Method and process for manufacturing local adhesion selective bactericide

Abstract

Disclosed herein are processes for preparing novel topically adhering selective fungicides and products produced thereby. More specifically, a method for producing a complex stable microbiome regulatory protein configured for topical application, and the use of the product as a medicament, which is effective in therapeutic effect against pathogenic bacteria but ineffective against symbiotic bacteria.

Description

Background Art

[0001] Bacteria are easy to attach to the surface and produce biofilm. Biofilm is usually found on all epithelial surfaces of humans and animals (including skin, intestinal tract, lungs, nasal cavity and oral epithelial surfaces). The bacteria contained in any biofilm may be benign or even beneficial (symbiotic), such as bacteria from the Lactobacillus (Lactobacillus) family; the bacteria may also be pathogenic, unfavorable to the host, causing disease and pain, such as Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) bacteria. In most cases, biofilm is beneficial, or at least benign, because the main component of biofilm is symbiotic bacteria. However, when the level of pathogenic bacteria in the film increases and overwhelms benign or symbiotic bacteria, the effect of biofilm may become unfavorable. For example, several skin diseases are the result of excessive proliferation of pathogenic bacteria on the skin, such as atopic dermatitis is the consequence of excessive number of pathogenic bacteria Staphylococcus aureus on the skin. In particular, if pathogenic bacteria become dominant in the biofilm (dental plaque) that covers the gums and teeth in an animal's mouth, tooth decay and / or gum and oral disease may result. For example, periodontal disease is one of the most commonly diagnosed oral diseases in dogs and cats, often due to adverse changes in dental plaque.

[0002] Dental prophylaxis has a significant impact on the oral and dental plaque microbiota, although prevention and treatment of dental plaque-related diseases may require more specific targeted therapies because general oral samples are not representative of dental plaque bacteria (which contribute to periodontal disease). Psychrobacter, while not the only cause, is an example of a genus that may be highly relevant due to its particular dominance in dog 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. https: / / doi.org / 10.1371 / journal.pone.0199676. Additionally, another study linked Psychrobacter to a common foul odor in dogs; Meason-Smith et al. Vet Dermatol 2018;29:465–e158. Physical dental cleaning of dental plaque significantly reduced psychrophilic bacilli levels, but the effect was transient; after cleaning, return to baseline levels was observed within five weeks. Therefore, both general and specific therapies targeting dental plaque are needed to promote effective treatment and / or maintenance of oral health in dogs, in addition to general oral therapies.

[0003] Applicants in the art have previously attempted to address this problem by providing a ductile product with a flexible matrix that provides a mechanical cleaning action to remove accumulated bacteria, such as US5407661. However, this does not selectively remove pathogenic bacteria. In order to prevent and avoid disease / illness, compositions for modifying biofilms typically include bactericides and other components in combination, such as phospholipids, surfactants or enzymes. These compositions bind well to epithelial surfaces to effectively destroy pathogens, but because the active agent is non-selective, it can destroy natural biofilms including beneficial commensal bacteria. Standard veterinary oral care products kill all bacteria present in the oral cavity of dogs.

[0004] Many strategies have been developed to modify biofilms on epithelial surfaces with the goal of disrupting or removing all biofilms, and / or reducing the levels of pathogenic bacteria in biofilms, usually by applying biocides.

[0005] For example, NZ763741 describes the use of phospholipids to disrupt biofilms and the use of antibiotics to non-selectively kill bacteria in biofilms.

[0006] NZ779091 describes the use of surfactants and biocides to disperse biofilms and non-selectively kill the bacteria that make up the biofilm.

[0007] NZ757876 describes the use of non-selective bactericides triacylpolyamines to kill and disperse bacteria in biofilms.

[0008] NZ732061 describes the use of oxidoreductases and substrates for these enzymes such as honey to kill pathogenic bacteria in chronic wounds and medical devices. The bactericide produced by the oxidoreductases is the non-selective bactericidal hydrogen peroxide.

[0009] NZ755166 describes the use of thiol-based antioxidants, enzymes that break down the extracellular matrix, and non-selective bactericides (such as antibiotics or preservatives).

[0010] In each of the above disclosures, the strategy involves:

[0011] - use of components that disrupt naturally occurring biofilms; and / or

[0012] - Use of bactericidal components that do not discriminate between helpful commensal bacteria and harmful pathogenic bacteria in naturally occurring biofilms.

[0013] While most biocides are generally non-selective, agents that selectively kill pathogenic bacteria without affecting the growth of commensal bacteria have recently been described. For example, PCT / NZ2017 / 050043 describes a combination of protein fractions from bovine milk (IDP TM) can effectively prevent the growth of pathogenic bacteria (such as Escherichia coli and Staphylococcus aureus) without affecting the growth of commensal bacteria from, for example, the Lactobacillus family. However, there is no evidence that IDP TM Binds to epithelial surfaces.

[0014] IDP TM Or "immune defense proteins" are protein fractions from bovine milk that modulate the microbiome. This fraction is described in at least US12 / 304108, NZ719276, NZ742157, PCT / NZ2017 / 050043 and NZ744458. According to the published literature, IDP TM It is a preparation based on milk bioactive protein extracted from breast milk. TM The components of IDP are naturally produced by cows as an immune defense response to infection and inflammation. TM It has anti-inflammatory, antioxidant and antimicrobial effects in vitro, selectively supporting "good" flora and killing "bad" bacteria. IDP TM It has been used for oral, throat, intestinal and dermal applications.

[0015] Despite their potential advantages, it has been demonstrated that such proteins are not able to effectively exploit their properties alone, and formulations containing these agents or similar types of functional agents do not appear to successfully deliver the desired effect at the intended location or provide activity over an extended period of time.

[0016] Fibraspect TM It is a known protein-based gel that can be used as a surfactant emulsifier substitute. The gel has the ability to bind active ingredients together, release the agent in a controlled manner, and has shear thinning rheology (thixotropy), which gives it good stability and skin-friendly feel. It has a semi-solid material composed of soluble native proteins, protein aggregates and protein fibrils produced from whey protein isolate (WPI) to form a modified WPI. The viscosity of the modified WPI gel is 0.25 poise to 4.5 poise.

[0017] To date, the problem of providing suitable oral products that are intrinsically effective in delivering and achieving specific effects in the oral mucosa of animals has remained largely unmet. In particular, adequately targeting dental plaque without harming the commensal flora is a challenge. As a result, effective solutions that are suitable and successful in maintaining or improving the oral health of companion animals have been extremely limited to date. Summary of the invention

[0018] A novel protein complex, a method for preparing and using the protein complex, and a composition comprising the protein complex are defined herein.

[0019] First, the present invention relates to a biocomplex comprising a unique combination of whey protein isolates; a semi-solid material consisting of soluble native proteins, protein aggregates and protein fibrils produced from whey protein isolates (WPI) to form modified WPIs. The adhesion of biopharmaceuticals that retain selective antimicrobial activity on epithelial surfaces enables the functional application of useful selective agents in biological environments that were previously impossible. The biocomplex is a complex protein formed by combining microbiome modulating proteins with modified WPIs through new and innovative processes.

[0020] The invention of new stable biocomplexes enables the functionality of selective agents potentially useful in biological environments. Specifically, the adhesion of agents that retain selective antimicrobial activity on epithelial surfaces is a further step towards a useful solution.

[0021] The new biocomplex is formed by combining microbiome modulating proteins with modified WPI in a new and unique way. This allows the selective bactericidal activity to be retained while developing epithelial adhesion characteristics in the composition. In vitro testing confirmed that these properties remain within the resulting powder.

[0022] Furthermore, formulation of the biocomplex may be more critical to achieving biological effect in certain applications, particularly in veterinary applications where the subject cannot follow instructions. For example, an antimicrobial may be used effectively in part by understanding and following instructions, such as rubbing a surface, washing / brushing in a cavity, rather than swallowing. In contrast, however, for effective veterinary use (e.g., pet dogs or cats), the formulation itself must inherently deliver and achieve biological activity regardless of the associated instructions.

[0023] Therefore, a new ability to adhere specific pharmaceutical agents to epithelial surfaces, such as mucosal surfaces, of animals, as well as useful formulations for this purpose, would be of great benefit, particularly in the veterinary field.

[0024] Therefore, the successful delivery of selective pharmaceutical agents in animals, including dogs and cats, by means of suitable formulations that inherently allow for effective use of the active substance would be a very useful advantage.

[0025] The methods and complexed proteins described herein have useful effects in terms of stability and, when applied topically, the ability to adhere the complexed proteins to epithelial surfaces and retain protein function.

[0026] The present invention provides a method of producing a composite stabilized microbiome modulating protein biocomplex configured for topical application, the method comprising the steps of:

[0027] Choose microbiome modulating proteins in powder form;

[0028] Selecting a modified whey protein isolate (WPI) from milk in gel form;

[0029] Dissolving the microbiome modulating protein in a saline solution having an ionic strength of 25 mM-200 mM NaCl;

[0030] The dissolved microbiome modulating protein and the modified WPI are mixed together;

[0031] Adjust pH to 2.0-6.0;

[0032] Optionally adjust the temperature to 18-37°C;

[0033] The mixture was maintained at a pH of 2.0-6.0 and a temperature of 18-37°C for at least 30 minutes.

[0034] The invention also encompasses complexed proteins produced by a method substantially as described above.

[0035] This process enables the production of new biocomposite compositions that retain selective bactericidal activity while benefiting from epithelial adhesion properties. In vitro testing of the biocomposite compositions has demonstrated that these beneficial properties are retained within the resulting powder.

[0036] The present invention also encompasses a complexed protein comprising a microbiome functional protein complexed with a modified whey protein isolate (WPI) from milk, the complexed microbiome functional protein being formulated for topical application and adhering to an epithelial surface upon application.

[0037] The present invention further relates to a method of selectively treating an animal for virulent pathogenic bacteria while minimizing any reduction in commensal flora, the method being performed by the step of topically applying a complexed protein comprising a microbiome functional protein complexed with a modified whey protein isolate (WPI) from milk, the complexed protein being configured for topical application and, when applied, being configured to adhere to an epithelial surface.

[0038] Additionally, the present invention provides the use of the complexed protein in the preparation of a medicament, wherein the complexed protein is configured for topical application and when applied is configured to adhere to an epithelial surface, wherein the medicament is used to topically treat virulent pathogenic bacteria on an animal's epithelial surface but minimize any reduction in commensal flora.

[0039] The inventors have identified a complexed protein that has useful effects in terms of stability and, when applied topically, in terms of the ability to adhere the functional protein to the epithelial surface and retain excellent protein / protein fraction function. This appears to be due, at least in part, to the important chemical and physical conditions described further below, under which the two components react together to form a complexed protein that can be controlled to optimize the functional activity of the two components. By carefully balancing these parameters, the useful effects produced and the extent of adhesion of the protein to the epithelial surface after complexing are more than twice that of each component used alone, and in the inventors' experience, there is a synergistic effect in terms of adhesion and function, or at least far beyond expectations.

[0040] The invention extends to a complex protein or biological complex as hereinbefore described for use as a medicament, optionally as a veterinary medicament.

[0041] In particular, the use is for the treatment of oral or dental infections mediated by an imbalance of pathogenic bacteria, optionally gum or periodontal disease.

[0042] In an embodiment, the composition comprises a therapeutically effective amount of a complexed protein or biological complex as described above.

[0043] Further reformulation of the biocomplex may be more critical to achieving biological effects in certain applications. Accordingly, the present invention extends to a composition comprising: a biocomplex comprising solubilized microbiome modulating proteins complexed with a modified whey protein isolate (WPI) from milk, wherein the biocomplex adheres to epithelial surfaces and is selectively toxic to pathogenic bacteria but not toxic to commensal bacteria. The biocomplex may include solubilized microbiome modulating proteins complexed with a modified whey protein isolate (WPI) from milk. In embodiments, one or more microbiome modulating proteins of the biocomplex are selected from lactoperoxidase, lactoferrin, lysosomal α-mannosidase, immunoglobulin G, angiogenin, ribonuclease 4, and restin sulfhydryl oxidase.

[0044] Therapeutic efficacy can be further improved by ensuring that the biocomposite is delivered to a precise location, such as a dog's gums. Because the biocomposite is in close proximity to the teeth and gum line, it can effectively adhere to the epithelial surfaces of the animal's mouth.

[0045] Other aspects and advantages of the methods and compositions and uses will become apparent from the following description which is provided by way of example only. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Other aspects of the method and composite protein will become apparent from the following description which is provided by way of example only and with reference to the accompanying drawings in which:

[0047] Figure 1 is a graph showing that the complex has no effect on the growth of the commensal bacterium Lactobacillus acidophilus;

[0048] Figure 2 is a graph showing the bactericidal effect of the complex on pathogenic bacteria Escherichia coli;

[0049] Figure 3 is a graph showing that the complex adheres well to the epithelial surface and retains its bactericidal activity against the pathogenic bacterium Staphylococcus aureus;

[0050] Figure 4 is a graph showing the effect of administering a protein biocomplex to dogs for 5 days on the oral pathogen Psychrophilus sp.;

[0051] Figure 5 is a graph showing the effect of using a protein biocomplex according to an embodiment of the present invention on canine intestinal microbiome after 5 days;

[0052] Figure 6 , 7 and 8 are graphs showing the effects of 28 days of application of the protein biocomplex on factors associated with canine periodontal disease;

[0053] Fig. 9 Demonstrating the effects of using the protein biocomplex over 12 weeks on the perception of breath odor scores; and

[0054] Fig.10 , 11 , 12 , and 13 show the effects of protein biocomplexes on factors associated with canine periodontal disease following alternative dosing regimens. DETAILED DESCRIPTION

[0055] As noted above, described herein are methods and complexed proteins having useful effects in terms of stability and the ability to adhere the complexed protein to epithelial surfaces and retain excellent protein function when topically applied.

[0056] For the purposes of this specification, the terms "about" or "approximately" and grammatical variations thereof mean a quantity, level, extent, value, number, frequency, percentage, dimension, size, amount, weight or length that differs by 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% from a reference quantity, level, extent, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0057] The term "substantially" or grammatical variations thereof refers to at least about 50%, such as 75%, 85%, 95% or 98%.

[0058] The terms "comprising" and their grammatical variations shall have an inclusive meaning, that is, they will be deemed to include not only the listed components they directly refer to, but also other unspecified components or elements.

[0059] For the purposes of this specification, the term "complex", "biocomplex" or grammatical variants thereof refers to the physical interaction between the microbiome modulating protein and the modified whey protein isolate (WPI) such that they bind to each other and adhere to the epithelial surface when topically applied, and when adhered, the microbiome modulating protein is functional. Without being bound by theory, the inventors understand that the physical interaction holding the complex together may be due to conjugation, physical capture, or a combination thereof. Other interactions may occur, and references to conjugation or physical capture should not be considered limited thereto.

[0060] 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).

[0061] For the sake of brevity of specification, the term "protein" or grammatical variations thereof as used herein is intended to cover one protein, multiple proteins, one protein fraction or multiple protein fractions, and reference to a single protein should not be considered limiting.

[0062] Methods of Producing Complexes

[0063] Described herein is a method of producing a composite, stable microbiome modulating protein according to the present invention, which is formulated for topical application, comprising the steps of: selecting a microbiome modulating protein in powder form; selecting a modified whey protein isolate (WPI) from milk in gel form; dissolving the microbiome modulating protein in a saline solution having an ionic strength of 25 mM-200 mM NaCl; mixing the dissolved microbiome modulating protein and the modified WPI together; adjusting the pH to 2.0-6.0; optionally adjusting the temperature to 18-37° C.; maintaining the mixture 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 if the temperature at any step of the process exceeds 37° C. and the pH drops below 2.0 or greater than 9.0, the selective bactericidal activity of the protein described herein may be adversely affected.

[0064] The viscosity of the modified whey protein isolate is important to facilitate compounding and the optimum temperature for the most suitable viscosity is in the range of 18-37°C.

[0065] The ionic strength of the mixture is important for the proper interaction of the protein groups on both components, and the optimal ionic environment is maintained by 25 mM-200 mM NaCl at pH 2.0-6.0.

[0066] Only through careful experimentation and balancing of these parameters can useful effects be achieved. The fact that the complexed proteins adhere to epithelial surfaces to a degree more than twice that of each component alone and that, in the inventors' experience, there is a natural synergy or at least far more than expected suggests that synergy is possible but that this could not have been anticipated or predicted before.

[0067] Compound form

[0068] The resulting composite protein from the above method can be a semisolid. The composite protein can be a gel. Prior to composite, the viscosity of the gel can be comparable to that of the modified WPI. The gel can have a viscosity of about 0.25 poise to 4.5 poise.

[0069] The complexed protein may be stable and the complexed protein may be retained in complexed form by the modified WPI until topical application, at which time the protein functionality is available to the epithelial surface to which the modified WPI is adhered.

[0070] Microbiome regulatory proteins

[0071] The microbiome modulating proteins may be a mixture of protein fractions in milk that are selectively toxic to pathogens but minimize any reduction in commensal flora. The microbiome modulating proteins may include: lactoperoxidase, lactoferrin, lysosomal α-mannosidase, immunoglobulin G, angiogenin, ribonuclease 4, restin sulfhydryl oxidase, and combinations thereof. The proteins may include all of the proteins listed above.

[0072] The microbiome modulating protein selected for the above methods may initially be in the form of a dry powder.

[0073] Modified WPI

[0074] The modified WPI selected for the above method may initially be in the form of an aqueous gel.The modified WPI selected may be derived from milk of bovine species, but may also be derived from modified WPI obtained from other animals such as goats and sheep.

[0075] Proportion

[0076] The ratio of microbiome modulated protein to modified WPI present in the protein used and / or complexed in the method can 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 can be 1:1 to 1:10; that is, 1 part microbiome modulated protein to 1-10 parts modified whey protein isolate. In one embodiment, the ratio can be a 1:1 ratio. According to the inventor's experience, it may be necessary to have a modified WPI:protein ratio that is at least as large as possible to ensure that the desired degree of complexation is achieved. Lower ratios can be used, but the method produces waste or non-complexed protein due to insufficient modified WPI present at the lower ratio.

[0077] Saline solution

[0078] The salt solution may comprise a mixture of water and chemical salts. In one embodiment, the chemical salt may be NaCl, although other salts such as MgCl may also be used. Salt may be added to the extent required to provide the ionic strength shown. The ionic strength of the salt solution may be approximately 25mM, or 50mM, or 75mM, or 100mM, or 125mM, or 150mM, or 175mM or 200mM NaCl. As described above, the ionic strength may vary between 25mM-200mM NaCl. In one embodiment, the ionic strength is approximately 75mM NaCl. During and after the retention period, this ionic strength is retained / maintained in the composite mixture of the protein and the modified WPI. According to the inventor's experience, this ionic strength range appears to be the best choice for compounding or dissolving the microbiome modulating protein into the modified WPI, although there may be some variations depending on the pH, temperature, and retention time used. It seems important to use a salt solution because this will produce the correct ionic characteristics of the solution to dissolve the powdered protein and to make the protein carry the correct charge to produce a complex.

[0079] mix

[0080] Mixing is performed gently so that no or minimal foam is generated during mixing. For example, mixing can be accomplished using a planetary mixer. Mixing can continue during pH adjustment, temperature adjustment, and / or holding. Gentle or slow mixing of proteins may be important to minimize or avoid denaturation and loss of function.

[0081] pH

[0082] The pH of the mixture before pH adjustment may be about 6.5 to 7.5, or about neutral pH 7.0. This starting point of pH may depend on the pH of the water used to form the salt solution. The pH of the mixture after pH adjustment can be about 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. In one embodiment as described above, the pH can be 2.0 to 6.0. In one embodiment, the pH can be about 4.0. This pH range and value seems to be the best pH for compounding or dissolving proteins into the modified WPI, although there may be some changes depending on the ionic strength, temperature and holding time used. The acidic conditions after adjustment seem to retain the appropriate charge on the protein to ensure the combination of the complex. pH can be adjusted by using acid. In one embodiment, the acid can be hydrochloric acid, although other acids can also be used. According to the experience of the inventors, once adjusted, no buffer is needed to maintain the reduced pH.

[0083] temperature

[0084] After any adjustment (if necessary), the temperature of the mixture can be about 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 described above, the temperature after any adjustment can be between 18-37°C. In one embodiment, the temperature after adjustment can be about 25°C. The temperature can be adjusted to maintain the modified WPI at a desired viscosity to achieve optimized complexation. The desired viscosity can be in the range of about 0.25 poise to 4.5 poise. The temperature ranges and values ​​described appear to be optimal temperatures for complexing or dissolving proteins into the modified WPI, although there may be some variation depending on the ionic strength, pH, and hold time used. The temperature can be adjusted according to ambient conditions (or maintained at ambient conditions if the ambient temperature is within the desired range). Depending on the difference between the desired temperature and the ambient temperature, the temperature regulation may go up or down.

[0085] Keep Time

[0086] The holding time may be at least 30 minutes. The holding time may be from 30 minutes to 24 hours. In one embodiment, the holding time may be about 60 minutes. According to the inventors' experience, based on the parameters selected, most complexation occurs within 30 minutes to 60 minutes. Based on the inventors' experience, extended durations may be used; holding the mixture for longer periods of time does not pose a risk of damage or loss of function. This time appears to be an optimal time for complexing or dissolving the protein into the modified WPI, although there may be some variation depending on the ionic strength, temperature, and pH used.

[0087] dry

[0088] Optionally, drying can be completed before subsequent formulation. In this embodiment, the dried complex can be a solid. It can be dried to a water activity of less than 0.6. In selected embodiments, the water activity can be as low as 0.2-0.3. The drying of the complex can be carried out under specific conditions of low temperature and low pressure for a period of time to ensure that the epithelial adhesion characteristics and selective bactericidal activity of the components of the complex are retained. The pressure during drying may be important because at low pressure, water evaporates from the gel at a lower temperature, so drying can be carried out at low temperatures. In addition, it may be important to reduce the pressure very slowly so that the complex protein solution in the dryer does not foam. Drying can be completed by freeze drying or any other gentle drying process using non-denaturing temperature / pressure (e.g., below 30°C, in a vacuum). When rehydrated after drying, the complex protein retains function.

[0089] Protein complex of the present invention

[0090] The present invention provides a composite protein or biocomplex produced by a method substantially as described above. In addition, the present invention provides a composite protein comprising a microbiome functional protein composited with a modified whey protein isolate (WPI) from milk, the composite microbiome functional protein being configured for topical application and adhering to the epithelial surface when applied. The composite protein can be a semisolid aqueous gel having a viscosity of about 0.25 poise to 4.5 poise. The composite protein can be further formulated into an emulsion, gel, paste or ointment for use in a dental composition. The microbiome modulating protein in the composite can be a mixture of proteins that are selectively toxic to pathogenic bacteria but minimize any reduction in commensal flora. The protein in the composite can include: lactoperoxidase, lactoferrin, lysosomal α-mannosidase, immunoglobulin G, angiogenin, ribonuclease 4, restin sulfhydryl oxidase and combinations thereof. The modified WPI in the composite can be derived from the milk of bovine species.

[0091] The ratio of microbiome modulating protein to modified WPI in the complexed protein can be 1:1 to 1:10. The complexed proteins produced by the methods disclosed herein or having the required characteristics have useful effects in terms of stability. In addition, when applied topically, the protein complex has the ability to adhere the functional protein to the epithelial surface and retain excellent protein function. This appears to be due, at least in part, to the important chemical and physical conditions under which the two components react together to form the complex, which can be controlled to optimize the functional activity of the two components.

[0092] Treatments and uses

[0093] Usefully, the composite protein prepared herein is suitable for use as a drug for the first time, and in particular as a veterinary drug. The increase in pathogenic bacteria generally leads to bad breath, dental plaque and gum disease in animals. Therefore, the protein biocomplex can be used to effectively treat poor health conditions, such as oral bacterial infections or dental diseases in animals, wherein the condition or disease is caused by the increase in pathogenic bacteria. The poor health of animals (cats or dogs) may also be due to the reduction of symbiotic bacteria, resulting in an imbalance of the microbial population in the oral mucosa or, in particular, dental plaque. This imbalance may lead to one or more conditions selected from bad breath (bad breath), dental plaque and / or gum disease, so the composition of the present invention can be used for treatment to restore the balance. In an embodiment, the pathogenic bacteria may be a psychrophilic bacillus. In particular, due to the selective function that can be delivered in the oral mucosa for the first time, the composite protein (or a composition comprising it) can be used to treat oral or dental infections. In some embodiments, the infection may be a gum or periodontal disease mediated by an imbalance of pathogenic bacteria. In addition, a method for selectively treating animal toxic pathogens but minimizing any reduction in symbiotic flora is described herein, the method being performed by the step of topically applying a composite protein, the composite protein comprising a microbiome functional protein composited with a modified whey protein isolate (WPI) from milk. The composite protein is configured for topical application, and when applied, the composite protein adheres to the animal epithelial surface. In the above method, the animal can be a non-human animal, optionally such as a cat or dog. The present invention provides the use of a composite protein in the preparation of a medicament, the composite protein comprising a microbiome functional protein composited with a modified whey protein isolate (WPI) from milk, the composite protein being configured for topical application, and when applied, being configured to adhere to the epithelial surface, the medicament is used for topical treatment of toxic pathogens in or on an animal in need but minimizing any reduction in symbiotic flora on the animal epithelial surface. In the above use, the animal can be a non-human animal.

[0094] Compositions comprising protein complexes

[0095] In an embodiment, the present invention relates to a composition comprising a protein complex prepared according to the disclosure herein. Such a composition may comprise at least 0.5% to 2% w / w, preferably 0.1% to 1.5% w / w of the biocomplex and at least one or more pharmaceutically acceptable excipients. The composition comprising the protein complex is suitable for promoting a selective antibacterial effect in the oral cavity of a companion animal. In the examples herein, the model is shown for the first time to be effective in the examples of the oral cavity of the described animals, such as canines. Examples of suitable compositions comprising the protein biocomplexes described according to the above embodiments were tested in suitable canine model tests to confirm the therapeutic effect. As required, a suitable composition comprising the biocomplex is provided that is shown to actually solve specific oral problems in dogs, such as dental disease and odor control.

[0096] Common excipients known in the art can be used to formulate such compositions; these excipients include fillers such as rice, oat flour, sweet potato flour and / or other plant-based flours. These can be selected alone in combination with other well-known excipients for veterinary food supplements or dental compositions. These excipients may also include: humectants, optionally 15% w / w-20% w / w; flavors, optionally 4% w / w-6% w / w; pH buffers, optionally 3% w / w-5% w / w; lubricants, optionally 1% w / w-3% w / w; emulsifiers, optionally 1% w / w-2% w / w; thickeners, optionally 0.5% w / w-1% w / w; and one or more preservatives, optionally up to 0.01% w / w.

[0097] To this end, compositions comprising protein biocomplexes can be used to effectively treat adverse health conditions, such as oral bacterial infections or dental diseases of animals, particularly in cases where the condition or disease may be caused by an increase in pathogenic bacteria. An increase in pathogenic bacteria typically results in bad breath, dental plaque, and gum disease in animals. The poor health of an animal (cat or dog) may be due to a reduction in symbiotic bacteria, resulting in an imbalance of microbiota in the oral mucosa or, in particular, dental plaque. This imbalance can result in one or more conditions selected from halitosis (bad breath), dental plaque, and / or gum disease, so the composition of the present invention can be used for treatment to restore the balance. In an embodiment, the pathogenic bacteria may be psychrophilic bacillus. This composition can also achieve a temporary physical barrier, which is configured to more effectively deliver the protein biocomplex at the desired site (on the gums and teeth of the animal) and provide the protein of the biocomplex and its antibacterial properties, while working with the oral microbiome to support the symbiotic flora required for the animal to maintain good oral health. In an embodiment, the animal is a cat or dog. In an embodiment, the disease may be periodontal disease.

[0098] The composition should be formulated to be gentle on the animal's digestive system to allow for high frequency use and / or as part of a long-term solution to maintain the animal's oral health over a reasonable period of months or years.

[0099] In embodiments, when the composition is used to treat and / or improve oral health, more specifically dental health, a suitable dosage regimen can be followed. In order to reduce the number of pathogens and significantly improve oral health, this composition has been shown to be effective, when used, including the minimum amount of biological complex required (effective for treatment)--daily application, at least 5 days per week, preferably continuing at least 4 weeks, 5-12 weeks or longer. Thereafter, the reduced frequency of administration every other day can be utilized to maintain the health of the appropriately rebalanced microbiome of the oral mucosa. However, regular application will more effectively prevent the recurrence of the accumulation of pathogens, and thereby maintain the appropriately rebalanced oral mucosa.

[0100] Without being bound by theory, such compositions may include other pharmaceutical excipients to enhance the protective function of the composition while the product is delivered and retained in the mouth of the subject. Such dental compositions may provide more effective and prolonged but precise release of bioactives at appropriate locations to achieve the desired efficacy. However, this may be a concern for other animals, particularly those with elongated jaws, particularly such as cats.

[0101] In embodiments, the protein of the biocomplex is uniformly distributed throughout the composition, or the composition may form an outer structure that completely surrounds the protein elements.

[0102] The inventors understand that the complexed proteins produced bind to other epithelial surfaces and can be used to maintain or reestablish a healthy microbiome on any epithelial surface, such as skin and nasal cavity, gut, etc. This can include products for humans and animals, skin care products for humans and animals, nasal spray products for humans and animals, and ingestible gut health products for humans and animals.

[0103] It is also contemplated that the complexed proteins can be used to maintain, establish or reconstitute a healthy microbiome on any protein-rich surface, such as: hair, including hair products for humans; lotions, creams, shampoos, conditioners, sprays; animal fur - lotions, creams, shampoos, conditioners, sprays; medical devices, including artificial skin, wound repair collagen scaffolds, bandages, etc. As described above, it should be understood that the topical surface can be an external surface (e.g., skin), or an internal surface (e.g., the intestine). References to the term "topical" in this specification are not limited to external applications of the complexed proteins.

[0104] The above-described embodiments may also be broadly composed of parts, elements and features mentioned or indicated in the specification of this application, either alone or together and in any or all combinations of any two or more of the parts, elements or features.

[0105] describe

[0106] The above methods, protein biocomplexes, compositions and uses and applications thereof are further exemplified in detail below.

[0107] Example 1 - provides an overview of the production method of the composite:

[0108]

[0109]

[0110] Example 2

[0111] The complex adheres to the epithelial surface

[0112] To determine that the complex adhered to the epithelial layer, a portion of pig skin was soaked in a 50 mg / mL solution of the complex (containing 50 mg / mL protein), washed twice in phosphate buffer at pH 7.4, and stained with Coomassie blue (to stain for protein). This resulted in intensely stained skin ( Figure 1 Treatment of pig skin with 50 mg / mL of the modified whey protein isolate fraction (containing 50 mg / mL protein) alone or the microbiome modulating protein from bovine milk (containing 50 mg / mL protein) alone, followed by washing, showed some binding of these components to the skin, but not as much as with 50 mg / mL of the complex of both components (Table 1).

[0113] Table 1. Pig skin sections treated without or with the complex and components of the complex were washed twice and stained for proteins attached to the epidermal layer of pig skin.

[0114]

[0115] The epithelial binding activity of the complex was much higher (more than two-fold) than that of the modified whey protein isolate alone or the microbiome modulating protein alone. The modified whey protein isolate has been specifically prepared to adhere to epithelial surfaces. Therefore, it was expected that the amount of binding of this component to porcine skin would be similar to that of the complex. However, the complex binding was 44% higher than the modified whey protein isolate alone, suggesting that the components have a synergistic effect, or that the process used to create the complex increases the adhesion ability of the complex.

[0116] Example 3

[0117] Retention of enzyme bioactivity of the complex

[0118] Lactoperoxidase is a protein related to microbiome regulatory proteins isolated from bovine milk (NZ547859 and PCT / NZ2017 / 050043). Lactoperoxidase activity was measured by adding the complex or components of the complex separately to a solution of hydrogen peroxide in 100 mM phosphate buffer at pH 5.5 and monitoring the oxidation of 2,2-azido-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by spectrophotometry at 436 nm.

[0119] When the microbiome modulating proteins were complexed with modified whey protein isolate, the lactoperoxidase activity of the combination of microbiome modulating proteins from bovine milk was retained (Table 2). As expected, the modified whey protein isolate had no peroxidase activity.

[0120] Table 2. Lactoperoxidase activity of the complexes compared to the components of the complexes.

[0121]

[0122] Specific bactericidal activity

[0123] Symbiotic bacteria

[0124] Microbiome modulating proteins isolated from bovine milk have been shown to have no effect on the growth of commensal bacteria, such as those from the Lactobacillaceae family (PCT / NZ2017 / 050043). To determine the effect of complexes containing these microbiome modulating proteins on commensal bacteria, Lactobacillus acidophilus bacteria were cultured anaerobically, the complexes were added to the cultures, and the number of bacteria was determined after 24 hours by optical density at 650 nm. The complexes of modified whey proteins and microbiome modulating proteins isolated from bovine milk had no effect on the growth of commensal bacteria Lactobacillus acidophilus ( Figure 1 )

[0125] Pathogens

[0126] Microbiome modulating proteins isolated from bovine milk have been shown to have a bactericidal effect on pathogenic bacteria such as E. coli (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 number of bacteria was determined after 24 hours by optical density at 650 nm. The complexes inhibited the growth of pathogenic E. coli to a similar extent as the microbiome modulating proteins from bovine milk ( Figure 2 ).Notice Figure 2The square data points in correspond to the results observed with the addition of microbiome modulating proteins from bovine milk, and the circular data points correspond to the results observed with the addition of microbiome modulating proteins in complex with modified whey protein isolate.

[0127] Effects of skin-bound complexes on the growth of pathogenic bacteria

[0128] Staphylococcus aureus (S. aureus) is a pathogenic bacterium commonly associated with skin diseases. Microbiome modulating proteins isolated from bovine milk have been shown to have a bactericidal effect on S. aureus grown in culture (PCT / NZ2017 / 050043). Pig skin samples treated with microbiome modulating proteins from bovine milk and then washed showed moderate inhibition of S. aureus growth. However, pig skin samples treated with the complex and then washed showed significantly greater inhibition of S. aureus growth ( Figure 3 ). This confirms the results shown in Table 1, demonstrating that the complex binds well to epithelial surfaces (such as skin) and also retains the bactericidal activity of the microbiome modulating proteins from bovine milk.

[0129] Example 4

[0130] The formulated composition should contain a minimum therapeutically effective amount of the protein biocomplex. In a formulated product, this may result in a final product of sufficient overall size that, for example, a dog can hold in its mouth. In an embodiment, this may include about 0.05%-2% w / w, preferably 0.13%-0.53% w / w of the protein biocomplex in the composition.

[0131] In this example, the compositions to be tested on animals (dogs) were formulated for lighter or toy dog ​​breeds (up to 15kg), medium weight breeds (15kg-31kg), and larger, heavier breeds (31kg+). Where required, the protein biocomplex in a given composition example included 0.13% w / w (where a larger size composition is required for heavier breeds) or 0.5% w / w (where a smaller size composition is required).

[0132] Such compositions can be prepared by mixing the protein biocomplex components with other biologically acceptable excipient components known for use in veterinary food supplement products (according to recognized techniques). For example, the known components and suitable ranges provided in the table below can illustrate the components used in the examples of compositions comprising the protein biocomplex.

[0133]

[0134] Selective fungicidal activity

[0135] The composition described in accordance with the above-referenced Example 4 was incubated with cultures of pathogenic and commensal bacteria isolated from a single canine oral source (according to methods well known in the art) to determine whether the composition retained its selective bactericidal activity.

[0136] The results showed that the number of canine pathogenic oral bacteria decreased from 10 to 10. 8 colony forming units (cfu) to 10 7 cfu.

[0137] After 18 hours of incubation, there was no reduction in the number of canine commensal oral bacteria. This demonstrates that the selective bactericidal activity required for use in animals is retained in the exemplary composition of the protein biocomplex.

[0138] This composition (as described in Example 4) was then used in the following canine studies:

[0139] Preliminary canine studies – short term (1 to 4 weeks)

[0140] Preliminary microbiome studies were conducted to evaluate the therapeutic effects of exemplary compositions comprising protein biocomplexes to assess their suitability for veterinary use.

[0141] Eleven (11) dogs were administered a composition (described in accordance with the invention as implemented in Example 4) daily for five (5) consecutive days (Day 2, 3, 4, 5 and 6 of the experimental protocol). Oral and fecal microbiota swabs were sampled from each dog before and after the above-mentioned continuous administration of the composition, and a final oral swab was collected 48 hours after the last administration of the composition. At the end of the experiment, oral and fecal swabs were collected and DNA was successfully extracted for analysis under appropriate laboratory conditions.

[0142] Complete microbiome profiling was performed using 16s amplicon sequencing and analysis (amplicon normalization, PCR reactions, amplicon barcoding, amplicon library QC, paired-end reads, 2x250bp read length). Deep amplicon sequencing incorporating principles from statistical ecology was used to survey the microbial community. Deep sequencing of the small ribosomal rRNA subunit (16S rRNA) gene was used to survey the bacterial community and gain insights into the dynamics of the flora, specifically the oral pathogen Psychrobacterium. Results for Psychrobacterium levels in oral samples are shown in Figure 2. Figure 4 The figure shows the mean and standard error of the relative abundance of Psychrophilic Bacteria.

[0143] According to the study protocol, there are 3 adjacent time bars in the oral swab showing Psychrophilus:

[0144] The first one is at time 1 = before administration of the composition comprising the protein biocomplex (day 2),

[0145] The second is time 2 = 5 days later (day 6),

[0146] The third bar is time 3 = 48 hours after the last administration of the composition (day 8).

[0147] from Figure 4 As can be seen on the far right, after 5 days of continuous application to dogs, there was a significant reduction in Psychrobacterium (a Proteus), which is known to cause dental diseases such as periodontal disease. Despite no further application after 2 days, the low level of this bacteria was maintained. Therefore, the results herein conclusively show that the composition containing a therapeutically effective amount of the protein biocomplex reduces the pathogenic bacteria specifically present in dental plaque, thereby enabling targeted and effective treatment (or prevention) of dental diseases associated therewith. Initially, a suitable dosage regimen for reducing pathogens may be once a day for at least 5 days. This may be followed immediately by a reduced frequency regimen of every other day, as regular continued application may be required to prevent the reappearance of high levels of pathogens, thereby specifically maintaining dental health, once the bacterial flora is properly rebalanced.

[0148] Furthermore, because Psychrobacterium is associated with, and may be responsible for, bad breath odor in (hound) dogs, it is possible to reduce the unpleasant and unpleasant symptoms of the disease, such as bad breath odor. In addition, according to the study, fecal samples obtained before and after the 5-day trial were also sent for microbiome profiling and analysis (as described above). Importantly, Figure 5 As can be seen, this showed no significant acute effects on the fecal microbiome in response to 5 days of use, indicating that the product is suitable for selectively targeting pathogenic bacteria in the oral cavity without affecting the microbiome of the intestine.

[0149] Following this, an extended version of the same trial over 28 days (4 weeks) confirmed these findings, demonstrating a statistical association:

[0150] After the 4th week, reduce oral pain associated with problems such as gum infection, tooth root infection, loose teeth, etc. (see Figure 6 )

[0151] · Reduction of tartar and plaque may improve oral hygiene (see Figure 7 ) and exhalation (see Figure 8 ) - Improved to Week 3 and Week 4 (After 1 week without using the composition, i.e., in Week 5, there was a decrease)

[0152] Canine Study 2 – Mid-Term (12 Weeks)

[0153] A 12-week dental trial was conducted to further evaluate the effects of the protein biocomplex as it relates to factors associated with periodontal disease in dogs.

[0154] A total of 17 dogs aged > 6 months with persistent dental problems such as gum disease, tartar, plaque, halitosis, and weighing between 15kg->30kg were enrolled. Dogs with healthy teeth were excluded.

[0155] Regimen: Use the composition (as described above) daily for 4 weeks, then use the composition every other day for 8 weeks.

[0156] Evaluation factors and results

[0157] Evaluations were performed at baseline (week 0), week 1, week 4, week 5, week 6, week 8, and week 12. The dogs reported significant improvement in breath freshness within 4 weeks, and continued use throughout the 12 weeks maintained breath freshness, even with reduced frequency of every other day application. It was observed that longer term use (approximately 8 weeks) was required to see more significant effects and improvements on other factors such as gum bleeding, plaque, and tartar.

[0158] Breath Odor and Cleanliness

[0159] like Fig. 9 As shown, a gradual decrease in breath odor score perception was reported, with the mean breath score decreasing from 2.2 at baseline to 1.5 by week 4 after daily use. Even after the frequency was changed (every other day), additional use (4 weeks+) showed a gradual decrease in breath scores. For example, the lowest mean breath score reported at week 6 was 0.8. Overall, mean breath scores below baseline (2.2) were reported at weeks 5, 8, 10, and 11 near the end of the trial, which were less than half of baseline (2.2) (between 1.0-1.1).

[0160] The percentage of responses for “slightly cleaner and fresher” and “much cleaner and fresher” was higher in Weeks 4-12 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.

[0161] Bleeding gums

[0162] Among owners who brushed their dogs' teeth, the proportion of bleeding gums decreased from 11.8% at baseline to 5.9% at week 8.

[0163] Dental plaque

[0164] By week 8, plaque was significantly reduced: owner-reported plaque scores decreased from 76.5% at baseline to 29.4%

[0165] Canine Study 3 – Alternative Dosing Regimen (12 Weeks)

[0166] Another 12-week trial has been completed to evaluate the efficacy of the protein biocomplex (formulated as above) administered every other day. In this trial, a larger sample size of dogs was involved: 35 dogs were started, including 13 small breeds (up to 15 kg), 11 medium breeds (15 kg-31 kg), and 11 large breeds (over 31 kg). This is important to evaluate and confirm the consistency of the effect in dogs of different weight categories.

[0167] Test process

[0168] Each dog was given a protein biocomplex composition of a size appropriate for its weight category (according to the example described in Example 4) every other day over a period of 10 weeks.

[0169] Factors and results of the assessment:

[0170] Assessment surveys were conducted at baseline, week 4, week 8, and week 12 (2 weeks after cessation of administration). The dogs’ microbiome and health were then assessed by their owners using a number of criteria:

[0171] like Fig.10 , 11 As can be seen from Figures 12 and 12, after 4 weeks, the breath odor score, tooth cleanliness, and dental plaque score were significantly improved. Fig.13 It can be seen that after 8 weeks, the tartar score improved significantly.

[0172] All changes observed through week 8 were maintained through week 12, but no significant changes were noted between weeks 8 and 12.

[0173] Statistical relevance - Participants with missing survey responses after baseline were included in the analysis. For each question, a mixed effects model was used to analyze significant differences between the mean scores for each survey during the trial. Post hoc multiple comparisons used Tukey's test to compare mean scores at baseline with those at Weeks 4 and 8 and at Weeks 8 with those at Week 12 (to account for missing values). Significant differences were defined at p < 0.05.

Claims

1. A complex protein comprising: A microbiome functional protein comprising lactoperoxidase, lactoferrin, lysosomal α-mannosidase, immunoglobulin G, angiogenin, ribonuclease 4 or restin sulfhydryl oxidase and / or a combination thereof, the microbiome functional protein being complexed with a modified whey protein isolate (WPI) from milk, wherein the complexed microbiome functional protein is configured for topical application and adheres to an epithelial surface upon application.

2. The complexed protein of claim 1, wherein the complexed protein is a semisolid aqueous gel having a viscosity of about 0.25 poise to 4.5 poise.

3. The complexed protein according to claim 1 or 2, wherein the microbiome functional protein is a mixture of protein fractions that are selectively toxic to pathogenic bacteria but minimize any reduction in commensal flora.

4. The complexed protein according to claims 1 to 3, wherein the modified WPI is derived from milk of bovine species. 5 . The composite protein according to claim 1 , wherein the ratio of the microbiome modulating protein to the modified WPI in the composite protein is 1:1 to 1:

10.

6. The complexed protein according to any one of claims 1 to 5 for use as a medicament, optionally as a veterinary medicament, in a therapeutically effective amount.

7. The complexed protein according to claim 6, for use in treating oral or dental infections mediated by pathogenic bacterial imbalance, optionally gum or periodontal disease.

8. Use of the complexed protein according to any one of claims 1 to 5 in the preparation of a medicament for topical treatment of virulent pathogenic bacteria or for use on an animal in need thereof while minimizing any reduction in the commensal flora on the epithelial surface of the animal.

9. A method of selectively treating virulent pathogens in an animal while minimizing any reduction in commensal flora, said method being performed by topically applying a complexed protein as claimed in any one of claims 1 to 5 and wherein upon application, said complexed protein adheres to the epithelial surface of the animal.

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

11. A topical composition comprising the complexed protein of claims 1 to 5 and further comprising at least one pharmaceutically acceptable excipient.

12. A method of producing a composite, stabilized microbiome modulating protein configured for topical application, the method comprising the steps of: selecting a microbiome modulating protein, wherein the protein comprises the following in powder form: lactoperoxidase, lactoferrin, lysosomal α-mannosidase, immunoglobulin G, angiogenin, ribonuclease 4 or quiescent sulfhydryl oxidase and / or combinations thereof; Selecting a modified whey protein isolate (WPI) from milk in gel form; dissolving the microbiome modulating protein in a saline solution having an ionic strength of 25-200 mM NaCl; The dissolved microbiome modulating protein and the modified WPI are mixed together; Adjust pH to 2.0-6.0; Optionally adjust the temperature to 18-37°C; The mixture was maintained at a pH of 2.0-6.0 and a temperature of 18-37°C for at least 30 minutes.

13. The method of claim 12, wherein the microbiome modulating proteins are a mixture of protein fractions that are selectively toxic to pathogenic bacteria but minimize any reduction in commensal flora.

14. The method of claim 12, wherein the resulting complexed protein is a semisolid gel having a viscosity of about 0.25 poise to 4.5 poise.

15. A method according to any one of claims 12 to 14, wherein the selected modified WPI is initially in the form of an aqueous gel.

16. The method according to any one of claims 12 to 15, wherein the selected modified WPI is derived from milk of bovine species.

17. The method of any one of claims 12 to 16, wherein the ratio of microbiome modulating protein used in the method and present in the composite protein to modified WPI is 1:1 to 1:

10.

18. The method of any one of claims 12 to 17, wherein the salt solution comprises a mixture of water and a chemical salt.

19. The method of any one of claims 12 to 18, wherein the saline solution has an ionic strength of about 75 mM NaCl.

20. The method of any one of claims 12 to 19, wherein the pH of the mixture prior to pH adjustment is about 6.5 to 7.

5.

21. The method of any one of claims 12 to 20, wherein the pH of the mixture after pH adjustment is about 4.

0.

22. The method of any one of claims 12 to 21, wherein the temperature of any conditioned mixture is about 25°C.

23. The method of any one of claims 12 to 22, wherein the holding time is about 60 minutes.

24. The method of any one of claims 12 to 23, wherein the complexed protein is dried to a water activity of less than 0.6 after holding.

25. A complexed protein produced by the method of any one of claims 12 to 24.

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