Improved casein peptide composition

Abstract

The current disclosure relates to a pharmaceutical composition comprising casein peptide, wherein said composition is substantially devoid of protease activity and uses thereof.

Description

P-635756-PCIMPROVED CASEIN PEPTIDE COMPOSITION BACKGROUND OF THE INVENTION

[0001] The casein protein comprises four main types of casein, aSl, a S2, P, and K, according to their sequence and electrophoretic mobility. Casein hydrolysate includes different casein peptides of different casein sequences and different lengths. It has been established that some of the casein peptides are bioactive, and play a significant role as immunomodulators, therefore can support the immune responses against microbial and viral-related infections.

[0002] There is an increasing interest in casein-derived peptides due to their wide potential applications in the food, drug, and cosmetic industries.

[0003] Trypsin is an enzyme in the first section of the small intestine, digesting protein and peptide molecules by cutting long chains of amino acids into smaller pieces. Trypsin is a serine protease found in the digestive system of many vertebrates. Trypsin cuts peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. Trypsin is available in significant quantities within the pancreas and can be easily purified. Due to its availability and effectiveness, trypsin is used in numerous biotechnological processes.

[0004] The use of trypsin for the hydrolysis of casein has many important advantages. Enzymatic cleavage is performed in mild conditions, therefore preserving the resulting peptides. Moreover, the activity of trypsin as serin protease may generate similar peptides as normally produced within milk.

[0005] In milk, several serine proteases (such as trypsin) play important roles in various biological processes, including digestion and the regulation of milk protein structure. The notable serine proteases found in milk are:Chymosin (Rennin): Primarily involved in milk coagulation; it's crucial for cheese-making.P-635756-PCPlasmin: A key enzyme that contributes to the breakdown of milk proteins, affecting flavor and texture in dairy products.Cathepsin D: Although primarily aspartic, some research indicates the presence of serine-type activity under specific conditions.Bacterial serine proteases: Some bacteria that contaminate milk or are involved in fermentation can produce serine proteases that affect milk properties.

[0006] This is the reason why casein peptides are always present in milk at some basal level and the bioactivity may be preserved. However, the elimination of residual trypsin activity from the product at the end of the hydrolysis is essential. The ongoing activity of residual active trypsin within the final product might damage the product by further changing the profile of the peptides over time. In addition, an active enzyme within the product is not desirable since introducing to the body a powerful protease such as trypsin, even in relatively small quantity, may substantially alter the biological environment and can even result in safety issues. Due to these potentially harmful effects, the presence of active enzymes within the product will be considered as an additional active pharmaceutical ingredient and needs to be addressed as such also from a regulatory perspective.

[0007] There are several ways to inactivate trypsin. Trypsin can be chemically inactivated by adding protease inhibitors such as al -antitrypsin and a2-macroglobulin or by using commercially available inhibitors. However, the use of inhibitors may become expensive, and the interactions may become reversible following the dilution after the injection into the body. Also, additional activity in the product makes it less practical from a regulatory perspective. Another additive that may block the activity of the trypsin is bivalent cationic ions such as magnesium and calcium. As with the chemical blocker, the use of ions may become reversible and therefore are less attractive for use in the product. Appreciating theP-635756-PClimitations of additives, heat inactivation of the trypsin protein is one of the common ways to inactivate trypsin. However, unexpectedly, and although large proteins such as enzymes are very sensitive to heat, the heat inactivation of the trypsin under conditions that will preserve the active peptides, may fail in some large-scale productions of casein hydrolysate. These failures in fully inactivating trypsin present with the casein peptides were evident even when relatively extreme inactivation conditions were used; high temperatures (80C°) over a significant time (up to one hour). This is a significant issue since heat inactivation of trypsin within a mixture of peptides, without harming the peptides, is a significant challenge.

[0008] To achieve a product made by trypsin degradation, that is free of trypsin activity is still a great need in the industry, representing the need for optimizing the production process and improving the quality of the final product.SUMMARY OF THE INVENTION

[0009] In some aspects, disclosed herein is a pharmaceutical composition comprising casein peptide, wherein the composition is substantially devoid of protease activity.

[0010] In some related aspects, the protease is trypsin.

[0011] In some related aspects, the composition further substantially does not contain residual full-length casein.

[0012] In some related aspects, the active peptides are casein fragments, manufactured in a process comprising protease.

[0013] In some aspects, the casein peptides comprise casein peptides originating from casein protease hydrolyzation. In some further aspects, the casein peptide is casein hydrolysate. In some further aspects, the casein peptides further comprises synthetic peptides, semi-synthetic peptides, or a combination thereof. In some further aspects, the synthetic casein peptide is a recombinant peptide. In some further aspects, the recombinantP-635756-PCcasein peptide is produced by fermentation, tissue culture, or a combination thereof. In some further aspects, the tissue culture comprises mammary gland tissue.

[0014] In some aspects, disclosed herein is a process for the manufacturing of a pharmaceutical composition comprising casein peptides, wherein said final composition does not contain protease activity comprising adjustment steps of the peptide solution to pH 7.3±0.1 (7.2-7.4) before the heat inactivation step performed at 80C° for at least 60 min and. In some related aspects, disclosed herein is a composition produced by the disclosed process. In some related aspects, disclosed herein is a process for manufacturing a pharmaceutical composition comprising casein hydrolysate, wherein said final composition does not contain trypsin activity, that is comprising steps to heat inactivation at 80°C° for at least 60 min and adjustment of pH to 7.2-7.4.

[0015] In some related aspects, disclosed herein is a method of treating, reversing, or preventing a condition or disease in a subject in need thereof, comprising administering a composition as described herein.

[0016] In some related aspects, the condition or disease comprises a microbiological infection of mammary gland tissue.

[0017] In some related aspects, the microbiological infection of mammary gland tissue is mastitis.

[0018] In some related aspects, disclosed herein is a method for increasing milk yield, increasing milk quality, accelerating involution, preventing, reversing or treating udder’s infections, or any combination thereof, comprising administering a composition as described herein.

[0019] In some related aspects, the method comprises administering between lOpg to 10,000 mg of said casein hydrolysate, per administration.P-635756-PC

[0020] In some related aspects, the administration comprises intramammary infusion to a single teat of polarity of teats, oral, intraoral, rectal, parenteral, topical, epicutaneous, transdermal, subcutaneous, intramuscular, intravenous, intranasal, intra rectal, intravaginal, sublingual, buccal, intradural, intrarespiratory, nasal inhalation, administration via intramammary intracanal, patch attached or and application via soaking to at least one infected udder quarter or and to at least one non-infected udder quarter, or any combination thereof.

[0021] In some related aspects, the pharmaceutical composition is administrated during a lactation period. In some further related aspects, the lactation period comprises the beginning of active lactation, middle of active lactation, end of active lactation, approaching, initiation of active lactation, end of the dry period or at the cessation of nursing.

[0022] In some related aspects, the subject is selected from the group consisting of a cow, a goat, a sheep, a buffalo, a camel, a donkey, a llama and a horse.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0024] Fig. 1 is a comparison of C18 profiles of trypsin inactivation for 30min boiling: Day 0 (black trace) vs Day 8 (blue trace) vs Day 13 (pink trace);

[0025] Fig. 2 is a C18 profile of STI reaction mixture on Day 3 (blue trace) vs Day 0 (black trace);P-635756-PC

[0026] Fig.3 is a Cl 8 profile of control reaction mixture on Day 3 (blue trace) vs Day 0 (black trace);

[0027] Fig. 4 is a C18 profile of STI reaction mixture on Day 7 (blue trace) vs Day 0 (black trace);

[0028] Fig. 5 is a Cl 8 profile of STI reaction mixture on Day 17 (blue trace) vs Day 0 (black trace);

[0029] Fig. 6 is a graphic illustration of pH values of different batches. Average of 6.99, median of 7.00. The maximum measured pH was 7.10 slightly basic, and the minimum was 6.89;

[0030] Fig. 7 is a graphic illustration of the correlation between the pH of the composition at the "end of hydrolyzation / starting of inactivation" and the final residual trypsin levels in our final product;

[0031] Figs. 8A, 8B and 8C are graphic illustrations of the residual concentration of trypsin in high-temperature inactivation of residual trypsin (80C°) at pH 4.7 (Fig.8A) or 7.4 (Fig. 8B). Fig.8C presents the same data used in Fig. 8A and Fig. 8B in the same figure to farther demonstrate that very low residual activity of trypsin following pH 7.41 (presented also in Fig. 8A) vs. the significant and positive presence of residual trypsin activity when acidic pH is used (pH=4.71, Fig. 8B).

[0032] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.P-635756-PCDETAILED DESCRIPTION OF THE PRESENT INVENTION

[0033] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.Composition

[0034] In some embodiments, disclosed herein is a pharmaceutical composition comprising casein peptide, wherein said composition is substantially devoid of protease activity. In one embodiment, the composition is manufactured using protease. In one embodiment, the composition is manufactured using trypsin.

[0035] As used herein, the term “substantially devoid” relates to the absence of protease activity, set below the acceptable level (11.7 BAEE), as calculated by the following formula: Activity of test solution BAEE unit ml) = (Slope for test solution - Slope of blank) * Factor.

[0036] In one embodiment, the protease is trypsin. In another embodiment, the protease is plasmin. In another embodiment, the protease is kallicrein. In another embodiment, the protease is cathepsin.

[0037] A skilled arctician would understand that a protease is used to break down peptide bonds. Trypsin is a protease enzyme that starts digestion in the small intestine by cutting long chains of amino acids into smaller pieces. It is a serine protease that is found in the digestive system of many vertebrates. Peptide fragments of a proteins, produced by either chemical synthesis, recombinant DNA technology, or subjecting the peptide or the protein amino acids chain to at least one cleaving agent. Hydrolysis of casein is typically performedP-635756-PCby digestion with trypsin. The advantages of using trypsin in commercial and industrial applications lie in its specificity, moderate heat stability, cost-effectiveness, and wide range of applications across different industries. Trypsin specifically cleaves peptide bonds at the carboxyl side of lysine and arginine residues, which can be advantageous for controlled proteolysis in various applications. This specificity allows for predictable and reproducible protein processing, which is critical in many industrial processes. The use of trypsin over other similar enzymes as the selected protease in the production of casein hydrolysate (bCNH) for veterinary use in the udder is beneficial due to three different advantages: First, trypsin is a serine protease, very similar to plasmin which is the natural most important protease in milk. Therefore, the results of casein peptides may be very similar to the naturally produced casein peptides and, therefore may become safer to use. Second, trypsin originated from mammals’ source, whereas many other commercially available with reasonable prices originating from microorganisms such as bacteria or fungal. Therefore, trypsin, either fully active or denatured, is expected to be less immunogenic. Third, the activity of trypsin on casein will be more like plasmin than the activity of chymotrypsin. Both plasmin and trypsin target lysine and arginine residues, resulting in similar cleavage patterns and peptide fragments. Chymotrypsin, with its different specificity for aromatic residues, will produce a distinctly different pattern of casein degradation. However, one of the most important disadvantages is that trypsin is relatively highly resistant to heat inactivation. Typically, temperatures of 60-65C0are used to fully denature this enzyme. Therefore, a temperature of 80C°, for 60 minutes, should be very effective in fully denaturing it.

[0038] In one embodiment, the heat inactivation method relies on the differential sensitivity of the full protein, trypsin or any other protease, and the relatively heat-resistant casein peptides. Due to the potential variations in the raw materials used in the preparationP-635756-PCof the casein peptides, probably the casein, as well as other potential variations, the peptide solution following the enzymatic degradation may end up slightly different between different preparations, affecting the efficiency of the subsequent step of inactivating the residual trypsin. One of the differences is the pH of the casein peptide solution following the enzymatic activity step and before the inactivation of the protease. It was demonstrated that the success in fully inactivating the protease activity in a given process was closely related to the level of the pH. It was even more surprising that the range of pH values that differentiated between sufficient inactivation of the trypsin and non-successful inactivation of the enzyme was very narrow.

[0039] In another embodiment, the pharmaceutical composition further substantially does not contain residual full-length casein.Casein peptides

[0040] Casein is a protein in mammal’ s milk, known to include the subgroups aSl, aS2, P, and K. Casein is defined according to the amino acid sequences of each of the subgroups aSl, aS2, P, and K. In the context of the present disclosure, when referring to casein, it is to be understood as also including acid casein, salts of casein, phosphorous-containing casein, and rennet casein.

[0041] The term "peptide" as used herein refers to amino acid residues, connected by peptide bonds. Peptides and protein sequences are generally reported from the N-terminal end containing the free amino group to the C-terminal end containing the free carboxyl group. Amino acids, as used herein, refer to naturally occurring and synthetic amino acids, as well as amino acid analogs, and amino acid mimetics, that function in a manner similar to the naturally occurring amino acids. Amino acids may be referred to herein by either theirP-635756-PCcommonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0042] The casein-derived peptide is a peptide that contains a sequence that appears within the sequence of casein proteins as defined herein. Casein-derived peptide may be a single peptide or a mixture of different peptides which may be independently selected from a naturally occurring casein peptide, a semi-synthetic peptide, a synthetic peptide, or a recombinant peptide. It should be further noted that the peptides according to the present disclosure may be produced synthetically, by recombinant DNA technology, or by any other technology. Methods for producing peptides are well-known in the art.

[0043] In some embodiments, the casein-derived peptide may comprise a casein peptide that is produced when casein protein is cleaved by enzymes or acids to peptide fragments (also known in the art by the term "casein hydrolysate"). A casein hydrolysate is to be understood as a peptide or peptides that resulted from the hydrolyzed form of casein (protein). Casein hydrolysate includes, for example, the active beta-, alpha S1-, alpha S2-, and kappa-casein-derived peptide. In some embodiments, the casein-derived peptide is or comprises a casein hydrolysate.

[0044] In some embodiments, the active peptides are casein fragments, manufactured in a process comprising protease. In one embodiment, the protease is trypsin. In the case of casein hydrolysis, the use of protease in large-scale production is beneficial due to its economic use and repeatability. In addition, the production of specific types of peptides that are also phosphorylated is very complicated and expensive, often even on a small production scale. It may be even not possible on a large scale or with some common methods.

[0045] In one embodiment, the casein peptides originate from casein protease hydrolyzation. In one embodiment, the casein peptide is casein that was hydrolysate.P-635756-PC

[0046] In one embodiment, the active peptides further comprise natural casein peptides, synthetic casein peptides, semi-synthetic casein peptides, homologues casein peptides or any combination thereof. In another embodiment, the active peptides comprise natural casein peptides, isolated from milk. In another embodiment, the active casein peptides comprise semi-synthetic casein peptides. In another embodiment, the active peptides comprise natural casein peptides and synthetic casein peptides. In another embodiment, the active casein peptides comprise natural casein peptides and semi-synthetic casein peptides. In another embodiment, the active peptides comprise synthetic casein peptides and semi-synthetic casein peptides. In another embodiment, the active peptides comprise natural casein peptides, synthetic peptides, and semi-synthetic peptides. In one embodiment, the natural casein peptides are isolated from milk. In another embodiment, the active peptide comprises homologues casein peptides

[0047] Natural casein-derived peptides are typically obtained following enzymatic hydrolysis, the enzyme may be any mammal peptidase, such as, without being limited thereto, plasmin, pancreatin, trypsin, chymotrypsin, neutrase, alcalase, elastase, pepsin, carboxypeptidase, cathepsin, as well as plant peptidase such as, without being limited thereto, papain, bromelain, as well as enzymes from microorganism source. For example, a naturally occurring casein-derived peptide may be the result of an enzyme activity such as plasmin on casein subunits P-casein, asl- and as2-casein or K-casein. In some embodiments, a casein hydrolysate is obtained by cleavage of the casein protein with trypsin.

[0048] A synthetic peptide may be obtained by any methods known in the art of peptide synthesis including chemical synthesis and recombinant DNA technology. For example, the peptides may be synthesized by using standard solid solid-phase techniques.P-635756-PC

[0049] In one embodiment, the synthetic peptide is a recombinant peptide.

[0050] A semi-synthetic casein-derived peptide may be obtained by chemical hydrolysis of casein, e.g. by prolonged boiling in a strong acid (acid-HVP) or strong base or using a chemical agent such as Cyanogen bromide (CNBr). The casein-derived peptide may also be obtained by molecular engineering, e.g. using recombinant DNA, in molecular techniques known in the art. In such embodiment, the casein-derived peptide is a recombinant peptide.

[0051] In one embodiment, the recombinant peptide is produced by fermentation, tissue culture or combination thereof. In another embodiment, the recombinant peptide is produced by fermentation. In another embodiment, the recombinant peptide is produced by tissue culture. In another embodiment, the recombinant peptide is produced by a combination of fermentation and tissue culture.

[0052] In one embodiment, the tissue culture comprises mammary gland tissue .

[0053] In some embodiments, the casein-derived peptide comprises one or more fragments of P-casein, aS 1 -casein, aS2-casein, K-casein or any combination thereof. In one embodiment, the casein derived peptide comprises one or more fragments of P-casein. In another embodiment, the casein derived peptide comprises one or more fragments of aSl-casein. In another embodiment, the casein derived peptide comprises one or more fragments of aS2-casein. In another embodiment, the casein derived peptide comprises one or more fragments of K-casein. In another embodiment, the casein derived peptide comprises one or more fragments of combination of P-casein, aS 1 -casein, aS2-casein and K-casein.

[0054] In one embodiment, the casein derived peptide comprises a casein hydrolysate

[0055] In one embodiment, the casein derived peptide comprises a phosphopeptide.

[0056] As used herein, the term "phosphopeptide" designates a phosphorylated peptide in the form of a conjugated peptide in which the non-peptide portion is a residue ofP-635756-PCphosphoric acid. The expression "phosphopeptide" or "phosphoserine" designates conjugated serine in which the non-peptide portion is a residue of phosphoric acid.

[0057] In some embodiments, the casein-derived peptide is a single peptide or mixture of a phosphopeptide, namely, which contains a single phosphorous group or is a phosphorus-enriched peptide. In some embodiments, the casein-derived peptide is any phosphoserine, phosphotyrosine, phosphothreonine, and / or phosphohystidine-enriched casein-derived peptides (casein phosphopeptide, CPP) and monovalent cation phosphocaseinates, such as sodium, potassium, calcium or ammonium phosphocaseinates.

[0058] In some embodiments, the casein-derived peptide is a phosphor-peptide.

[0059] The phosphor-peptide may be genetically engineered casein-derived peptides as well as peptidomimetics of casein-derived peptides. For example, phosphorylation of amino acids such as at least one serine residue may be performed by any method as is known in the art. The term "casein-derived peptide" also encompasses peptide fragments or peptidomimetic products obtained from or corresponding to one or more sections of casein protein. The peptidomimetic peptide may be for example a peptoid or a semipeptoid, which are peptide analogs, having, for example, modifications such as, but not limited to, cyclization, N-terminus modification, C-terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S-O, O-C-NH, CH2-O, CH2-CH2, S-C-NH, CH-CH or CF-CH, backbone modification and residue modification.

[0060] As used herein, the term “casein-derived peptide” further encompasses any derivatives, analogues, variants or homologues of any of the peptides. The term "derivative" is used to define amino acid sequences (peptide), with any insertions, deletions, substitutions and modifications to the amino acid sequences (peptide) that do not alter the activity of the original peptides. By the term “derivative” it is also referred to homologues, variants andP-635756-PCanalogues thereof, as well as covalent modifications of a polypeptides made according to the present invention.

[0061] In some embodiments, the modified, synthetic, semi-synthetic or other types of analogs of the naturally occurring casein-derived peptides are in some embodiments at least 75%, at times 85%, 90%, 95% and even 99% identical (in sequence) to a naturally occurring casein-derived peptide when the two sequences are optimally aligned. Further, any non-naturally occurring casein-derived peptide to be used in accordance with the present disclosure may retain at least part of the biological activity of the naturally occurring casein peptide.

[0062] The present disclosure also encompasses homologues of the casein-derived peptide. The term "homologues” is used to define amino acid sequences (peptide) which maintain a minimal homology to the amino acid sequences defined by the invention, e.g. have at least about 65%, at least about 75%, at least about 85%, or at least about 95% overall sequence homology with the amino acid sequence of any of the peptide as structurally defined above, e.g. of a specified sequence.

[0063] In some embodiments, the casein-derived peptide may also include a chemical modification of a naturally occurring peptide, e.g. where one or more amino acids are deleted, substituted or modified, e.g. by removal of a side group, substitution of a side group or the introduction of a chemical group. Without being limited thereto, the chemical modification may include acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristoylation, pegylation, prenylation, phosphorylation, ubiquitination, or any similar process. When referring to replacement of an amino acid sequence by another, it is likely that the replacement is a conservative substitution. For example, one or moreP-635756-PCamino acid residues within a casein sequence is substituted by another amino acid of a similar polarity or charge. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Nonetheless, non-conservative substitutions may also take place as long as it does not significantly change the desired (casein like) biological activity of the resulting caseinderived peptide analog.

[0064] A casein-derived peptide in accordance with the present disclosure is characterized by a molecular weight of between about an average 100 to an average 10,000 Dalton (e.g. between 2 to 100 amino acids) at times between about an average 100 to an average 7,000 Dalton and at times between an average 1,000 to an average 5,000 Daltons.

[0065] A casein-derived peptide in accordance with the disclosure is characterized by a length of from 2 to 200, from 2 to 100 amino acids, at times between 4 amino acids to 40 amino acids, at times from 4 amino acids to 30 amino acids, at times 4 amino acids to 10 amino acids, at times between 10 amino acids to 50 amino acids.

[0066] In one embodiment, the composition is free of non-casein peptide antimicrobials and comprises an acceptable carrier.

[0067] In one embodiment, the milk-derived protein is measured by UV at a range from 204 to 220 nm.Process

[0068] In some embodiments, disclosed herein is a process for the manufacturing a pharmaceutical composition comprising casein hydrolysate, wherein said composition doesP-635756-PCnot contain trypsin activity comprising steps of adjustment of the pH to 7.3±0.1 followed by deactivation of the enzyme at 80°C + / - 5°C for at least 60 min.

[0069] In one embodiment, the inactivation step is for 60 min. In another embodiment, the inactivation step is for 65 min. In another embodiment, the inactivation step is for 70 min. In another embodiment, the inactivation step is for 75 min. In another embodiment, the inactivation step is for 80 min. In another embodiment, the inactivation step is for 85 min. In another embodiment, the inactivation step is for 90 min. In another embodiment, the inactivation step is for 95 min. In another embodiment, the inactivation step is for 100 min. In another embodiment, the inactivation step is for 105 min. In another embodiment, the inactivation step is for 110 min. In another embodiment, the inactivation step is for 115 min. In another embodiment, the inactivation step is for 120 min.

[0070] In one embodiment, the pH during the inactivation step is 7.2. In another embodiment, the pH is 7.22. In another embodiment, the pH is 7.24. In another embodiment, the pH is 7.26. In another embodiment, the pH is 7.28. In another embodiment, the pH is 7.3. In another embodiment, the pH is 7.32. In another embodiment, the pH is 7.34. In another embodiment, the pH is 7.36. In another embodiment, the pH is 7.38. In another embodiment, the pH is 7.4.

[0071] In some embodiment, disclosed herein is a composition comprising casein hydrolysate, produced by the described process.Method of treatment

[0072] In some embodiments, disclosed herein is a method of treating reversing, or preventing a condition or disease in a subject in need thereof comprising administering a composition as previously described.P-635756-PC

[0073] In one embodiment, disclosed herein is a method of treating a condition or disease in a subject in need thereof comprising administering a composition as previously described.

[0074] In another embodiment, disclosed herein is a method of reversing a condition or disease in a subject in need thereof comprising administering a composition as previously described.

[0075] In another embodiment, disclosed herein is a method of preventing a condition or disease in a subject in need thereof comprising administering a composition as previously described.

[0076] In one embodiment, the condition or disease comprises a microbiological-related infection of mammary gland tissue. In one embodiment, the infection can be caused by bacterial, yeast, fungal, or viral contamination.

[0077] In one embodiment, the microbiological infection of mammary gland tissue is mastitis.

[0078] A skilled arctician would understand that the two principal causes of mastitis, which is inflammation of the breast tissues, are milk stasis (non-infection) and pathogen-related infection, both can contribute to the inflammation of the mammary gland. Milk stasis is usually the secondary cause, which may or may not be accompanied by or progress to an infection. In dairy cows, infection is by far the more common cause of mastitis, especially bacterial infection. However, milk stasis (non-infectious) can still play a significant role, particularly as a predisposing factor for infection. If milk is not properly removed, the stagnant milk provides an ideal environment for bacterial growth, and this can lead to infection. Infected cows may develop swelling, redness, heat, and pain in the affected udder, with a reduction in milk quality (e.g., high somatic cell count, which indicates inflammation). The disease caused by intramammary infection (IMI) is related to pathogens,P-635756-PCmostly bacteria, but also yeast, viruses, fungi, or even algae. Mastitis can be clinical with local (and in some cases general) clinical signs and milk abnormalities, or subclinical with production loss and lowered milk quality. Clinical and subclinical mastitis produce significant economic losses due to rejected milk (less farm production), degraded milk quality (less revenue) early culling of cows (loss of genetic potential) drug costs, veterinary expenses, and increased labor costs for the farmer. Mastitis is the most debilitating disease in dairy herds, costing the US dairy industry alone about 2 billion $ annually.

[0079] A skilled arctician would understand that reversing a condition or disease means changing a high degree of a disease to a low degree. For example - High Degree of Clinical Mastitis (e.g., Severe Clinical Mastitis) is reversed to Low Mastitis.

[0080] In some embodiments, disclosed herein is a method for increasing milk yield, increasing milk quality, accelerating involution, or any combination thereof, comprising administering a composition as previously described.

[0081] A skilled arctician would understand that increasing milk yield can be measured, for example, by the average volume of milk produced by the cows per day versus non-treated cows, the delta elevated average daily milk volume between the pre-lactation and the pot treatment lactation versus the non-treated cows, all of a matched age and / or parity.

[0082] A skilled arctician would understand that increasing milk quality can be demonstrated by the percentage of either butterfat, protein, lactose or the combination thereof.

[0083] A skilled arctician would understand that accelerating involution can be demonstrated by faster elevation of somatic cells, lactoferrin or any other known biomarkers for involution.P-635756-PC

[0084] In one embodiment, disclosed herein is a method for increasing milk yield. In another embodiment, disclosed herein is a method for increasing milk quality. In another embodiment, disclosed herein is a method for accelerating involution. In another embodiment, disclosed herein is a method for increasing milk yield, increasing milk quality, and accelerating involution.

[0085] In some embodiments, the involution comprises permanent involution or transient involution. In one embodiment, the involution comprises permanent involution. In another embodiment, the involution comprises transient involution.Dosage and administration

[0086] In some embodiments, the method comprises administering between lOpg to 10,000 mg of the casein hydrolysate, per administration. In one embodiment, the method comprises administering lOpg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering lOOpg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering Img of the casein hydrolysate, per administration. In another embodiment, the method comprises administering lOOmg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 150mgof the casein hydrolysate, per administration. In another embodiment, the method comprises administering 500mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 600mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 700mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 800mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 900mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering lOOOmg of the casein hydrolysate, per administration. In another embodiment, the method comprisesP-635756-PCadministering 1 lOOmg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 1200mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 1300mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 1400mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 1500mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 1600mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 1700mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 1800mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 1900mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 2000mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 2 lOOmg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 2200mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 2500mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 2700mg of the casein hydrolysate, per administration.

[0087] In another embodiment, the method comprises administering 3000mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 4000mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 5000mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 6000mg of the casein hydrolysate, per administration. In another embodiment, the method comprisesP-635756-PCadministering 7000mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering 8000mg of the casein hydrolysate, per administration.

[0088] In another embodiment, the method comprises administering 9000mg of the casein hydrolysate, per administration. In another embodiment, the method comprises administering lOOOOmg of the casein hydrolysate, per administration.

[0089] In some embodiments, the administration comprises inframammary infusion to a single teat of a polarity of teats, oral, intraoral, rectal, parenteral, topical, epicutaneous, transdermal, subcutaneous, intramuscular, intravenous, intranasal, intra rectal, intravaginal, sublingual, buccal, intradural, intrarespiratory, nasal inhalation, or any combination thereof.

[0090] In one embodiment, the administration comprises inframammary infusion to a single teat of a polarity of teats. In another embodiment, the administration comprises oral administration. In another embodiment, the administration comprises intraoral administration. In another embodiment, the administration comprises rectal administration. In another embodiment, the administration comprises parenteral administration. In another embodiment, the administration comprises topical administration. In another embodiment, the administration comprises epicutaneous administration. In another embodiment, the administration comprises transdermal administration. In another embodiment, the administration comprises subcutaneous administration. In another embodiment, the administration comprises intramuscular administration. In another embodiment, the administration comprises intravenous administration. In another embodiment, the administration comprises intranasal administration. In another embodiment, the administration comprises intra rectal administration. In another embodiment, the administration comprises intravaginal administration. In another embodiment, the administration comprises sublingual administration. In another embodiment, theP-635756-PCadministration comprises buccal administration. In another embodiment, the administration comprises intradural administration. In another embodiment, the administration comprises intrarespiratory administration. In another embodiment, the administration comprises nasal inhalation.

[0091] In some embodiments, the treatment or prevention comprises between one to thirty (30) administrations. In one embodiment, the treatment or prevention comprises one administration. In another embodiment, the treatment or prevention comprises two administrations. In another embodiment, the treatment or prevention comprises five administrations. In another embodiment, the treatment or prevention comprises ten administrations. In another embodiment, the treatment or prevention comprises fifteen administrations. In another embodiment, the treatment or prevention comprises twenty administrations. In another embodiment, the treatment or prevention comprises twenty -five administrations. In another embodiment, the treatment or prevention comprises thirty administrations.

[0092] In some embodiments, the treatment or prevention is repeated after a period of lapse time. In one embodiment, the lapse time comprises between 10 minutes and 46 hours. In another embodiment, the lapse time is between 1 hour and 40 hours. In another embodiment, the lapse time is between 10 hours and 30 hours. In another embodiment, the lapse time is between 20 hours and 25 hours. In another embodiment, the lapse time is 10 minutes. In another embodiment, the lapse time is 30 minutes. In another embodiment, the lapse time is 60 minutes. In another embodiment, the lapse time is 2 hours. In another embodiment, the lapse time is 5 hours. In another embodiment, the lapse time is 10 hours. In another embodiment, the lapse time is 15 hours. In another embodiment, the lapse time is 20 hours. In another embodiment, the lapse time is 25 hours. In another embodiment, theP-635756-PClapse time is 30 hours. In another embodiment, the lapse time is 35 hours. In another embodiment, the lapse time is 40 hours. In another embodiment, the lapse time is 45 hours. In another embodiment, the lapse time is 46 hours.

[0093] In another embodiment, the lapse time comprises several days. In another embodiment, the lapse time comprises several weeks. In another embodiment, the lapse time comprises several months. In another embodiment, the lapse time comprises several years.

[0094] In some embodiments, the treatment, reversing or prevention comprises intervals of from 1 hour to about 72 hours. In one embodiment, the administrations comprise intervals of about 1 hour. In one embodiment, the administrations comprise intervals of about 4 hours. In one embodiment, the administrations comprise intervals of about 5 hours. In one embodiment, the administrations comprise intervals of about 8 hours. In one embodiment, the administrations comprise intervals of about 10 hours. In one embodiment, the administrations comprise intervals of about 12 hours. In one embodiment, the administrations comprise intervals of about 15 hours. In one embodiment, the administrations comprise intervals of about 16 hours. In one embodiment, the administrations comprise intervals of about 20 hours. In one embodiment, the administrations comprise intervals of about 24 hours. In one embodiment, the administrations comprise intervals of about 25 hours. In one embodiment, the administrations comprise intervals of about 28 hours. In one embodiment, the administrations comprise intervals of about 30 hours. In one embodiment, the administrations comprise intervals of abouthours. In one embodiment, the administrations comprise intervals of about 36 hours. In one embodiment, the administrations comprise intervals of about 40 hours. In one embodiment, the administrations comprise intervals of about 45 hours.one embodiment, theP-635756-PCadministrations comprise intervals of about 50 hours. In one embodiment, the administrations comprise intervals of about 55 hours. In one embodiment, the administrations comprise intervals of about 60 hours. In one embodiment, the administrations comprise intervals of about 65 hours. In one embodiment, the administrations comprise intervals of about 70 hours. In one embodiment, the administrations comprise intervals of about 72 hours.

[0095] In another embodiment, the treatment or prevention comprises intervals of from 1 hour to about 24 hours.

[0096] In one embodiment, the pharmaceutical composition is administrated during a lactation period.

[0097] In some embodiments, the lactation period comprises the beginning of active lactation, middle of active lactation, end of active lactation, approaching, initiation of active lactation, end of the dry period or at the cessation of nursing.

[0098] In one embodiment, the lactation period comprises the beginning of active lactation. In another embodiment, the lactation period comprises middle of active lactation. In another embodiment, the lactation period comprises the end of active lactation. In another embodiment, the lactation period comprises approaching, initiation of active lactation. In another embodiment, the lactation period comprises the end of the dry period. In another embodiment, the lactation period comprises the cessation of nursing.

[0099] In one embodiment, the subject is selected from a group consisting of a cow, a goat, a sheep, a buffalo, a camel, a donkey, a llama and a horse. In another embodiment, the subject is a cow. In another embodiment, the subject is a goat. In another embodiment, the subject is a sheep. In another embodiment, the subject is a buffalo. In another embodiment,P-635756-PCthe subject is a camel. In another embodiment, the subject is a donkey. In another embodiment, the subject is a llama. In another embodiment, the subject is a horse.[000100] In one embodiment, the method comprises administration of one or more additional pharmaceutically active agents.[000101] In one embodiment, the additional pharmaceutically active agent is antibiotics. In another embodiment, the additional pharmaceutically active agent is steroids.[000102] The term “casein” as used herein generally refers to a family of related proteins (aSl, aS2, P, K) commonly found in mammalian milk.[000103] The term "treatment" concerns improvement of at least one undesired manifestation of the disease such as increase in disease free periods, decrease in acute disease periods (in time and severely), decrease in severity of the disease, improvement in life quality, improvement in comfort and welfare, decreased mortality, decrease in the rate of disease progression as well as prophylactic treatment before disease occurs. More specifically, the term “treatment or prevention” as used herein, refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction, alleviation and relief from a disorder or any related condition and illness, symptoms or undesired side effects or related disorders. It should be appreciated that the term “reduction” or as referred to herein, relate to the retardation, restraining or reduction of a process by any one of about 1% to 99.9%, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.P-635756-PC[000104] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. As used herein the term "about" refers to ± 10 %.[000105] As used herein, the term “average molecular weight” refers to the mean plus or minus standard deviation of the molecular weight of the peptide or protein as measured by a method known to a person skilled in the art. Such methods include, for example, SDS-gel electrophoresis and size exclusion chromatography in an apparatus such as HPLC, wherein the sample is run against Standards with known molecular weight.ExamplesExample 1 - Process for manufacturing a composition.[000106] The purpose of this experiment was to achieve a complete inactivation of trypsin activity. In the current trypsin inactivation process, the trypsin is not always completely deactivated across different production batches. In some of the cycles, it was found that the final product contained -1.24% of residual trypsin activity (i.e. 98.86% of the inserted enzyme was inactivated). This level of inactivation is not sufficient, which motivated the inventors to raise the inactivation temperature and increase the inactivation time to achieve complete inactivation.Methods for measuring trypsin activity:[000107] Method for measurement of trypsin activity in casein hydrolysate (bCNH)[000108] The determination of residual trypsin activity in the final product and the activity of trypsin in the product solution were tested using BAEE substrate. This substrate can be detected at 253nm only following the hydrolysis by trypsin.P-635756-PC[000109] Materials:• BAEE (Sigma)• 25mM Tris pH 7.4• 4M HC1 In-house• 2MNaOH• Sodium dihydrogen phosphate monohydrate• Trypsin SST (Sigma)Assay Procedure[000110] All measurements were performed in duplicate (except for the blank measurements). All samples were run in the same testing session using the same 0.25 mM Tris buffer BAEE solution. The temperature of the solution in the test was set to 25 °C.[000111] lOOOpl 0.25 mM BAEE were placed in a quartz cuvette with a plastic cover. The next steps were performed as fast as possible. 67 pl bCNH were added to the test item, the cuvette was covered, inverted to mix (twice), and placed in the cuvette holder. The overall duration of the follow-up was 300 seconds.[000112] The activity of the trypsin sample solutions was calculated according to the following general formula:Activity of test solution BAEE unit ml) = (Slope for test solution - Slope of blank) * Factor[000113] For Trypsin SST: the value for the “slope of blank” was obtained from the blank 1 mM HC1 slope which was performed at the beginning of each assay day.[000114] For the bCNH test sample: the value for the “slope of blank” was obtained from the blank 25mM Tris pH 7.4 slope which was performed after the SST run.P-635756-PC[000115] The acceptable criteria for the absence of protease activity were set below the acceptable level (11.7 BAEE) representing a level below the reliable detection level of this method.[000116] In the first experiment, the composition was heated to 80°C for 30 minutes. As can be seen in Fig. 1, comparing the C 18 chromatography profiles on Day 0, Day 8, and Day 13, the C 18 profile does not remain constant over time. This proves that the protein digestion reaction continues and that the trypsin is not fully deactivated. Therefore, the process was amended by changing the heating time from 30 minutes to 60 minutes.[000117] In the next step, the inventors used Soybean trypsin inhibitor (STI). As can be seen below (Figs. 2-5), the use of STI was not successful in blocking the potential residual activity of trypsin. STI is a 20kD protein purified from soybean. The STI was added to the reaction mixture after the 80°C incubation. The reaction mixture was incubated at 26°C and analyzed by HPLC on Days 0, 3, 7, and 17. Comparison of the Day 3 profile with the Day 0 profile showed that the profile of the reaction mixture containing STI is substantially stable over 3 days (Fig. 2) except for changes in the peak at 17.1 min, whereas the profile of the reaction mixture without STI (Fig.3) shows changes after 3 days. The ability of the specific protease inhibitor to block the demonstrated changes in the profile of the peptides further supports the assumption that the residual protease activity is the main cause of these changes.[000118] Thus, it appears that the ability of STI to inhibit trypsin is short-lived and reversible, lasting between 3-7 days. It can be concluded that the addition of STI, a specific trypsin inhibitor, to the final composition, is not sufficient to totally halt the hydrolysis reaction for an extended period.P-635756-PC[000119] Next, the inventors examined the correlation between the residual activity of trypsin and pH at the end of the hydrolysis in large-scale production.[000120] As can be seen in Fig. 6, when different batches were tested, the range of pH at the end of the hydrolysis was very narrow and not expected to affect the neutralization of the residual trypsin (between 7.1 to 6.89, a delta of only 0.21 pH units).[000121] Next, the inventors examined the pH correlation to the residual activity of the trypsin following the extended (60 minutes) heat inactivation step (80°C). As can be seen in Fig- 7, there is a correlation between the final pH in the solution and the residual activity of trypsin following heat inactivation steps (80±2°C for 60 min), ranging from 0-20 units, although the difference in the final pH is very narrow; between 6.89 to 7.10.[000122] Next, the inventors examined the residual activity of trypsin in high-temperature inactivation of residual trypsin (80°C) at pH 7.4 or 4.7 (Fig. 8A and Fig. 8B respectively). As can be seen in Fig. 8C, which presents the data from Fig. 8A and Fig. 8B together, Trypsin inactivation was completed when using a basic pH; of 7.4 (non-linear results with a maximum OD of <0.8, R2>0.76) and not an acidic; pH of 4.7 (linear elevated OD up to, R2>0.999).[000123] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

P-635756-PCCLAIMS1. A pharmaceutical composition comprising casein peptide, wherein said composition is substantially devoid of protease activity.

2. A pharmaceutical composition according to claim 1 , wherein said protease is trypsin.

3. A pharmaceutical composition according to claims 1-2, further substantially does not contain residual full-length casein protein.

4. A pharmaceutical composition according to claims 1-3, wherein said active peptides are casein fragments, manufactured in a process comprising protease.

5. The pharmaceutical composition according to any of the previous claims, wherein said casein peptide originated from casein hydrolyzation.

6. The pharmaceutical composition according to claim 5, wherein said composition further comprises synthetic casein peptides, semi -synthetic casein peptides, natural casein peptides, homologues casein peptides, or any combination thereof.

7. The pharmaceutical composition according to claim 6, wherein said synthetic casein peptide is a recombinant peptide.

8. A process for manufacturing a pharmaceutical composition comprising casein hydrolysate, wherein said final composition does not contain trypsin activity, that is comprising steps to heat inactivation at 80°C° for at least 60 min and adjustment of pH to 7.2-7.4.

9. A composition comprising casein hydrolysate, produced by the process of claim 8.

10. A method of treating, reversing, or preventing a condition or disease in a subject in need thereof, comprising administering a composition according to claims 1-8 and 9.P-635756-PC11. The method according to claim 10, wherein said condition or disease comprises a microbiological infection of mammary gland tissue.

12. A method of increasing milk yield, increasing milk quality, accelerating involution, or any combination thereof, comprising administering a composition according to claims 1-9.

13. The method according to claim 12, wherein said involution comprises permanent involution or transient involution.

14. A method according to claims 10-13, comprising administering between lOpgto 10,000mg of said casein hydrolysate, per administration.

15. A method according to claims 10-14, wherein said administration comprises intramammary infusion to a single teat of a polarity of teats, oral, intraoral, rectal, parenteral, topical, epicutaneous, transdermal, subcutaneous, intramuscular, intravenous, intranasal, intra rectal, intravaginal, sublingual, buccal, intradural, intrarespiratory, nasal inhalation, administration via intramammary intracanal, patch attached or and application via soaking to at least one infected udder quarter or and to at least one non-infected udder quarter, or any combination thereof.

16. A method according to claims 10-15, wherein said administration comprises between one to 30 administrations.

17. A method according to claims 10-16, wherein said administration is repeated after a period of lapse time.

18. A method according to claims 10-17, wherein said administration comprises intervals of from 1 hour to about 72 hours.

19. A method according to claims 10-18, wherein said administration comprises intervals of from 1 hour to about 24 hours.P-635756-PC20. The method according to claims 10-19, wherein said pharmaceutical composition is administrated during a lactation period.

21. The method according to claim 20, wherein said lactation period comprises the beginning of active lactation, middle of active lactation, end of active lactation, approaching, initiation of active lactation, end of the dry period or at the cessation of nursing.

22. The method according to claims 10-22, wherein said subject is selected from the group consisting of a cow, a goat, a sheep, a buffalo, a camel, a donkey, a llama, a horse.

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