Method and process for producing topically adhesive selective fungicides

Abstract

Disclosed herein are methods for the production of novel topically-adherent selective bactericides and products resulting therefrom, more specifically, methods for the production of complexed stable microbiome-regulating proteins configured for topical administration, and the use of such products as pharmaceuticals effective in treating pathogenic bacteria, but not commensal bacteria.

Description

[Technical field]

[0001] Bacteria easily attach to surfaces and form biofilms. Biofilms are commonly found on all epithelial surfaces of humans and animals, including the skin, intestines, lungs, nasal cavity, and oral cavity. The bacteria contained in biofilms can be benign or even beneficial (probiotic), such as bacteria of the Lactobacillus genus, or they can be pathogenic and harmful to the host, causing disease and illness, such as E. Coli and S. aureus. In most cases, biofilms are beneficial or at least benign, since the main components of the biofilm are probiotic bacteria. However, the effects of biofilms can become harmful if the levels of pathogenic bacteria in the film increase and overwhelm the benign or probiotic bacteria. For example, some skin diseases are the result of an overgrowth of pathogenic bacteria on the skin, such as atopic dermatitis, which is the result of an excess of the pathogenic bacteria Staphylococcus aureus on the skin. In particular, the predominance of pathogenic bacteria in the biofilm (dental plaque) that covers the gums and teeth in an animal's oral cavity can result in dental caries and / or gum and oral disease. For example, periodontal disease is one of the most commonly diagnosed oral diseases in dogs and cats and is often due to deleterious changes in dental plaque. [Background technology]

[0002] Dental prophylaxis has a profound effect on the oral and plaque microbiota, but general oral samples are poor surrogates for plaque bacteria (and the causative bacteria of periodontal disease), and more specific targeted therapies may be needed to prevent and treat plaque-related diseases. Although not the sole culprit, Psychrobacter is one example of a bacterial genus that is likely to be strongly associated given its specific dominance in canine dental plaque (Flancman R, Singh A, Weese JS (2018) Evaluation of the impact of dental prophylaxis on the oral microbiota of dogs. PLoS ONE 13(6): e0199676).

[0003] https: / / doi.org / 10.1371 / journal.pone.0199676. Additionally, another study has linked Psychrobacter as a common malodor-causing bacterium in dogs (Meason-Smith et al. Vet Dermatol 2018; 29: 465-e158). Physical dental cleaning of plaque significantly reduces Psychrobacter levels, but the effect is temporary, with a return to baseline levels observed within 5 weeks of cleaning. Therefore, regular and specific treatments targeting plaque are needed compared to general oral therapy to promote effective treatment and / or maintenance of oral health in dogs.

[0004] Applicants in the art have previously attempted to solve this problem by providing malleable products with a flexible matrix, such as in US Pat. No. 5,407,661, which provide a mechanical cleansing action to remove accumulated bacteria. However, this does not selectively remove pathogenic bacteria. To prevent and avoid disease / illness, compositions for modifying biofilms often contain a combination of bactericides and other ingredients, such as phospholipids, surfactants, or enzymes. These compositions bind well to epithelial surfaces and effectively destroy pathogens, but the active agents are non-selective and therefore act to destroy natural biofilms, including beneficial commensal bacteria. Standard animal oral care products kill all bacteria present in the dog's oral cavity.

[0005] A number of strategies have been developed to modify biofilms on epithelial surfaces, usually by administering bactericidal agents, with the goal of completely destroying or removing the biofilm and / or reducing the levels of pathogenic bacteria in the biofilm.

[0006] For example, New Zealand Patent No. 763741 describes the use of phospholipids to disrupt biofilms and antibiotics to non-selectively kill bacteria in the biofilm.

[0007] New Zealand Patent No. 779091 describes the use of surfactants and bactericides to disperse biofilms and non-selectively kill the bacteria that make up the biofilm.

[0008] New Zealand Patent No. 757876 describes the use of non-selective bactericidal triacylpolyamines to kill and disperse bacteria in biofilms. New Zealand Patent No. 732061 describes the use of oxidoreductase enzymes and the application of substrates for these enzymes such as honey to kill pathogenic bacteria in chronic wounds and medical devices. The germicide produced by the oxidoreductase enzymes is hydrogen peroxide, a non-selective germicide.

[0009] New Zealand Patent No. 755166 describes the use of thiol-based antioxidants, enzymes which degrade the extracellular matrix, and non-selective germicides such as antibiotics or disinfectants.

[0010] In each of the above publications, strategies include: - Use of ingredients that disrupt natural biofilms; and / or - Use of bactericidal components that do not distinguish between beneficial commensal bacteria in natural biofilms and harmful pathogenic bacteria.

[0011] Most bactericides are generally non-selective, but recently, agents that selectively kill pathogenic bacteria without affecting the growth of symbiotic bacteria have been reported.For example, International Application PCT / NZ2017 / 050043 describes that a combination of protein fractions (IDP™) derived from bovine dairy milk can be effective in preventing the growth of pathogenic bacteria such as Escherichia coli and Staphylococcus aureus, while not affecting the growth of symbiotic bacteria, such as Lactobacillus.However, there is no evidence that IDP™ binds to epithelial surfaces.

[0012] IDP™ or “Immune Defense Protein” is a microbiome-regulating fraction of proteins derived from milk. This fraction is described in at least U.S. Patent Application 12 / 304108, New Zealand Patent 719276, New Zealand Patent 742157, International Application PCT / NZ2017 / 050043, and New Zealand Patent 744458. According to published literature, IDP™ is a formulation based on milk-derived bioactive proteins extracted from milk. The components of IDP™ are naturally produced by cows as an immune defense response to infection and inflammation. IDP™ has been reported to exhibit anti-inflammatory, antioxidant, and antimicrobial effects in vitro that selectively support “good” flora and kill “bad” bacteria. IDP™ is already in use for administration to the oral cavity, throat, intestine, and skin.

[0013] Despite the potential advantages, it has been shown that the above proteins cannot be used alone to effectively utilize their properties, and formulations that include these agents or similar types of functional agents do not appear to deliver the required effect well to the intended location or provide their activity for an extended period of time.

[0014] Fibraspect™ is a known protein-based gel that can be used as an alternative to surfactant emulsifiers. The gel has the ability to bind active ingredients together, release drugs in a controlled manner, and has a shear-thinning rheology (thixotropy) that gives it good stability and good skin feel. It is a semi-solid material composed of soluble native proteins, protein aggregates, and protein fibrils produced from whey protein isolate (WPI) to form modified WPI. The modified WPI gel has a viscosity of 0.25 to 4.5 poise.

[0015] The problem of providing suitable oral products that are essentially effective in providing and enabling specific effects on the oral mucosa of animals has remained largely unsolved to date. In particular, it is difficult to adequately target dental plaque without harming the commensal flora. Thus, to date, there are very limited effective solutions suitable for maintaining or improving the oral health of companion animals. Summary of the Invention

[0016] Provided herein are novel protein complexes, methods for their production, and uses and compositions comprising the protein complexes. First, the invention relates to a biocomposite comprising a unique combination of whey protein isolate; a semi-solid material composed of soluble native proteins, protein aggregates, and protein fibrils produced from whey protein isolate (WPI) to form modified WPI. The attachment of biological agents that retain selective antimicrobial activity to epithelial surfaces allows the application of useful selective agent functionality to biological environments where it was not possible before. The biocomposite is a complexed protein formed by combining microbiome-regulating proteins with modified WPI through a novel and innovative process.

[0017] The invention of novel stable bioconjugates allows selective drug functionality that may be useful in biological environments. Specifically, the attachment of drugs that possess selective antimicrobial activity to epithelial surfaces is a further step towards a useful solution.

[0018] The novel biocomplex is formed by combining microbiome-regulating proteins with modified WPI in a novel and unique manner, which allows the composition to retain selective bactericidal activity while providing epithelial adhesion properties. In vitro testing confirmed that the resulting powder retained these properties.

[0019] Furthermore, in some applications, especially veterinary applications where the subject is unable to follow instructions, formulating the bioconjugate to enable the biological effect may be more important. For example, effective use of an antimicrobial agent is possible, in part, by understanding and following instructions, such as rubbing on surfaces and washing / rinsing the mouth, rather than swallowing. In contrast, however, for effective veterinary use (e.g., pet dogs or cats), the formulation itself must inherently provide and enable the biological activity, regardless of the associated instructions.

[0020] Thus, the novel ability to attach specific drugs to epithelial surfaces in animals, such as mucosal surfaces, as well as useful formulations of drugs for this purpose, would be of great value, particularly to the veterinary field.

[0021] Therefore, successful delivery of selective drugs in animals, including dogs and cats, by appropriately formulated means that essentially allow for effective use of the active agent, would be a highly useful advantage.

[0022] Described herein are methods and conjugated proteins that have useful effects in terms of stabilization and the ability to attach the conjugated proteins to epithelial surfaces and retain protein functionality when administered topically.

[0023] A method for producing a complexed stable microbiome-regulating protein, bioconjugate, configured for topical administration, is provided, the method comprising: selecting a microbiome-regulating protein in powder form; selecting a modified whey protein isolate (WPI) from milk in the form of a gel; Dissolving the microbiome-regulating protein in an aqueous solution of a salt having an ionic strength of 25 to 200 mM NaCl; blending the dissolved microbiome controlling protein and the modified WPI together; adjusting the pH to 2.0 to 6.0; If desired, adjusting the temperature to 18-37°C. maintaining the blend at a pH between 2.0 and 6.0 and at a temperature between 18 and 37° C. for at least 30 minutes; Includes.

[0024] The invention further includes a conjugated protein produced by a method substantially as described above. This process allows the production of novel bioconjugate compositions that retain the benefits of epithelial adherence characteristics while maintaining selective bactericidal activity. In vitro testing of the bioconjugate compositions confirmed that the resulting powder maintained these advantageous properties.

[0025] The invention also includes a complexed protein comprising a microbiome functional protein complexed with a modified milk-derived whey protein isolate (WPI), the complexed microbiome functional protein configured for topical administration and upon administration to adhere to an epithelial surface.

[0026] The present invention further relates to a method of selectively treating an animal against harmful pathogenic bacteria while minimizing the reduction in probiotic bacterial populations by locally administering a complexed protein, the complexed protein comprising a microbiome functional protein complexed with a modified milk-derived whey protein isolate (WPI), the complexed protein configured for topical administration and configured to adhere to an epithelial surface upon administration.

[0027] Further provided is the use of a complexed protein configured for topical administration and configured to adhere to an epithelial surface upon administration, comprising a microbiome functional protein complexed with a modified milk-derived whey protein isolate (WPI), in the manufacture of a medicament for the localized treatment of harmful pathogenic bacteria, but with minimal reduction in commensal bacterial populations on an epithelial surface of an animal.

[0028] The inventors have identified complexed proteins that have beneficial effects in terms of stabilization and the ability to attach the described functional proteins to epithelial surfaces when administered topically, and retain the functionality of the protein / protein fractions to a great extent. This is believed to be due, at least in part, to key chemical and physical conditions, further described below, where the two components react together to form a complexed protein that can be controlled to optimize the functional activity of both components. By carefully balancing these parameters, the beneficial effects obtained and the degree of attachment of the protein to the epithelial surface after complexation were more than twice that of each component alone, and in the inventors' experience, were synergistic in attachment and functionality, or at least greatly exceeded expectations.

[0029] The present invention extends to conjugated proteins or bioconjugates as described above for use as pharmaceuticals, and, if desired, for use as veterinary pharmaceuticals. In particular, the use may be for the treatment of oral or dental infections mediated by an imbalance of pathogenic bacteria, gingival or periodontal disease as desired.

[0030] In embodiments, the composition comprises a therapeutically effective amount of a conjugated protein or bioconjugate as described herein above. In some applications, further reformulation of the biocomplex may be more important to enable the biological effect. Thus, the present invention extends to a composition comprising a biocomplex comprising a dissolved microbiome-regulating protein complexed with a modified whey protein isolate (WPI) from milk, said biocomplex attaching to an epithelial surface and selectively detrimental to pathogenic bacteria but not to commensal bacteria. The biocomplex may comprise a dissolved microbiome-regulating protein complexed with a modified whey protein isolate (WPI) from milk. In an embodiment, one or more of the microbiome-regulating proteins of the biocomplex are selected from lactoperoxidase, lactoferrin, lysomal alpha-mannosidase, immunoglobulin G, angiogenin, ribonuclease 4, and quiescin sulfhydryl oxidase.

[0031] The therapeutic effect can be further improved by ensuring that the biocomposite is delivered to a precise location, such as the gums of a dog, where the biocomposite is placed near the teeth and gum line and therefore effectively adheres to the epithelial surfaces within the animal's oral cavity.

[0032] Further aspects and advantages of the described methods and composites and uses will become apparent from the following description, which is given by way of example only. Further aspects of the described methods and complexed proteins will become apparent from the following description, given by way of example only, and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 is a graph showing that the complex has no effect on the growth of the commensal bacterium L. acidophilus. [Diagram 2] FIG. 2 is a graph showing the bactericidal effect of the complex against Escherichia coli, a pathogenic bacterium. [Diagram 3] FIG. 3 is a graph showing that the complex adheres well to epithelial surfaces and retains its bactericidal activity against the pathogenic bacterium Staphylococcus aureus. [Figure 4] FIG. 4 is a graph showing the efficacy of a protein bioconjugate against the oral pathogen Psychrobacter after five days of use in canaines, according to an embodiment of the present invention. [Diagram 5] FIG. 5 is a graph showing the effect of a protein bioconjugate according to an embodiment of the present invention on the gut microbiome of a dog after five days of use. [Figure 6] FIG. 6 shows the effect of the protein biocomplex on factors related to periodontal disease in dogs after 28 days of use. [Figure 7] FIG. 7 shows the effect of the protein biocomplex on factors related to periodontal disease in dogs after 28 days of use. [Figure 8] FIG. 8 shows the effect of the protein biocomplex on factors related to periodontal disease in dogs after 28 days of use. [Figure 9] FIG. 9 shows the effect on breath odor perception scores after 12 weeks of using the protein biocomplex. [Figure 10] FIG. 10 shows the effect on factors related to periodontal disease in dogs following use of the protein biocomplex according to an every other day dosing regimen. [Figure 11] FIG. 11 shows the effect on factors related to periodontal disease in dogs following use of the protein biocomplex according to an every other day dosing regimen. [Figure 12] FIG. 12 shows the effect on factors related to periodontal disease in dogs following use of the protein biocomplex according to an every other day dosing regimen. [Figure 13] FIG. 13 shows the effect on factors related to periodontal disease in dogs following use of the protein biocomplex according to an every other day dosing regimen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] As noted above, methods and conjugated proteins are described herein that have useful effects in terms of stabilization and the ability to attach the conjugated proteins to epithelial surfaces and retain excellent protein functionality when administered locally.

[0035] For purposes of this specification, the terms "about" or "approximately" and grammatical variations thereof mean a quantity, level, degree, value, number, frequency, percent, dimension, size, amount, weight, or length that varies by at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference quantity, level, degree, value, number, frequency, percent, dimension, size, amount, weight, or length.

[0036] The term "substantially" or grammatical variations thereof means at least about 50%, such as 75%, 85%, 95%, or 98%. The term "comprise" and its grammatical variations are intended to have an inclusive meaning, i.e., to mean including not only the recited component to which it directly refers, but also other unspecified components or elements.

[0037] For purposes of this specification, the term "complex", "biocomplex", or grammatical variations thereof, refers to a physical interaction between a microbiome controlling protein and a modified whey protein isolate (WPI) such that they bind to each other and adhere to an epithelial surface when administered topically, whereby the microbiome controlling protein functions. Without wishing to be bound by theory, the inventors understand that the physical interaction that holds the complex together may be due to conjugation, physical encapsulation, or a combination thereof. Additional interactions may occur, and references to conjugation or physical encapsulation should not be construed as limiting.

[0038] As used herein, the term "modified whey protein isolate" or grammatical variations thereof refers to soluble native proteins, protein aggregates, and protein fibrils produced from whey protein isolate (WPI).

[0039] For brevity, as used herein, the word "protein" or grammatical variations thereof is intended to encompass a protein, a plurality of proteins, a protein fraction, or a plurality of protein fractions, and reference to a singular protein should not be construed as limiting.

[0040] Method for producing the composite Described herein is a method for making a complexed stable microbiome controlling protein configured for topical administration according to the invention, the method comprising the steps of selecting a microbiome controlling protein in powder form, selecting a modified whey protein isolate (WPI) from milk in gel form, dissolving the microbiome controlling protein in an aqueous solution of salt having an ionic strength of 25-200 mM NaCl, blending the dissolved microbiome controlling protein and the modified WPI together, adjusting the pH to 2.0-6.0, optionally adjusting the temperature to 18-37° C., and holding the blend at a pH of 2.0-6.0 and a temperature of 18-37° C. for at least 30 minutes. It has been observed that the selective bactericidal activity of the proteins described herein may be adversely affected if the temperature of any step in this process exceeds 37° C. and the pH falls below 2.0 or exceeds 9.0.

[0041] The viscosity of the modified whey protein isolate is important to promote complexation, and the optimum temperature for best viscosity is in the range of 18-37°C. The ionic strength of the mixture is important for the protein groups of both components to interact correctly, and the optimal ionic environment is maintained at 25-200 mM NaCl at pH 2.0-6.0.

[0042] Only by careful experimentation and balancing of these parameters was a useful effect obtained. Indeed, the degree of adhesion of the protein to the epithelial surface after complexation was more than twice that of each component alone, which, in our experience, was synergistic in nature, or at least greatly exceeded expectations, suggesting that a synergistic effect was possible, which could not have been anticipated or predicted heretofore.

[0043] Complex form The complexed protein obtained from the above method may be semi-solid. The complexed protein may be a gel. The gel may have a viscosity similar to that of the modified WPI before complexation. The gel may have a viscosity of approximately 0.25 to 4.5 poise.

[0044] The complexed protein can be stable, and the complexed protein can remain in a complexed form with the modified WPI until topical administration, at which point the function of the protein is made available to the epithelial surface to which the modified WPI is attached.

[0045] Microbiome Regulatory Proteins The microbiome-regulating protein may be a blend of milk-derived protein fractions that are selectively detrimental to pathogenic bacteria but minimize the reduction of commensal bacterial populations. The microbiome-regulating protein may include lactoperoxidase, lactoferrin, lysomal α-mannosidase, immunoglobulin G, angiogenin, ribonuclease 4, quiesin sulfhydryl oxidase, and combinations thereof. The protein may include all of the proteins listed above.

[0046] The microbiome controlling protein selected for the above methods may initially be in the form of a dry powder. Modified WPI The modified WPI selected for the above method may be initially in the form of an aqueous gel. The modified WPI selected may be derived from the milk of bovine species, but may also be derived from modified WPI obtained from other animals, such as goats and sheep.

[0047] ratio The ratio of microbiome-regulating protein to modified WPI used in the method and / or present in the complexed protein may be 1:1, or 1:2, or 1:3, or 1:4, or 1:5, or 1:6, or 1:7, or 1:8, or 1:9, or 1:10. In one embodiment, the ratio may be 1:1 to 1:10, i.e., 1 part microbiome-regulating protein to 1 to 10 parts modified whey protein isolate. In one embodiment, the ratio may be 1:1. In the inventor's experience, at least the same amount of modified WPI to protein may be necessary to ensure that the desired degree of complexation is reached. Lower ratios may be used, but lower ratios may result in wasted protein or uncomplexed protein from the method due to insufficient modified WPI present.

[0048] Salt solution The salt solution may include a mixture of water and chemical salt. In one embodiment, the chemical salt may be NaCl, but other salts such as MgCl may be used. Salt may be added to the extent required to obtain the indicated ionic strength. The ionic strength of the salt solution may be approximately 25, or 50, or 75, or 100, or 125, or 150, or 175, or 200 mM NaCl. As noted above, the ionic strength may vary in the range of 25-200 mM NaCl. In one embodiment, the ionic strength is approximately 75 mM NaCl. This ionic strength is unchanged / maintained in the complexed blend of protein and modified WPI during and after the hold. In the inventor's experience, this ionic strength range appears to be optimal for complexation or dissolution of microbiome-regulating proteins in modified WPI, but may vary to some extent depending on the pH, temperature, and hold time used. The use of a salt solution appears to be important as it allows for the dissolution of the powdered protein and creates the correct ionicity of the solution to properly charge the protein to form complexes.

[0049] blend Blending is performed gently such that no or minimal foaming of the mixture occurs during blending. Blending may be completed using, for example, a planetary mixer. Blending may continue during pH adjustment, temperature adjustment, and / or holding. It may be important to blend the protein gently or slowly to minimize or avoid denaturation and loss of function.

[0050] pH The pH of the blend before pH adjustment may be approximately 6.5-7.5, or around 7.0, which is a neutral pH. The starting point of this pH may depend on the pH of the water used to form the aqueous salt solution. The pH of the blend after pH adjustment may be approximately 2.0, or 2.5, or 3.0, or 3.5, or 4.0, or 4.5, or 5.0, or 5.5, or 6.0. As noted above, in one embodiment, the pH may be 2.0-6.0. In one embodiment, the pH may be approximately 4.0. This pH range and value appears to be optimal for complexation or dissolution of the protein in the modified WPI, but may vary to some extent depending on the ionic strength, temperature, and holding time used. The acidic conditions after adjustment appear to maintain the proper charge of the protein and ensure binding of the complex. The pH may be adjusted using an acid. In one embodiment, the acid may be hydrochloric acid, but other acids may be used. In the inventor's experience, the reduced pH, once adjusted, does not require a buffer to maintain it.

[0051] temperature The temperature of the blend after any adjustment (if necessary) may be approximately 18°C, or 19°C, or 20°C, or 21°C, or 22°C, or 23°C, or 24°C, or 25°C, or 26°C, or 27°C, or 28°C, or 29°C, or 30°C, or 31°C, or 32°C, or 33°C, or 34°C, or 35°C, or 36°C, or 37°C. As noted above, the temperature after any adjustment may be between 18 and 37°C. In one embodiment, the temperature after adjustment may be approximately 25°C. The temperature may be adjusted to maintain the modified WPI at a desired viscosity to allow for optimized complexation. This desired viscosity may be in the range of approximately 0.25 to 4.5 poise. The temperature ranges and values ​​listed are believed to be optimal for complexation or dissolution of proteins into the modified WPI, but may vary to some extent depending on the ionic strength, pH, and hold time used. The temperature may be adjusted from ambient conditions (or may be kept at ambient conditions if the ambient temperature is within a desired range). The temperature adjustment may be up or down depending on the difference between the desired temperature and the ambient temperature.

[0052] retention time The holding time may be at least 30 minutes. The holding time may be between 30 minutes and 24 hours. In one embodiment, the holding time may be approximately 60 minutes. In the inventor's experience, most of the complexation occurs within 30 to 60 minutes, based on the parameters selected. Longer times may be used, and based on the inventor's experience, there is no harm or risk of loss of functionality in holding the mixture for a longer period of time. This time appears to be optimal for protein complexation or dissolution in the modified WPI, but may vary to some extent depending on the ionic strength, temperature, and pH used.

[0053] Drying If desired, drying may be completed prior to subsequent formulation. In this embodiment, the dried complex may be solid. Drying may be to a water activity of less than 0.6. In selected embodiments, the water activity may be as low as 0.2-0.3. Drying of the complex may be performed under specific conditions of low temperature and pressure for a time period that ensures that the epithelial adhesiveness and selective bactericidal activity of the components of the complex are maintained. The pressure during drying may be important in that the drying can be performed at a lower temperature because the water evaporates from the gel at a lower temperature at low pressure. In addition, it may be important to reduce the pressure very slowly so that the solution of the complexed protein in the dryer does not foam. Drying may be completed by lyophilization or any other gentle drying process using a non-denaturing temperature / pressure, e.g., less than 30°C, in a vacuum. The complexed protein remains functional when rehydrated after drying.

[0054] Protein Complex of the Invention There is provided a complexed protein or biocomplex produced by a method substantially as described above. The invention further provides a complexed protein comprising a microbiome functional protein complexed with a modified whey protein isolate (WPI) derived from milk, the complexed microbiome functional protein configured for topical administration and for adhering to an epithelial surface upon administration. The complexed protein may be a semi-solid aqueous gel having a viscosity of approximately 0.25 to 4.5 poise. The complexed protein may further be formulated into an emulsion, gel, paste, or putty for use in a dental composition. The microbiome control protein in the complex may be a blend of proteins that are selectively detrimental to pathogenic bacteria but cause minimal reduction in commensal bacterial populations. The proteins in the complex may include lactoperoxidase, lactoferrin, lysomal alpha-mannosidase, immunoglobulin G, angiogenin, ribonuclease 4, quiesin sulfhydryl oxidase, and combinations thereof. The modified WPI in the complex may be derived from the milk of a bovine species.

[0055] The ratio of microbiome-regulating protein to modified WPI in the complexed protein may be 1:1 to 1:10. The complexed protein produced by the methods disclosed herein or having the claimed features has a useful effect in terms of stabilization. Moreover, when administered topically, the protein complex has the ability to attach the described functional protein to the epithelial surface and retain very good protein functionality. This is believed to be due, at least in part, to the key chemical and physical conditions under which the two components react together to form a complex that can be controlled to optimize the functional activity of both components.

[0056] Treatment Methods and Uses Advantageously, the complexed proteins produced herein are suitable for the first time for use as pharmaceuticals, particularly for use as veterinary pharmaceuticals. An increase in pathogenic bacteria typically causes bad breath, dental plaque, and gum disease in animals. The protein bioconjugates can therefore be used to effectively treat poor health in animals, such as oral bacterial infections or dental diseases, where the condition or disease is due to an increase in pathogenic bacteria. The poor health of an animal, cat or dog, may also result from a decrease in commensal bacteria, thereby creating an imbalance in the bacterial flora in the oral mucosa or in particular in dental plaque. Such an imbalance may result in one or more conditions selected from bad breath (halitosis), dental plaque, and / or gum disease, and the compositions of the invention are therefore useful in treatments to restore that balance. In embodiments, the pathogenic bacteria may be Psychrobacter. In particular, due to the selective functionality that can be delivered for the first time to the oral mucosa, the complexed proteins (or compositions comprising same) are useful for treating oral or dental infections. In some embodiments, the infection may be a gingival or periodontal disease mediated by an imbalance of pathogenic bacteria. Further described herein is a method of selectively treating an animal against harmful pathogenic bacteria, but with minimal reduction in the commensal bacterial population, by locally administering a complexed protein, the complexed protein comprising a microbiome functional protein complexed with a modified whey protein isolate (WPI) derived from milk, the complexed protein configured for local administration, and upon administration, the complexed protein adheres to an epithelial surface of the animal. In the above method, the animal may be a non-human animal, such as a cat or a dog, as desired. Provided is the use of a complexed protein comprising a microbiome functional protein complexed with a modified whey protein isolate (WPI) derived from milk, configured for local administration, and configured for adherence to an epithelial surface upon administration, in the manufacture of a medicament for local treatment of harmful pathogenic bacteria in or on an animal in need thereof, but with minimal reduction in the commensal bacterial population on an epithelial surface of the animal. In the above use, the animal may be a non-human animal.

[0057] Compositions Comprising Protein Complexes In an embodiment, the present invention relates to a composition comprising a protein complex, produced according to the disclosure herein. Such a composition may comprise at least 0.5%-2% (w / w), preferably 0.1-1.5% (w / w) of the biocomplex, and at least one or more pharma- ceutically acceptable excipients. The composition comprising the protein complex is suitable for promoting a selective antibacterial effect in the oral cavity of a companion animal. This model has been shown for the first time in the examples herein to be effective in the described example of the oral cavity of an animal, such as a dog. An example of a suitable composition comprising a protein biocomplex according to the above embodiment has been tested in a suitable dog model test to confirm therapeutic efficacy. It has been shown that by providing a suitable composition comprising the claimed biocomplex, specific oral problems in dogs, such as dental disease and halitosis control, are practically addressed.

[0058] To formulate such compositions, common excipients known in the art may be used, including bulking agents such as rice, oat flour, sweet potato flour, and / or other plant-based flours. These may be selected alone or in combination with other known excipients useful in animal supplements or dental compositions. Such excipients may further include optionally 15-20% (w / w) of a humectant, optionally 4-6% (w / w) of a flavoring, optionally 3-5% (w / w) of a pH buffer, optionally 1-3% (w / w) of a lubricant, optionally 1-2% (w / w) of an emulsifier, optionally 0.5-1% (w / w) of a thickener, and optionally up to 0.01% (w / w) of one or more preservatives.

[0059] To this end, compositions comprising the protein biocomplex can be used to effectively treat poor health conditions, such as oral bacterial infections or dental diseases in animals, especially when the condition or disease can be attributed to an increase in pathogenic bacteria. An increase in pathogenic bacteria typically causes bad breath, plaque, and gum disease in animals. The poor health of an animal, cat, or dog can be attributed to a decrease in commensal bacteria, thereby creating an imbalance of the bacterial flora in the oral mucosa or especially in plaque. Such an imbalance can result in one or more conditions selected from bad breath (halitosis), plaque, and / or gum disease, and the compositions of the present invention are useful in treatment to restore that balance. In an embodiment, the pathogenic bacteria can be Psychrobacter. Such compositions can also allow for a temporary physical barrier configured to more effectively deliver the protein biocomplex to the site where it is needed (the gums and teeth of the animal) and provide the biocomplexed protein and its antibacterial properties, while simultaneously working with the oral microbiome to support the commensal bacterial flora that the animal needs to maintain good oral health. In an embodiment, the animal is a cat or a dog. In an embodiment, the disease may be periodontal disease.

[0060] The composition should be formulated to be gentle on the animal's digestive system, intended for frequent use and / or for adoption as part of a long-term viable solution for maintaining an animal's oral health over a reasonable period of months or years.

[0061] In embodiments, when the composition is used for the treatment and / or improvement of oral health, more specifically dental health, a suitable dosing regimen may be followed. To reduce pathogen counts and significantly improve oral health, such compositions have been shown to be effective when used with the minimum necessary (therapeutically effective amount) of the bioconjugate and administered daily for at least 5 days per week, preferably for at least 4 weeks, 5-12 weeks or more. Thereafter, reduced frequency of administration to every other day may maintain a healthy state with a properly balanced microbiome of the oral mucosa. However, regular administration will more effectively prevent recurrence of pathogen accumulation and thus maintain the proper balance of the oral mucosa.

[0062] Without being bound by theory, such compositions may contain additional pharmaceutical excipients to enhance the protective function of the composition during the product is delivered and then retained in the oral cavity of a subject.Such dental compositions may more effectively and over a long period of time, but accurately release bioactive substances at the right place to achieve desired efficacy.However, it is plausible that such compositions may be suitable for other animals, especially animals with particularly long jaws, such as cats.

[0063] In embodiments, the bioconjugated protein may be distributed evenly throughout the composition, or the composition may form an outer structure that completely surrounds the protein element. The inventors understand that the conjugated proteins produced may be used to bind to other epithelial surfaces and maintain or re-establish a healthy microbiome on any epithelial surface, such as the skin and nasal passages, gut, etc. This may include human and animal products, human and animal skin care products, human and animal nasal spray products, and ingestible gut health products for humans and animals.

[0064] It is also envisioned that the complexed proteins may be used to maintain, establish, or re-establish a healthy microbiome on any protein-rich surface, such as hair, including human hair products, lotions, creams, shampoos, conditioners, sprays; animal coats - lotions, creams, shampoos, conditioners, sprays; medical devices, including artificial skin, collagen scaffolds for wound repair, bandages, and the like. As should be understood from the above, the topical surface may be an external surface, for example the skin, or an internal surface, for example the gut. Reference herein to the term "topical" is not limited to only external administration of the complexed protein.

[0065] The above-described embodiments may also be broadly described as consisting of the parts, elements, and features referred to or suggested in the specification of this application, individually or collectively, as well as any or all combinations of any two or more of such parts, elements, or features.

[0066] Description The above methods, protein bioconjugates, compositions, and uses and applications thereof are exemplified in further detail below.

[0067] Example 1 - A method for producing the composite is outlined.

[0068] [ka]

[0069] [ka]

[0070] Example 2 Attachment of the complex to epithelial surfaces To determine whether the complex adhered to the epithelial layer, pieces of porcine skin were immersed in a 50 mg / mL solution of the complex (containing 50 mg / mL of protein), washed twice with pH 7.4 phosphate buffer, and stained with Coomassie blue, which stains proteins. This resulted in more intensely stained skin compared to porcine skin that was not immersed in the complex (Figure 1). Treatment of porcine skin with 50 mg / mL of modified whey protein fraction isolate (containing 50 mg / mL of protein) or bovine milk-derived microbiome control protein (containing 50 mg / mL of protein) alone, followed by washing, showed some binding of these components to the skin, but not as much as the 50 mg / mL complex of both components (Table 1).

[0071] [Table 1]

[0072] The epithelial binding activity of the complex was much higher (>2-fold) than either modified whey protein isolate or the microbiome control protein alone. Modified whey protein isolate is specifically manufactured to adhere to epithelial surfaces. Therefore, the amount of binding of this component to porcine skin would be expected to be similar to that of the complex. However, the binding of the complex was 44% higher than modified whey protein isolate alone, suggesting a synergistic effect of the components or that the process used to manufacture the complex enhanced the adhesion ability of the complex.

[0073] Example 3 Retention of enzymatic bioactivity of the complex The enzyme lactoperoxidase is a protein that has been linked to microbiome-regulating proteins isolated from bovine milk (New Zealand Patent No. 547859 and International Application No. PCT / NZ2017 / 050043). Lactoperoxidase activity was measured by adding the complex, or the components of the complex separately, to a solution of hydrogen peroxide in 100 mM phosphate buffer (pH 5.5) and monitoring the oxidation of 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) spectrophotometrically at 436 nm.

[0074] The lactoperoxidase activity of the combination of bovine milk-derived microbiome-regulating proteins was retained when the microbiome-regulating proteins were complexed with modified whey protein isolate (Table 2). As expected, the modified whey protein isolate had no peroxidase activity.

[0075] [Table 2]

[0076] Specific bactericidal activity symbiotic bacteria It has been shown that microbiome-regulating proteins isolated from bovine milk do not affect the growth of symbiotic bacteria such as bacteria of the genus Lactobacillus (International Application PCT / NZ2017 / 050043). To identify the effect of complexes containing these microbiome-regulating proteins on symbiotic bacteria, Lactobacillus acidophilus was cultured anaerobically, the complexes were added to the culture, and the number of bacteria after 24 hours was determined as an optical density at 650 nm. The complexes of modified whey proteins and microbiome-regulating proteins isolated from bovine milk did not affect the growth of the symbiotic bacteria Lactobacillus acidophilus (Figure 1).

[0077] pathogenic bacteria Microbiome-regulating proteins isolated from bovine milk have been shown to be bactericidal against pathogenic bacteria such as E. coli (International Application PCT / NZ2017 / 050043). To determine the effect of complexes containing these proteins on E. coli, these bacteria were cultured, the complexes were added to the cultures, and the bacterial count after 24 hours was determined as optical density at 650 nm. The complexes inhibited the growth of the pathogenic bacteria E. coli to the same extent as the microbiome-regulating proteins from bovine milk (Figure 2). It should be noted that in Figure 2, the square data points correspond to the results seen when the microbiome-regulating proteins from bovine milk were added, and the circle data points correspond to the results seen when the complexes of the microbiome-regulating proteins and modified whey protein isolate were added.

[0078] Effect of skin-bound complexes on the growth of pathogenic bacteria Staphylococcus aureus is a pathogenic bacterium that is often associated with skin diseases. Microbiome-regulating proteins isolated from bovine milk have been shown to be bactericidal against cultured Staphylococcus aureus (International Application PCT / NZ2017 / 050043). Samples of porcine skin treated with microbiome-regulating proteins from bovine milk and subsequently washed inhibited the growth of Staphylococcus aureus to a lesser extent. However, samples of porcine skin treated with the complex and subsequently washed inhibited the growth of Staphylococcus aureus to a much greater extent (Figure 3). This confirms the results shown in Table 1, confirming that the complex binds well to epithelial surfaces such as skin and also retains the bactericidal activity of the microbiome-regulating proteins from bovine milk.

[0079] Example 4 The formulated composition should contain a minimum therapeutically effective amount of protein bioconjugate. In a formulated product, this may result in a final product of sufficient overall size that it can be retained, for example, by a dog in its oral cavity. By way of example, this may include approximately 0.05%-2% (w / w) of protein bioconjugate in the composition, preferably 0.13%-0.53% (w / w).

[0080] In this example, the compositions tested on animals (dogs) were formulated for light or small breeds (up to 15 kg), medium weight breeds (15-31 kg), and larger weight breeds (over 31 kg). The protein biocomplex in the given example composition was included as appropriate at 0.13% (w / w) (if a larger sized composition was required for heavy breeds) or 0.5% (w / w) (if a smaller sized composition was required).

[0081] Such compositions can be made by blending (according to well-established techniques) protein biocomplex ingredients with other biologically acceptable excipient ingredients known for use in animal supplements. For example, the known ingredients and suitable ranges provided in the table below can be exemplary of ingredients utilized in embodiments of compositions that include protein biocomplexes.

[0082] [Table 3]

[0083] Selective bactericidal activity The composition according to Example 4 described above was incubated (according to methods known in the art) with cultures of both pathogenic and commensal bacteria isolated from a single canine oral source to determine whether its selective bactericidal activity was retained.

[0084] As a result, after 18 hours of incubation, the number of pathogenic bacteria in the dog's oral cavity was confirmed to have increased from 108 colony forming units (cfu) to 107 cfu compared to the control.

[0085] The number of canine oral commensal bacteria was not reduced after 18 hours of incubation, confirming that the exemplary protein bioconjugate compositions retained the selective bactericidal activity required for administration to animals.

[0086] This composition (according to Example 4) was then used in the following dog study. Canine Preliminary Study - Short Term (1-4 weeks) A preliminary microbiome study was conducted to evaluate the therapeutic efficacy of an exemplary composition comprising the protein bioconjugate and therefore its suitability for veterinary use.

[0087] Each of the eleven dogs was given a composition (a composition according to the invention embodied in Example 4) daily for five consecutive days (days 2, 3, 4, 5, and 6 of the test regimen). Oral and fecal microbiome swabs were taken for each dog before and after the test, and a final oral swab was taken 48 hours later. After completion of the test, oral and fecal swabs were collected and DNA was successfully extracted for analysis under appropriate laboratory conditions.

[0088] A complete microbiome profile analysis was performed using 16s amplicon sequencing and analysis (amplicon normalization, PCR reactions, amplicon barcoding, amplicon library QC, paired-end reads, 2 × 250 bp read length). Deep amplicon sequencing was used in combination with principles of statistical ecology to investigate the microbiome community. Deep sequencing of the small ribosomal rRNA subunit (16S rRNA) gene was used to investigate the bacterial community and specifically to gain insight into the population dynamics of the oral pathogenic bacterium Psychrobacter. The results of Psychrobacter levels in the oral samples are shown in Figure 4 by graphical analysis. The plot shows the mean and standard error of the relative abundance of Psychrobacter.

[0089] Three adjacent time bar graphs are shown for Psychrobacter in oral swabs collected according to the study protocol. The first is time 1 = before the composition containing the protein bioconjugate was given (day 2). The second is time 2 = 5 days later (day 6). The third bar is time 3 = 48 hours after the last composition was given (day 8).

[0090] Looking towards the right of FIG. 4, it can be seen that Psychrobacter (Proteobacteria), a known cause of dental diseases such as periodontal disease, is greatly reduced after five consecutive days of administration to dogs. After two days, the level of this bacteria remains low despite the absence of further administration. Thus, the results herein conclusively demonstrate that a composition comprising a therapeutically effective amount of a protein bioconjugate reduces the pathogenic bacteria specifically present in dental plaque, thus allowing for targeted and effective treatment (or prevention) of the associated dental diseases. Initially, a suitable administration regimen to reduce pathogenic bacteria may be once daily for at least five days. This may be quickly followed by a less frequent regimen of every other day, since once the balance of the microflora is properly restored, regular continued administration is likely to be required to prevent the reoccurrence of high levels of pathogens, and thus to maintain particularly good dental health.

[0091] Furthermore, since Psychrobacter is associated with and likely causes malodour in dogs (bloodhounds), it is plausible that harmful and unpleasant symptoms of disease such as halitosis would be reduced. Furthermore, in this study, fecal samples obtained before and after the 5-day study were also sent for microbiome profiling and analysis (as detailed above). Importantly, as can be seen in Figure 5, this showed no significant acute effects on the fecal microbiome over the 5-day period, suggesting that this product is suitable for selectively targeting disease-causing bacteria in the oral cavity without affecting the gut microbiome.

[0092] A subsequent extension of the same study to 28 days (4 weeks) confirmed these findings and demonstrated a statistical association with: Reduction in oral pain after 4 weeks associated with problems such as gum infection, root infection and loose teeth (see Figure 6) Reduction in tartar and plaque which may have improved oral cleanliness (see Figure 7) and space (see Figure 8) - improved by weeks 3 and 4 (worsened at week 5 after one week without using the composition).

[0093] Dog Study 2 - Medium Term (12 Weeks) A 12-week dental trial was conducted to further evaluate the efficacy of the protein biocomplex as it relates to factors associated with periodontal disease in dogs.

[0094] A total of 17 dogs over 6 months of age, weighing 15-30 kg and with persistent dental problems such as gum disease, tartar, plaque or bad breath were enrolled in the study. Dogs with healthy teeth were excluded.

[0095] Regimen: The composition (as previously disclosed) was used daily for 4 weeks, followed by every other day for 8 weeks. Factors assessed and outcomes Evaluations were performed at baseline (week 0), weeks 1, 4, 5, 6, 8, and 12. Significant improvement in the dogs' breath was reported within 4 weeks, and the fresh breath was maintained with continued use for the full 12 weeks, even when the frequency of administration was reduced to every other day. Longer periods of use (around 8 weeks) were found to be necessary to see greater effects and improvements on other factors such as bleeding gums, plaque, and tartar.

[0096] Breath and cleanliness As can be seen from Figure 9, the halitosis perception scores gradually decreased, with a reduction in baseline mean halitosis score of 2.2 reported to 1.5 by week 4 after daily use. Additional use (over 4 weeks) showed a gradual reduction in halitosis scores, even after changing frequency (every other day). For example, the lowest mean halitosis score was 0.8 reported at week 6. Overall, mean halitosis scores less than half of baseline (2.2) were reported at weeks 5, 8, 10, and 11 (1.0-1.1) toward the end of the study.

[0097] The response rates for "slightly cleaner and fresher" and "much cleaner and fresher" were higher from weeks 4 to 12 compared to week 1. After just 4 weeks, over 75% of owners reported that their dog's mouth was (slightly or much) cleaner and fresher.

[0098] Bleeding gums Among owners who brushed their dogs' teeth, the rate at which dogs had bleeding gums decreased from 11.8% at baseline to 5.9% by week 8.

[0099] Dental plaque By week 8, plaque reduction was evident, with owner-reported plaque scores decreasing from 76.5% to 29.4% of baseline.

[0100] Dog Study 3 - Every other day dosing regimen (12 weeks) An additional 12-week study was completed to evaluate the efficacy of the protein biocomplex (previously formulated) when administered every other day. The study included an increased sample size of dogs. Thirty-five dogs were started, including 13 small breeds (up to 15 kg), 11 medium breeds (15-31 kg), and 11 large breeds (>31 kg). This was important to evaluate and confirm the consistency of the effect on dogs in different weight categories.

[0101] Testing Process Each dog was fed a protein biocomplex composition (following the example set out in Example 4) in a size appropriate for their weight category every other day for a period of 10 weeks.

[0102] Factors and outcomes assessed: Assessments were conducted at baseline, 4 weeks, 8 weeks, and 12 weeks (2 weeks after treatment was stopped), after which the dogs' owners rated their microbiomes and health status using several criteria.

[0103] After 4 weeks, there was a significant improvement in halitosis scores, tooth cleanliness, and plaque scores, as can be seen from Figures 10, 11, and 12. After 8 weeks, there was a significant improvement in tartar scores, as can be seen from Figure 13.

[0104] All changes seen by week 8 were maintained through week 12, but no significant changes were observed between weeks 8 and 12. Statistical Associations - Participants without post-baseline survey responses were included in the analysis. For each question, a mixed effects model was used to analyze significant differences between mean scores at each survey during the study. Multiple comparisons were performed using Tukey's test to compare baseline mean scores with weeks 4 and 8, and week 8 mean scores with week 12 (to account for missing values). Significant differences were defined as p<0.05.

Claims

1. It is a complexed protein, A complexed protein comprising a microbiome functional protein containing lactoperoxidase, lactoferrin, lysomal α-mannosidase, immunoglobulin G, angiogenin, ribonuclease 4, or kyesin sulfhydryl oxidase, and / or combinations thereof, which is complexed with modified whey protein isolate (WPI) derived from milk, wherein the complexed microbiome functional protein is configured for topical administration and is configured to adhere to the epithelial surface upon administration.

2. The complexed protein according to claim 1, which is a semi-solid aqueous gel having a viscosity of approximately 0.25 to 4.5 poise.

3. The complexed protein according to claim 1 or 2, wherein the microbiome functional protein is a blend of protein fractions that are selectively harmful to pathogenic bacteria but minimize the reduction of the symbiotic bacterial population.

4. The complexed protein according to claim 1 or 2, wherein the modified WPI is derived from bovine milk.

5. The complexed protein according to claim 1 or claim 2, wherein the ratio of microbiome regulatory protein to modified WPI in the complexed protein is 1:1 to 1:

10.

6. A therapeutically effective amount of the complexed protein according to claim 1 or claim 2, for use as a pharmaceutical, and optionally for use as a veterinary pharmaceutical.

7. The complexed protein according to claim 6, for use in the treatment of oral or dental infections transmitted by an imbalance of pathogenic bacteria, optionally in the treatment of gingival or periodontal disease.

8. The use of the complexed protein according to claim 1 in the manufacture of a pharmaceutical product for the local treatment of harmful pathogenic bacteria on the body of an animal requiring treatment, while minimizing the reduction of the symbiotic bacterial population on the epithelial surface of the animal.

9. A method for selectively treating an animal against harmful pathogenic bacteria by locally administering the complexed protein described in claim 1, wherein the reduction of the symbiotic bacterial population is minimized, and the complexed protein adheres to the epithelial surface of the animal at the time of administration.

10. The use or method according to claim 8 or 9, wherein the animal is a non-human animal, and optionally the animal is a dog or a cat.

11. A topical composition comprising the complexed protein described in claim 1 or claim 2, and further comprising at least one pharmaceutically acceptable excipient.

12. A method for producing a complexed, stable microbiome-regulating protein configured for local administration, A step of selecting a microbiome-regulating protein in powder form, comprising lactoperoxidase, lactoferrin, lysomal α-mannosidase, immunoglobulin G, angiogenin, ribonuclease 4, or kyesin sulfhydryl oxidase, and / or a combination thereof. A step of selecting modified whey protein (WPI) derived from milk in gel form, The steps include dissolving the microbiome regulatory protein in an aqueous solution of a salt having an ionic strength of 25 to 200 mM NaCl, The process of blending the dissolved microbiome control protein and modified WPI together, A process to adjust the pH to 2.0 to 6.

0. A process to adjust the temperature to 18-37°C as desired. The process involves maintaining the blend at a pH of 2.0 to 6.0 and a temperature of 18 to 37°C for at least 30 minutes. Methods that include...

13. The method according to claim 12, wherein the microbiome regulatory protein is a blend of protein fractions that are selectively harmful to pathogenic bacteria but minimize the reduction of the symbiotic bacterial population.

14. The method according to claim 12, wherein the complexed protein produced is a semi-solid gel having a viscosity of approximately 0.25 to 4.5 poise.

15. The method according to claim 12, wherein the selected modified WPI is initially in the form of an aqueous gel.

16. The method according to claim 12, wherein the selected modified WPI is derived from bovine milk.

17. The method according to claim 12, wherein the ratio of microbiome regulatory protein to modified WPI present in the complexed protein is 1:1 to 1:10, as used in the above method.

18. The method according to claim 12, wherein the salt solution comprises a mixture of water and a chemical salt.

19. The method according to claim 12, wherein the ionic strength of the salt solution is approximately 75 mM NaCl.

20. The method according to claim 12, wherein the pH of the blend before pH adjustment is approximately 6.5 to 7.

5.

21. The method according to claim 12, wherein the pH of the blend after pH adjustment is approximately 4.

0.

22. The method according to claim 12, wherein the temperature of any of the adjusted blends is approximately 25°C.

23. The method according to claim 12, wherein the holding time is approximately 60 minutes.

24. The method according to claim 12, wherein the complexed protein is dried to a water activity of less than 0.6 after retention.

25. A complexed protein produced by the method described in claim 12.

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