the coated enzyme preparations by weight. In some aspects the uncoated pancreatic/digestive enzyme particles have a size range of about 105-425 m including 105, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, and 425 um along with all values in-between.

[0181] In one embodiment, the disclosure relates to a method of preparing an encapsulated digestive enzyme preparation, the method comprising (a) screening uncoated digestive enzyme particles to obtain particles of a suitable size for encapsulation; and (b) coating the screened digestive enzyme particles with a crystallizable lipid to form coated or encapsulated digestive enzymes containing a core which contains the pancreatic/digestive enzyme and a coating which contains the crystallizable lipid. In some embodiments, the encapsulated digestive enzyme preparation is a controlled release digestive enzyme preparation, which may have enhanced flow properties.

[0182] Screening of the particles may include quality control steps to improve the activity, appearance or particle size of the digestive enzyme. For example, the particles may be analyzed to determine enzyme activity content, and/or visualized using chromatographic, microscopic or other analytical methods. The particles may also be screened to obtain particles of a suitable size for encapsulation by removing particles that are too fine or too large. For example, the particles may be sieved to obtain particles of a suitable size or more uniform size range for encapsulation. As a further example, the particles may be sieved through USSS #40 mesh and through USSS #140 mesh. Particles that pass through the #40 mesh but are retained by the #140 mesh are of an appropriate size range for coating or encapsulation. Particles may also be screened by sieving through USSS #140, #120, #100, #80, #70, #60, #50, #45, or #40 mesh, or any combination thereof.

[0183] In some embodiments, the lipid should be present in the preparation at a minimum amount of about 5% by weight of the encapsulated composite, preferably about 30%, and more preferably about 50% by weight of the encapsulated composite. The maximum amount of pancreatic/digestive enzyme present in the encapsulated composite is about 95% by weight of the composite, preferably about 90%, and more preferably about 85% of the encapsulated composite. The crystallizable lipid can be any lipid or lipid-derived material that creates a solid coating around the digestive enzyme substrate. This lipid coating releases the digestive enzyme in the GI tract. The lipid coating may be emulsifiable in the GI tract, releasing the digestive enzyme.

[0184] The crystallizable lipid can be derived from animal or vegetable origins, such as, for example, palm kernel oil, soybean oil, cottonseed oil, canola oil, and poultry fat, including hydrogenated type I vegetable oils and waxes. In some embodiments, the lipid is hydrogenated. The lipid can also be saturated or partially saturated. Examples of crystallizable lipids include, but are not limited to, monoglycerides, diglycerides, triglycerides, fatty acids, esters of fatty acids, phospholipids, salts thereof, and combinations thereof. The crystallizable lipid can be an emulsifiable lipid.

[0185] Emulsifiable lipids as used herein means those lipids which contain at least one hydrophilic group and at least one hydrophobic group, and have a structure capable of forming a hydrophilic and hydrophobic interface. These chemical and/or physical properties, mentioned above, of an emulsifiable lipid permit emulsification. Examples of interfaces include, for example, micelles and bilayers. The hydrophilic group can be a polar group and can be charged or uncharged.

[0186] The crystallizable lipid which is emulsifiable is preferably a food grade emulsifiable lipid. Some examples of food grade emulsifiable lipids include sorbitan monostearates, sorbitan tristcaratcs, calcium stearoyl lactylates, and calcium stearoyl lactylates. Examples of food grade fatty acid esters which are emulsifiable lipids include acetic acid esters of mono- and diglycerides, citric acid esters of mono- and di-glycerides, lactic acid esters of mono- and di-gylcerides, polyglycerol esters of fatty acids, propylene glycol esters of fatty acids, and diacetyl tartaric acid esters of mono- and diglycerides. Lipids can include, for example, hydrogenated soy oil.

[0187] Any emulsifiable lipid may be used in the methods and products of this disclosure. In certain embodiments the emulsifiable lipid used will produce non-agglomerating, non-aerosolizing enzyme preparation particles.

[0188] The coating of the enzyme with the lipid allows for the enzyme to become more uniform in size and shape, but reduces the jagged edges associated with the raw enzyme, and allows for ease of administration and ease of manufacturing, as the flow properties associated with the covered enzyme will allow for the manufacturing machinery to easily fill the appropriate containers.

[0189] The inclusion of one or more additives with an emulsifiable lipid of the present disclosure is used to control emulsification of the coating and release of the enzyme. For example, the additive, triglyceride, can be blended with monoglycerides (e.g., an emulsifiable lipid), to control emulsification of the coating and thus control (e.g., decrease) the rate of release of the enzyme from the composite. As a further example, one or more additives, such as a diglyceride and a triglyceride can be blended with the emulsifiable lipid to control the rate of release of the enzyme. Hydrogenated vegetable oils may contain emulsifying agents, such as soy lecithin or other components.

[0190] Properties including mechanical strength, melting point, and hydrophobicity can be considered when choosing a suitable lipid coating for the digestive enzyme. Lipids having lower melting points or more polar, hydrophilic properties were generally less suitable for encapsulation because they resulted in product that would cake under accelerated storage stability conditions. Enzyme preparations made using, for example, hydrogenated soy oil, hydrogenated castor wax, and carnauba wax all demonstrated good pouring and no caking.

[0191] The wax can be paraffin wax; a petroleum wax; a mineral wax such as ozokerite, ceresin, or montan wax; a vegetable wax such as, for example, camuba wax, bayberry wax or flax wax; an animal wax such as, for example, spermaceti; or an insect wax such as beeswax.

[0192] Additionally, the wax material can be an ester of a fatty acid having 12 to 31 carbon atoms and a fatty alcohol having 12 to 31 carbon atoms, the ester having from a carbon atom content of from 24 to 62, or a mixture thereof. Examples include myricyl palmitate, cetyl palmitate, myricyl cerotate, cetyl myristate, ceryl palmitate, ceryl certate, myricyl melissate, stearyl palmitate, stearyl myristate, and lauryl laurate.

[0193] The solvent in which a lipid emulsifies can be an aqueous solvent. The aqueous solvent interacts with the hydrophilic groups present in the emulsifiable lipid and disrupts the continuity of the coating, resulting in an emulsion between the aqueous solvent and the lipids in the coating, thus releasing the bioactive substance from the composites.

[0194] Other additives for inclusion in the compositions described herein can be determined by those having ordinary skill in the art, and will be based on a number of features, including intended application, e.g., human vs. veterinary applications; desired release profile; desired pharmacokinetics; safety; stability; physical characteristics (smell, color, taste, pour, aerosilization). Suitable formulation ingredients, excipients, binders, bulking agents, flavorants, colorants, etc. can be determined and evaluated by methods known to those having ordinary skill.

[0195] In one aspect of the disclosure, the method comprises using the enzyme formulations to treat individuals that have an enzyme deficiency. The enzyme deficiency could be determined by any method used in determining or diagnosing an enzyme deficiency. In one aspect the determination or diagnosis may be made by evaluating symptoms, including eating habits, self-imposed dietary restrictions, symptoms of eating disorders and/or gastrointestinal disorders. In other aspects, the determination may be made on the basis of a biochemical test to detect, for example, levels or activities of enzymes secreted, excreted or present in the GI tract, and/or by determining the presence of a mutation in a gene or aberrant expression of a gene encoding one or more digestive enzymes. The enzyme deficiency may also be determined, for example, by detecting a mutation or aberrant expression of a gene encoding a product regulating or otherwise affecting expression or activity of one or more digestive enzymes.

[0196] The present disclosure also provides viricidal and/or viristatic compositions comprising one or more digestive enzymes for use as or in disinfectants, sanitizers, detergents, and antiseptics, e.g., in hospitals, nursing homes, nurseries, daycares, schools, work environments, public transportation and restroom facilities, to reduce and/or destroy influenza viruses present in such settings. The surfaces can be large (e.g., operating room tables, doors, changing tables) or small (e.g., medical devices, door handles); inanimate (tables) or animate (hands, e.g., detergents for hand-washing). The compositions include one ore more digestive enzymes, and optionally additives customarily used in antiseptics, disinfectants, sanitizers and detergents. The viricidal and viristatic compositions can be encapsulated enzyme compositions, as described previously, to increase stability and shelf-life.

[0197] Disinfectants are antimicrobial agents that are applied to non-living objects to destroy microorganisms, the process of which is known as disinfection. Disinfectants should generally be distinguished from antibiotics that destroy microorganisms within the body, and from antiseptics, which destroy microorganisms on living tissue. Sanitizers are substances that reduce the number of microorganisms to a safe level. One official and legal version states that a sanitizer must be capable of killing 99.999%, known as a 5 log reduction, of a specific test population, and to do so within 30 seconds. The main difference between a sanitizer and a disinfectant is that at a specified use dilution, the disinfectant must have a higher kill capability compared to that of a sanitizer. Very few disinfectants and sanitizers can sterilize (the complete elimination of all microorganisms), and those that can depend entirely on their mode of application.

[0198] The choice of the disinfectant to be used depends on the particular situation. Some disinfectants have a wide spectrum (kill nearly all microorganisms), whilst others kill a smaller range of disease-causing organisms but are preferred for other properties (they may be non-corrosive, non-toxic, or inexpensive).

[0199] The relative effectiveness of disinfectants can be measured by comparing how well they do against a known disinfectant and rate them accordingly. Phenol is the standard, and the corresponding rating system is called the Phenol coefficient. The disinfectant to be tested is compared with phenol on a standard microbe (usually Salmonella typhi or Staphylococcus aureus). Disinfectants that are more effective than phenol have a coefficient >1. Those that are less effective have a coefficient <1. For example, the Rideal-Walker method gives a Rideal-Walker coefficient and the U.S. Department of Agriculture method gives a U.S. Department of Agriculture coefficient.

[0200] To calculate phenol coefficient, the concentration of the test compound at which the compound kills the test organism in 10 minutes, but not in 5 minutes, is divided by the concentration of phenol that kills the organism under the same conditions. The phenol coefficient may be determined in the presence of a standard amount of added organic matter or in the absence of organic matter.

[0201] A detergent is a material intended to assist cleaning. The term is sometimes used to differentiate between soap and other surfactants used for cleaning where soap is a surfactant cleaning compound, typically used for personal or minor cleaning.

[0202] Detergents and soaps are used for cleaning because pure water can't remove oily, organic soiling. Soap cleans by acting as an emulsifier. Basically, soap allows oil and water to mix so that oily grime can be removed during rinsing. Detergents were developed in response to the shortage of the animal and vegetable fats used to make soap during World War I and World War II. Detergents are primarily surfactants, which could be produced easily from petrochemicals. Surfactants lower the surface tension of water, essentially making it wetter so that it is less likely to stick to itself and more likely to interact with oil and grease.

[0203] Modern detergents contain more than surfactants. Cleaning products may also contain enzymes to degrade protein-based stains, bleaches to de-color stains and add power to cleaning agents, and blue dyes to counter yellowing. Like soaps, detergents have hydrophobic or water-hating molecular chains and hydrophilic or water-loving components. The hydrophobic hydrocarbons are repelled by water, but are attracted to oil and grease. The hydrophilic end of the same molecule means that one end of the molecule will be attracted to water, while the other side is binding to oil. Neither detergents nor soap accomplish anything except binding to the soil until some mechanical energy or agitation is added into the equation. Swishing the soapy water around allows the soap or detergent to pull the grime away from clothes or dishes and into the larger pool of rinse water. Rinsing washes the detergent and soil away. Warm or hot water melts fats and oils so that it is easier for the soap or detergent to dissolve the soil and pull it away into the rinse water. Detergents are similar to soap, but they are less likely to form films (soap scum) and are not as affected by the presence of minerals in water (hard water).

[0204] Detergents, especially those made for use with water, often include different components such as Surfactants to cut (dissolve) grease and to wet surfaces, abrasives to scour, substances to modify pH or to affect performance or stability of other ingredients, acids for descaling or caustics to break down organic compounds, water softeners to counteract the effect of hardness ions on other ingredients, oxidants (oxidizers) for bleaching, disinfection, and breaking down organic compounds, non-surfactant materials that keep dirt in suspension, enzymes to digest proteins, fats, or carbohydrates in stains or to modify fabric feel, ingredients that modify the foaming properties of the cleaning surfactants, to either stabilize or counteract foam, ingredients to increase or decrease the viscosity of the solution, or to keep other ingredients in solution, in a detergent supplied as a water solution or gel, ingredients that affect aesthetic properties of the item to be cleaned, or of the detergent itself before or during use, such as optical brighteners, fabric softeners, colors, perfumes, etc., ingredients such as corrosion inhibitors to counteract damage to equipment with which the detergent is used, ingredients to reduce harm or produce benefits to skin, when the detergent is used by bare hand on inanimate objects or used to clean skin, and preservatives to prevent spoilage of other ingredients.

[0205] There are several factors that dictate what compositions of detergent should be used, including the material to be cleaned, the apparatus to be used, and tolerance for and type of dirt.

[0206] Modern detergents may be made from petrochemicals or from oleochemicals derived from plants and animals. Alkalis and oxidizing agents are also chemicals found in detergents. The most used disinfectants are those applying: active chlorine (i.e., hypochlorites, chloramines, dichloroisocyanurate and trichloroisocyanurate, wet chlorine, chlorine dioxide etc.): active oxygen (peroxides, such as peracetic acid, potassium persulfate, sodium perborate, sodium percarbonate and urea perhydrate); iodine (iodpovidone (povidone-iodine, Betadine), Lugol's solution, iodine tincture, iodinated nonionic surfactants); concentrated alcohols (mainly ethanol, 1-propanol, called also n-propanol and 2-propanol, called isopropanol and mixtures thereof; further, 2-phenoxyethanol and 1- and 2-phenoxypropanols are used); phenolic substances (such as phenol (also called carbolic acid), cresols (called Lysole in combination with liquid potassium soaps), halogenated (chlorinated, brominated) phenols, such as hexachlorophene, triclosan, trichlorophenol, tribromophenol, pentachlorophenol, Dibromol and salts thereof); cationic surfactants, such as some quaternary ammonium cations (such as benzalkonium chloride, cetyl trimethylammonium bromide or chloride, didecyldimethylammonium chloride, cetylpyridinium chloride, benzethonium chloride) and others, non-quarternary compounds, such as chlorhexidine, glucoprotamine, octenidine dihydrochloride etc.); strong oxidizers, such as ozone and permanganate solutions; heavy metals and their salts, such as colloidal silver, silver nitrate, mercury chloride, phenylmercury salts, copper sulfate, copper oxide-chloride etc. Heavy metals and their salts are the most toxic, and environment-hazardous bactericides and therefore, their use is strongly oppressed or canceled; further, also properly concentrated strong acids (phosphoric, nitric, sulfuric, amidosulfuric, toluenesulfonic acids) and alkalis (sodium, potassium, calcium hydroxides), such as of pH<1 or >13, particularly under elevated temperature (above 60 C.), kills bacteria.

[0207] Antiseptics (from Greek {acute over (.Math.)}anti, against+.Math.septikos, putrefactive) are antimicrobial substances that are applied to living tissue/skin to reduce the possibility of infection, sepsis, or putrefaction. They should generally be distinguished from antibiotics that destroy bacteria within the body, and from disinfectants, which destroy microorganisms found on non-living objects.

[0208] A composition for use as a sanitizer, detergent, disinfectant, or antiseptic can, in some cases, be diluted in a suitable diluent such as saline, phosphate buffered solutions, and other pH stabilized aqueous solutions, and can be used in combination with other sanitizers, detergents, disinfectants, or antiseptics, such as alcohols, quaternary ammonium compounds, boric acid, chlorhexidine gluconate, iodine, octenidine dihydrochloride, and sodium chloride.

[0209] Recent USDA Studies in Swine

[0210] Recent experimental test results from the Unites States Department of Agriculture serve to verify the efficacy of porcine intestinal immune systems against various H1N1 and other influenza diseases.

[0211] Experiment 1:

[0212] Four Pig Pathogenesis Study with the 2009 A/H1N1 Influenza A Virus.

[0213] Purpose of Study:

[0214] An important concern is to address whether meat, blood and tissue from pigs infected with the new 2009 H1N1 Influenza A Virus is free of infectious virus.

[0215] Experiment:

[0216] Four 5-week-old cross-bred pigs from a herd free of swine influenza virus (SIV) and porcine reproductive and respiratory syndrome virus (PRRSV) were housed in containment facilities and cared for in compliance with the Institutional Animal Care and Use Committee of the National Animal Disease Center (NADC).

[0217] Pigs were inoculated intra-tracheally with an infective dose of the 2009 HIN1 Influenza A Virus isolated from persons in California (A/CA/04/2009) obtained from the Center for Disease Control and Prevention (CDC).

[0218] Pigs were observed daily for clinical signs of disease. Nasal swabs were taken on 0, 1, 2, 3, 4, and 5 days post infection (dpi) to evaluate nasal shedding. Pigs were humanely euthanized on 5 dpi, which is considered the peak of infection in the NADC porcine SIV challenge model, to evaluate lung lesions and viral load in the lung and tissues. Fresh samples were taken from lung, tonsil, inguinal lymph node, liver, spleen, kidney, skeletal muscle (ham), and colon contents (feces), and examined using both real time RT-PCR and virus isolation (VI) techniques, which are the most sensitive and specific tools to detect the presence of viral nucleic acid and live virus, respectively.

[0219] Results:

[0220] Tissues outside the respiratory tract were found to be negative by VI at 5 days post infection. Only respiratory tract samples were positive by both methods (real time RT-PCR and VI). The inguinal lymph node from one pig and serum from two pigs were positive by real time RTPCR. However, lymph node and serum samples from all pigs were negative by VI. By contrast, all day 5 post infection nasal swabs and lung lavage fluids were positive by real time RT-PCR and VI, and lung tissue homogenates from all four pigs were positive by real time RT-PCR and 2/4 samples positive by VI.

[0221] Conclusion:

[0222] Live 2009 A/H1N 1 Influenza A Virus was only detected in the respiratory tract of infected pigs and the virus does not appear to spread and replicate in other tissues based on the day 5 post infection samples.

[0223] In addition, while not bound by theory, the efficacy of porcine enzymes may be enhanced by vaccinating pancreatic porcine donors with Trivalent inactivated influenza vaccine (TIV) or Live attenuated influenza vaccine LAIV. Current test results suggest that vaccinated pigs demonstrate a pre-existing immunity to certain currently circulating H1N1 SW strains may protect against an outbreak virus.

[0224] Experiment 11

[0225] Recent Results from Studies with the 2009 A/H1N1 Influenza A VirusProject 1: Serologic cross-reactivity of serum samples from U.S pigs against the new 2009H1N1 influenza virus.

[0226] Purpose of Study:

[0227] An important concern is to address whether U.S commercial swine herds are susceptible to the 2009 A/H1N1 influenza viruses isolated from persons in California, New York, and Mexico.

[0228] Experiment:

[0229] Three 2009 A/H1N1 influenza A viruses isolated from persons in 2009 in California A/CA/04/2009), New York (A/NY/18/2009), and Mexico (A/Mexico/4108/2009) were obtained from the Centers for Disease Control and Prevention (CDC) and grown in vitro (i.e., in a permissive cell line).

[0230] A standard hemagglutination inhibition (HI) test was used to investigate antigenic relatedness between these three 2009 A/H1N1 influenza A viruses and 19 H1 Swine Influenza Virus (SIV) strains known to be circulating in U.S. swine herds or with SW strains used for five licensed U.S H1N1 SW vaccines. Antigenic relatedness would be predicted on the basis of how well these antisera could inhibit the three 2009 A/H1N1 influenza A viruses from agglutinating (clumping) red blood cells. This test indicates the presence of antibodies that prevent the influenza virus from attaching to red blood cells and is therefore indicative that the animal may have protective antibodies. The CDC and USDA-APHIS-Center for Veterinary Biologics report an 8-fold or greater reduction in HI titer a significant reduction in cross reactivity between virus hemagglutinin variants.

[0231] Thirty-eight serum samples from pigs vaccinated with 19 H1 SIV isolated from U.S commercial swine operations between 1999-2008 (NADC H1 serum reference panel) were tested in the standard H1 test. The 19 H1 SIV in the NADC H1 serum reference panel used in this study represent all four phylogenetic (genetically characterized) clusters (, , , and ) of all the endemic H1 swine influenza viruses known to circulate in the U.S. An additional 14 serum samples from pigs vaccinated with five different commercial products used to vaccinate pigs against H1 swine influenza viruses in the U.S were tested by the standard H1 test.

[0232] Results: Eleven of the thirty-eight serum samples from pigs inoculated with U.S H1N1 SW had a measurable H1 titer against the A/CA/04/2009 H1N1 influenza virus. The same experiment with the A/NY/18/2009 H1N1 influenza virus had similar results. In contrast, twenty two of the thirty-eight serum samples from pigs inoculated with U.S H1 SIV had a measurable H1 titer against the (A/Mexico/4108/2009) H1N1 influenza virus. Serologic cross-reactivity with anti-sera from 5 commercially-available SW H1 vaccines was additionally assessed by H1 with the three 2009 A/H1N1 strains. Cross reactivity was consistently low between the vaccine antisera and all 2009 A/H1N1 novel strains tested, although titers were slightly higher with the isolate from Mexico. This suggests that currently available vaccines may provide only limited protection against challenge with the novel H1N1.

[0233] Conclusion:

[0234] Results of this experiment suggest that pre-existing immunity induced by swine influenza viruses circulating in the U.S swine herd may not protect pigs against the new 2009 A/H1N1 influenza viruses presently circulating in people. Importantly, vaccines currently used to protect pigs in U.S swine operations against swine influenza virus may not be effective against the new 2009 H1N1 influenza viruses.

[0235] Limited cross-reactivity of serum samples from the NADC H1 SIV antiserum reference panel or sera from pigs vaccinated with commercial vaccines was demonstrated against the 2009 A/H1N1 influenza virus (A/CA/04/2009) isolated in California as measured by a standard H1 test. A second 2009 A/H1N1 strain from New York, A/NY/18/2009, was also used with the NADC H1 antiserum reference panel with very similar results to A/CA/04/2009. However, a third strain, A/Mexico/4108/2009, demonstrated broader cross-reactivity with the NADC H1 antiserum reference panel. This was especially apparent in the Hly phylogenetic cluster. The cross-reactivity with the Hly phylogenetic cluster is important since this is the genetic group in which the HA from the 2009 A/H1N1 originated. This would suggest that pre-existing immunity to certain currently circulating H1N1 SIV strains may protect against the outbreak virus. However, the differences between the novel H1N1 isolates suggest that there may be biologic variation in host and/or virus properties responsible for the variation in serologic crossreactivity.

[0236] It remains unknown whether this variation would have any effect on protection from live challenge in pigs from circulating strains of the 2009 A/H1N1 from the human population. Serologic cross-reactivity with anti-sera from 5 vaccines was also assessed by H1 with the three 2009 A/H1N1 strains. Cross reactivity was consistently low between the vaccine antisera and all 2009 A/H1N1 novel strains, although titers were slightly higher with the isolate from Mexico. This suggests that currently available vaccines may provide only limited protection against challenge with the 2009 H1N1.

EXAMPLES

[0237] Proposed Experiments

[0238] Experiment 1In Vitro Testing

[0239] Reactivity of A/H1N1 influenza A virus to uncoated pancreatic porcine enzyme dilutions in a pH neutral medium.

[0240] Purpose of Study

[0241] To address the efficacy of uncoated pancreatic porcine enzymes in eradicating or inhibiting the spread of various strains of A/H1N1 influenza virus by measuring the degradation in viral RNA integrity following exposure to an enzymatic preparation.

[0242] Experiment:

[0243] Influenza virus type A, H1N1, isolated from persons in various time periods and geographic locations will be obtained from the Centers for Disease Control and Prevention (CDC) or World Health Organization (WHO) and grown in vitro (i.e., in a permissive cell line). Appropriate viral containment facilities will be employed.

[0244] A/H1N1 virus will be grown in culture using infection into an appropriate cell host such as MDCK anchorage dependant cells cultures or CACO-2 cell lines. A/H1N1 viral particles shed into the culture medium will then be collected and the viral particle concentration determined using a real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR) based assay for a/H1N1 specific RNA. Porcine Enzyme Concentrate will then be added to a mixture of the viral particles prepared in standard cell culture growth media used with either of the above two named cell lines. Various dilutions will be prepared including 1:25, 1:50, 1:100, 1:200. Materials will be incubated for a period of time ranging from 30 minutes to 6 hours at 37 C. Virus particles will then be separated from the Enzyme preparation, reconstituted in growth medium and plated onto the CACO-2 or MDCK cell lines. Cells will be allowed to grow and titer of released viral particles over a period of 3 days will be determined. Controls will be prepared by exposing A/H1N1 viral particles to phosphate buffered saline solution at 37 C for identical times. Control particles will then be prepared and plated as per experimentally treated virus cultures.

[0245] Samples at 12 hour periods over the course of 3 days will be assayed using laboratory diagnostic testing for the presence of influenza viruses in the specimens. In addition to utilization of a Real-time Reverse Transcriptase Polymerase Chain Reaction based assay (rRT-PCR) that has been shown capable of detecting the A/H1N1 specific RNA, other tests may be used including direct antigen detection tests such as ELISA based assays for viral antigens and virus isolation in cell culture may be used.

[0246] Other rRT-PCR assays such as laboratory developed tests, not approved by FDA, may also be able to detect novel influenza A (H1N1) viruses. Public health laboratories in the U.S. are currently able to perform the CDC rRT-PCR Swine Flu Panel assay.

[0247] Anticipated Results:

[0248] Replication of A/H1N1 in a permissive cell line should be inhibited (static), reduced or eradicated (-cidal) by exposure to the pancreatic porcine enzyme dilutions as compared to control samples.

[0249] Experiment 2In Vivo Testing

[0250] Effectiveness of coated pancreatic porcine enzyme on A/H1N1 influenza A virus symptoms or viral load in humans.

[0251] Purpose of Study

[0252] To address the efficacy of coated pancreatic porcine enzymes in eradicating or decreasing the symptoms or viral load of A/H1N1 influenza virus.

[0253] Experiment:

[0254] Candidate test subjects are those individuals who exhibit symptoms of A/H1N1 influenza A viruses and meet other test inclusion or exclusion criteria will be tested for presence of the virus using rapid A/H1N1 testing. Test Groups may be selected by age including infant and elderly, general health including healthy subjects and immuno-compromised subjects, geographic location, or other selection/exclusion criteria.

[0255] Rapid influenza diagnostic testing is utilized to identify candidate test subjects in a clinically relevant time period. Rapid influenza diagnostic tests (RIDTs) are typically antigen detection tests that detect influenza viral nucleoprotein antigen. The present commercially available test can provide results within 30 minutes or less. These assays may be referred to as point-of care tests since CLIA-waived RIDTs (not all RIDTs are CLIA waived) may be used in facilities with a certificate of waiver or in locations outside a central laboratory. Commercially available RTDTs can either: i) detect and distinguish between influenza A and B viruses; ii) detect both influenza A and B but not distinguish between influenza A and B viruses; or, iii) detect only influenza A viruses. None of the currently FDA approved RIDTs can distinguish between influenza A virus subtypes (e.g. seasonal influenza A (H3N2) versus seasonal influenza A (H1N1) viruses), and RTDTs cannot provide any information about antiviral drug susceptibility.

[0256] Once a positive result is obtained further swabs or test specimens are obtained from the test subjects for detailed laboratory analysis to validate positive test subject and isolate specific viral subtype(s). Laboratory tests may include direct antigen detection tests, virus isolation in cell culture, or detection of influenza-specific RNA by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR).

[0257] Laboratory tests typically differ in their sensitivity and specificity in detecting influenza viruses as well as in their commercial availability, the amount of time needed from specimen collection until results are available, and the tests' ability to distinguish between different influenza virus types (A versus B) and influenza A subtypes (e.g. novel H1N1 versus seasonal H1N1 versus seasonal H3N2 viruses). At the present time, there are only two FDA authorized assays for confirmation of novel influenza A (H1N1) virus infection, including the CDC rRT-PCR Swine Flu Panel assay.

[0258] Other rRT-PCR assays such as laboratory developed tests, not approved by FDA, may also be able to detect novel influenza A (H1N1) viruses. Public health laboratories in the U.S. are currently able to perform the CDC rRT-PCR Swine Flu Panel assay

[0259] Stool samples may be collected from the subjects both prior to the start of clinical testing and periodically during testing. This may especially important when testing immune-compromised individuals who may have low levels of chymotrypsin or other pancreatic secretions in their stool.

[0260] Coated Porcine Enzyme Concentrate will be administered to test subjects at appropriate doses with each major meal for a minimum of 3 dosings per day over a period of 7 to 10 days. Swabs or test specimens and, optionally, stool samples are collected on a daily basis for subsequent laboratory analysis over a clinically relevant time period such as 7 to 10 days. Patients will be observed for progression of symptoms, viral related sequelae and the presence of viral particles in collected samples over the course of treatment.

[0261] Placebos arc also administered to a control sub group of test subjects at the same time intervals and swabs or test specimens and optionally stool samples are collected on a daily basis for subsequent laboratory analysis over a clinically relevant time period such as 7 to 10 days.

[0262] Anticipated Results:

[0263] Administration of coated pancreatic porcine enzymes to test subjects positive for A/H1N1 should decrease the severity and/or duration of associated symptoms. Additionally, compared to placebo treated patients, there should be an accelerated decrease in detectable virus obtained from physiological samples.

[0264] The foregoing description of the embodiments of the disclosure has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Many modifications and variations are possible in light of this disclosure. It is intended that the scope of the disclosure be limited not by this detailed description, but rather by the claims appended hereto.