Combination, therapeutic uses and prophylactic uses

Abstract

This invention relates to the therapeutic and prophylactic use of a combination including lactoperoxidase and at least one other component, with an isoelectric point of, or substantially above 6.8, and which are extracted from milk, to modulate the microbiome of an animal by selectively against at least one pathogenic microoganism.

Claims

1. A method of modulating a microbiome by selectively inhibiting growth or killing pathogenic micro-organism Streptococcus pyogenes without a comparative inhibition of at least one commensal micro-organism, the method comprising the step of administering to a human tissue including a microbiome a combination including: lactoperoxidase present at a minimum amount of at least 5% w/w; angiogenin; lactoferrin; lysozyme-like protein; quiescin; and jacalin-like protein, wherein the lactoperoxidase, angiogenin, lactoferrin, lysozyme-like protein, quiescin, and jacalin-like protein all have an isoelectric point of or above 6.8 and are extracted from milk from a non-human species.

2. The method according to claim 1 wherein the combination is a composition.

3. The method according to claim 2 wherein the lactoperoxidase and angiogenin are in intimate admixture in the milk and remain in intimate admixture in the formation of the composition.

4. The method according to claim 1 wherein the combination is applied externally.

5. The method according to claim 1 wherein the combination is applied internally.

6. The method according to claim 1 wherein the combination further includes proteins isolated from milk which have an isoelectric point of or above 6.8.

7. The method according to claim 1 wherein the commensal micro-organism is selected from: Staphylococcus epidermidis, Streptococcus pneumonia, Staphylococcus hominis, Lactobacillus bulgaricus, Lactobacillus casei, Porphyromonas gingivalis, Streptococcus mitis and Streptococcus salivarius.

8. The method according to claim 1 wherein the combination further includes one or more components selected from substrates.

9. The method according to claim 8 wherein the one or more component substrates includes a peroxidase substrate.

10. The method according to claim 1 wherein the combination further includes thiocyanate.

11. The method according to claim 1 wherein the combination further includes one or more components selected from: cathelicidin 1; N-acetyl glucosaminidase; serum amyloid A; ? Defensin; Peptidoglycan recognition protein; Xanthine dehydrogenase; Immunoglobulin(s) IgA, IgD, IgG, IgM, IgA, and/or IgE; and/or Growth factors EGF, IGF 1, TGF B1 and TGF B2.

12. The method according to claim 1 wherein the combination selectively inhibits growth of Streptococcus pyogenes by a multiple of at least 1.1 compared with the degree of inhibition of at least one commensal micro-organism.

13. The method according to claim 1 wherein the human tissue is at least part of skin, conjunctiva, nose, pharynx, lower gastrointestinal tract, anterior urethra, or vagina.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which:

(2) FIG. 1 shows the general elution profile of all the fractions from cation exchange. This represents all the protein peaks (as detected at 280 nm) that would be present in a single fraction eluted in a gradient from 80-100 mS. The main components in the cationic fraction are immunoglobulin, lactoperoxidase, lactoferrin, and a group of minor components that include angiogenin.

(3) FIG. 2 shows the fractions separated on SDS-PAGE, and indicates the band that was excised for Mass Spectroscopy and identified as bovine angiogenin. The immunoglobulin fraction shows PIGR (76 kDa) as the predominant band, and the heavy (52 kDa) and light chains of immunoglobulin. The Lp fraction is mainly lactoperoxidase with a small amounts of heavy and light chains of immunoglobulin and angiogenin. The intermediate fraction has a prominent band of lactoperoxidase and lactoferrin (80 kDa) and a band at around 15 kDa that was identified by Mass Spectrometry as angiogenin, a band at approximately 13 kDa that was identified by Mass Spectrometry as jacalin-like. The Lf fraction is predominantly lactoferrin (80 kDa).

(4) FIG. 3 shows a graph of pathogenic Escherichia coli inhibition using the cationic fraction alone, and with 40 ppm of sodium thiocyanate,

(5) FIG. 4 shows a graph of pathogenic Streptococcus uberis inhibition using the cationic fraction alone, and with 75 ppm of sodium thiocyanate and 150 ppm of ascorbate,

(6) FIG. 5 shows a graph of pathogenic Escherichia coli growth using various sub-fractions of the cationic fraction, a recombined cationic fraction and an unfractionated (whole) cationic fraction,

(7) FIG. 6 shows a graph of pathogenic Staphylococcus aureus growth using various sub-fractions of the cationic fraction, a recombined cationic fraction and an unfractionated (whole) cationic fraction,

BEST MODES FOR CARRYING OUT THE INVENTION

Example 1: Assessment of the Proteins in the Composition (i.e. the Cationic Fraction) Via Mass Spectrometry

(8) The process of producing the cationic fraction involved fractionating milk through a cation exchange resin, eluting the bound components from the resin using a salt solution, which can be either a one-step high molarity (>1M) salt or a gradient elution from a lower molarity up to over 1M, collecting the eluted components in a single fraction, and then desalting and purifying the collected fraction.

(9) The cationic fraction was analysed for its constituent components, and the results shown in Table 3. This shows a typical result for yield and identity of the major proteins identified in the cationic protein fraction.

(10) This particular cationic fraction was captured from raw, whole milk.

(11) TABLE-US-00003 TABLE 3 Sub-fractions from the cationic fraction, as measured by Mass Spectrometry (MS). (Lactoperoxidase was determined via extinction coefficient rather than MS.) Total Protein Isoelectric Identity from MS (mg/ml) % of total point lactoperoxidase 4.2 8.0% 8.3 quiescin 1.6 3.0% 8.69 jacalin-like protein 1.4 2.7% 8.71 chitinase-like protein 0.4 0.8% 8.74 angiogenin 10.0 19.0% 9 Lactoferrin 35.0 66.5% 8.7 TOTAL 52.6 100.0%

Example 2: Inhibition Trials on Pathogens Vs Commensals

(12) The Applicant used the methodology described herein to prepare a cationic fraction isolated from bovine milk as described in Table 3. The composition was tested in vitro against a number of micro-organisms using micro-titre plates and some in agar diffusion tests. The results are shown in Table 4 (shown below) identify the MIC (mg/ml) of the cationic fraction against the different micro-organisms.

(13) TABLE-US-00004 TABLE 4 Inibitory analysis of cationic fraction against range of commensals and pathogens. MIC mg/ml Pathogens Propionibacterium acnes 0.1 Trichophyton mentagrophytes 0.1 Trichophyton rubrum 0.1 Escherichia coli 0.1 Streptococcus pyogenes 0.1 Malassezia furfur 0.2 Commensal/Opportunistic pathogen Candida albicans 0.5 Streptococcus mutans 2.5 Staphylococcus aureus-coagulase negative 3 Commensals and Probiotics Streptococcus saliyarius (probiotic strain) >5 Streptococcus pneumonia >10 Staphylococcus epidermidis >10 Staphylococcus hominis >10 Lactobacillus bulgaricus >10 Lactobacillus casei >10 Porphyromonas gingivalis >10

(14) The conclusions that can be reached from this preliminary work are: 1. Micro-organisms that frequently cause infections (i.e. pathogens) are killed by the lowest concentrations of the combination (provided as a composition). 2. Micro-organisms that are common, harmless commensals and/or are used as probiotics were not killed by the highest concentrations tested in these trials. (100? greater than the concentration that killed pathogens). 3. Intermediate organisms, such as Candida albicans, which are frequently found as harmless commensals and only cause infection (i.e. become opportunistic) when the conditions change in the local environment so that growth is enhanced (e.g an increase in sugar concentration) show moderate to high MIC values.

Example 3: Comparative Selectivity Between Isolated and/or Recombined Proteins, Sub-Cationic Fractions, and Whole Cationic Fraction

(15) Additional studies were conducted which show the individual proteins in the cationic fraction (i.e. lactoperoxidase, lactoferrin, quiescin-like, jacilin-like, chitisase-like, angiogenin) have poor anti-microbial effectiveness against pathogens, and therefore will not be able to provide the selectivity offered by the combination of proteins in the preferred compositions (most preferably the full suite of proteins in the cationic fraction). The results are shown in FIGS. 5 and 6.

(16) Informal results also showed that the middle cationic fraction (not containing lactoperoxidase or lactoferrin) has relatively poor selectivity, supporting that lactoperoxidase is an important component of the composition, but requires other protein(s) from milk in order to develop the selectivity profile observed.

(17) Also, the results shown in FIGS. 5 and 6 showed that with there is a benefit of retaining the proteins together during the extraction process, rather than isolating them and recombining to form the composition. Nonetheless, the recombined fraction provides useful inhibition of E. coli and S. aureus.

(18) The fact that anti-microbial activity is enhanced when the cationic fraction remains intact also strongly suggests the selectivity will be better if the components are not individually separated from one another before recombining. That is, the combination is preferably provided as a composition which preferably includes the cationic fraction of milk.

(19) Based on these results, the following schematic representation is provided to illustrate the effectiveness of the present invention towards selectivity.

(20) TABLE-US-00005 Low High MIC (mg/ml) <0.1 0.5 1.0 2.0 5.0 >10.0 Lactoferrin (Lf) Pathogenic-Opportunistic- Commensals (no selectivity) Lactoperoxidase Pathogenic-Opportunistic-Commensals (no (Lp) selectivity) Middle Cationic Pathogenic-Opportunistic-Commensals Fraction* (no selectivity) Whole cationic Pathogenic Opportunistic commensals Commensals fraction** such as C albicans *e.g. angiogenin, quiescin, jacalin-like protein, and chitinase-like protein - no Lp or Lf **as seen in Table 4.

Example 4: Effect of Additional Substrates

(21) FIGS. 3 and 4 illustrate that anti-microbial effects (and hence potentially selectivity too) are improved in the presence of substrates. If such substrates are not present at the site of infection, it may be beneficial to include suitable substrates within the combination. The extent of growth of the micro-organism is indicated by the height of the bars. The shortest bars show maximum inhibition of growth. For this figure, the left-hand bars indicate that some inhibition of growth is achieved with the cationic fraction alone at a concentration of 1 mg/ml. However, adding 40 ppm of sodium thiocyanate to the cationic fraction allowed total growth inhibition to occur at a cationic fraction concentration of 2 mg/ml. This indicates that lactoperoxidase contributes to the antimicrobial activity when its substrate (thiocyanate) is included.

(22) FIG. 4 shows the results of a different formulation of the cationic fraction against Streptococcus uberis, this time using sodium thiocyanate (75 ppm) and ascorbate (150 ppm) as substrates. Against Streptococcus uberis, there is no inhibition in vitro using the cationic fraction alone up to 0.8 mg/ml. However, adding sodium thiocyanate and ascorbate shows an inhibitory effect occurring as low as 0.2 mg/ml of the cationic fraction. This confirms that in the absence of milk (or another natural source of substrates) the addition of thiocyanate (as substrate) and ascorbate (as a source of peroxide) may be useful for increasing inhibition (and perhaps selectivity) towards Streptococcus uberis.

(23) Note that in FIG. 4, none of the additives were totally inhibitory on their own. The samples labeled 0 in the figure are buffer-only and additive-only samples.

(24) TABLE-US-00006 TABLE 5 Example formulations used for comparative testing are provided below Activated Combination (as Cationic cationic a composition) Lactoperoxidase Lactoferrin fraction fraction Lactoferrin 0% 92.9% 64.3% 61.3% Lactoperoxidase 97.7%* 0% 22.8% 26.6% Other protein <2.3%* (n.m.) 5.1% 9.3% 8.1% Glucose 0% 0% 0% 0.845% Glucose oxidase 0% 0% 0% 0.015% Thiocyanate 0.004% 0% 0.004% 0.004% Monolaurin 0% 0% 0% 0.25% *total protein value as no specific assay for lactoperoxidase is reported for this material, hence other protein level cannot be estimated. Note: for each sample, the remainder of material to 100% is largely inorganic (measured as ash) or residual moisture. Note: n.m. was not measured Note: there are hundreds or probably thousands of minor proteins (other protein) within the cationic whey fraction.

(25) It will be noted that the activated cationic fraction includes glucose, glucose oxidase and monolaurin which are not typically found in milk, let alone the cationic fraction. Glucose oxidase can use glucose as a substrate to generate peroxide in situ. Other peroxide generating systems may include percarbonate or peracetate, which may be encapsulated or coated to control the release rates of the peroxides. These components may be considered to act as adjuvants.

(26) Thiocyanate is present in the activated cationic fraction and is an example of a substrate. Other examples of substrates include iodide or chloride, having countercations of sodium, potassium or calcium.

(27) The innate lactoperoxidase system protects the eyes, nose, mouth and airways from invasion by harmful microbes and requires presence of the lactoperoxidase enzyme, peroxide and thiocyanate or halide.

(28) H.sub.2O.sub.2 is naturally present in internal biological environments as it is a by-product of various oxidative processes. For example, neutrophils produce large amounts of free peroxy radicals (O.sub.2.sup.?) of which the steady state concentration has been estimated to be in the micromolar range. (Ref. Hampton, M B, Kettle A J, Winterbourn C C. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 1998; 92:3007-17)

(29) Peroxidases (such as lactoperoxidase) are present in biological secretions and catalyse H.sub.2O.sub.2 dependent oxidation of halides (thiocyanate, iodide, bromide, chloride) that can react with and kill microbes. (Ref. Klebanoff S J. Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin. Hemotol 1975; 12:117-42)

(30) Thiocyanate is naturally present in lymph and blood, in the mammary, salivary and thyroid glands and their secretions, in synovial, cerebral, cervical and spinal fluids and in organs such as stomach and kidney. For example, thiocyanate levels measured in human trachea-bronchial secretions from intubated adult patients were 0.46+/?0.19 mM or 26.7+/?11 ppm (range 16-38 ppm). (Ref. Wijkstrom-Frei, C., El-Chemaly, S., Ali-Rachedi, R., Gerson, C., Cobas, M. A., Forteza, R., Salathe, M. and G. E. Conner. 2003. Lactoperoxidase and human airway host defense, Am. J. Respir. Cell Mol. Biol., 29:206-12).

(31) As such, in some circumstances in some circumstances, such as where the combination is being applied internally, to an open wound, or to a mucosal membrane it may not be necessary, or even preferable to provide an adjuvant and/or a substrate as part of the combination since that substrate will be provided endogenously by the tissue to which the combination is applied.

(32) In other circumstances the endogenous concentration of the adjuvant and/or substrate may be too low or non-existent to have an appreciable effect on the activity of the combination.

(33) In those circumstances it may be preferable to include an adjuvant and/or substrate in the combination.

(34) For in vitro testing, the assay medium will not typically contain the adjuvant and/or substrate and the improved results for the activated cationic fraction compared with the cationic fraction may be partially explained by the beneficial effect of the substrate and adjuvants contained in the activated cationic fraction. However, the cationic fraction may still provide useful selectivity when applied, for example, to an area of the body where the substrate and/or adjuvants (halides and peroxide generation, for example) are already present such as application internally, to an open wound, or to a mucosal membrane.

Example 5: Bacterial Selectivity

(35) The activity and selectivity of a range of test compounds/compositions were determined against a range of pathogenic and commensal organisms.

(36) The methodology for each of the following test compositions is described below: Lactoperoxidase (sample 3); Lactoferrin (sample 2); Cationic fraction (sample 4); and Activated cationic fraction (sample 1)

(37) Each sample was prepared as a stock solution at 5 mg/ml. Samples 1, 3 and 4 were dissolved in HBSS which contains potassium isothiocyanate at 40 ppm (40 ?g/ml). Sample 2 was dissolved in HBSS at 5 mg/ml (Table 4).

(38) Aerobic Testing

(39) Experiment Protocol for C. albicans, S. aureus, S. epidermidis, S. mitis, S. mutans and S. salivarius 1. For C. albicans Sabouraud dextrose broth powder was added to distilled water at 30 g/L and stirred. For S. aureus and S. epidermidis tryptic soy broth powder was added to distilled water at 30 g/L and stirred. For S. mitis, S. mutans and S. salivarius 5% sheep blood broth was prepared by diluting the sheep blood with double distilled water. 2. The broth solutions were then boiled for 1 minute with stirring to completely dissolve the powder. 3. The broth media were then autoclaved at 121? C. for 20 minutes. 4. A small quantity of each pure micro-organism was taken and used to inoculate 40 ml of broth medium. The inoculated broth was incubated for approximately 66 hours at 37? C. 5. The broth culture was diluted with fresh, sterile broth medium to an OD.sub.650nm of approximately 0.1, equivalent to approx 10.sup.5 CFU/ml prior to commencement of MIC testing. This is the inoculant which will be used to inoculate the test wells in each plate. The inoculant was held at 4? C. until required for plating. 6. Stock solutions of the test sample were prepared such that the concentration is 5 mg/ml in the appropriate broth. 7. The reference antibiotic was dissolved in broth to give a final concentration of 100 ?g/ml. 8. 96 well microtitre plates were then set up as indicated in the plate layout diagrams below: 200 ?l of the appropriate sample stock solution of test sample in the appropriate broth, antibiotic standards and vehicle control were added to the relevant wells in Column 1 on Plates 1 to 3. 9. To all other wells 100 ?l of the appropriate sterile broth was added. 10. Using a multichannel pipette, 100 ?l of the sample and antibiotic was sampled from the wells of column 1 on each plate and transferred to wells in column 2, mixed thoroughly by pipetting up and down 5 times. Fresh tips were added to the pipette and 100 ?l of solution was transferred from the wells of column 2 to those of column 3, mixed thoroughly by pipetting up and down 5 times and then discarding the tips. This process was continued through to the wells of column 11 on each plate. This process will result in serial double-dilutions that range from 5 mg/ml to 0.005 mg/ml for samples, and 100 ?g/ml to 0.098 ?g/ml for the antibiotic standard. The 12th and final well in each row (Plates 1 to 3) contain wells of broth only with inoculants and broth only without inoculants (Plates 4). These wells serve as sterility control blanks and test substance free control blanks respectively. 11. Wells A1-12, 81-12, C1-12, D1-12, E1-12, F1-12, G1-12 and H1-12 on Plate 4 contain broth only and were not inoculated with seed culture. These wells served as sterility controls and blank for each row. Wells A12-F12 (Plates 1-2) and A12-C12 (Plate 3) contained the cells and serve as the negative control. 12. 100 ?l of inoculant or broth were added to each well as indicated in the plate layouts below. The addition of inoculant or broth halve the extract concentration in each well giving final well concentrations ranging from 2.5 mg/ml to 0.0025 mg/ml for samples (including vehicle control) and 50 ?g/ml to 0.049 ?g/ml for the antibiotic standard. 13. The plates were gently tapped to ensure even mixing of the inoculant with the sample solutions. 14. The OD.sub.650nm of each well were read using a Versamax microtitre plate reader. This will be recorded as the zero time reading. 15. The plates were incubated for 3 hours at 37? C. at which time the OD.sub.650nm of each well were read and recorded as the 3 hour reading. 16. The plates were returned to the incubator for a further 13 hours and the OD.sub.650nm of each well was read and recorded as the 16 hour reading. 17. The microtitre plates were returned to the incubator for a further 8 hours and the OD.sub.650nm was read and recorded as the 24 hour reading. 18. Once the OD.sub.650nm of the plates was read, the wells containing the highest dilution of each sample (lowest concentration of test extract) without a detectable change in OD.sub.650nm in comparison to the initial reading at time zero were noted.

(40) Anaerobic Testing

(41) Experiment Protocol for B. bifidum, B. breve, C. difficile, C. perfringens and P. acnes 1. For each of these bacteria, Brain Heart Infusion Blood broth was used. It was prepared by adding it to distilled water at 37 g/L. 2. The broth solution was then boiled for one minute with stirring to completely dissolve the powder. 3. The broth media was then autoclaved at 121? C. for 20 minutes. 4. A small quantity of each organism was used to inoculate 40 ml of the Brain Heart Infusion broth that was de-aerated by bubbling nitrogen into it. This sealed tube is then incubated at 37C. 5. During this incubation, the samples were prepared. Stock solutions of the samples were prepared at 5 mg/ml in the broth. 6. The reference antibiotic was dissolved in broth to give a final concentration of 100 ?g/ml. 7. 96 well microtitre plates was then set up as indicated in the plate layout diagrams below: 200 ?l of the appropriate sample stock solution of test sample in the appropriate broth, antibiotic standards and vehicle control) was added to the relevant wells in Column 1 on Plates 1 to 3. 8. To all other wells 100 ?l of the appropriate sterile broth was added. 9. Using a multichannel pipette, 100 ?l of the sample and antibiotic was sampled from the wells of column 1 on each plate and transferred to wells in column 2, mixed thoroughly by pipetting up and down 5 times. Fresh tips were added to the pipette and 100 ?l of solution was transferred from the wells of column 2 to those of column 3, mixed thoroughly by pipetting up and down 5 times and then discarding the tips. This process was continued through to the wells of column 11 on each plate. This process results in serial double-dilutions that range from 5 mg/ml to 0.005 mg/ml for samples, and 100 ?g/ml to 0.098 ?g/ml for the antibiotic standard. The 12th and final well in each row (Plates 1 to 3) contain wells of broth only with inoculants and broth only without inoculants (Plates 4). These wells serve as sterility control blanks and test substance free control blanks respectively. 10. Wells A1-12, B1-12, C1-12, D1-12, E1-12, F1-12, G1-12 and H1-12 on Plate 4 contain broth only and are not inoculated with seed culture. These wells serve as sterility controls and blank for each row. Wells A12-F12 (Plates 1-2) and wells A12-C12 (Plate 3) contain the cells and serve as the negative control. 11. 100 ?l of inoculant or broth are added to each well as indicated in the plate layouts below. The addition of inoculant or broth halve the extract concentration in each well giving final well concentrations ranging from 2.5 mg/ml to 0.0025 mg/ml for samples (including vehicle control) and 50 ?g/ml to 0.049 ?g/ml for the antibiotic standard. 12. The plates are gently tapped to ensure even mixing of the inoculant with the sample solutions. 13. The OD.sub.650nm of each well were read using a Versamax microtitre plate reader. This was recorded as the zero time reading. 14. The plates are then placed in a sealed container along with one or more anaerobic pouches. The sealed container is then incubated at 37? C. 15. After 3 hours, the plates are removed from the container and the OD.sub.650nm of each well are read in the platereader immediately, one plate at a time. The plates were then returned to the container along with new anaerobic pouches and plates incubated at 37? C. 16. At 16 hours after commencing the study, step 15 was repeated. 17. After 24 hours, the OD.sub.650nm of each well was measured. 18. Once the OD.sub.650nm of the plates was read, the wells containing the highest dilution of each sample (lowest concentration of test extract) without a detectable change in OD.sub.650nm in comparison to the initial reading at time zero was noted.

(42) The results of the testing of the anaerobic species and the aerobic species are presented in Table 6.

(43) TABLE-US-00007 TABLE 6 MIC values @ 3 hrs (mg/ml) Organism Candida albicans Commensal/ Staphylococcus Staphylococcus Opportunistic aureus epidermidis Type Pathogen Pathogen Commensal Sample 1 Activated 0.005 0.002 0.01 cationic fraction Selectivity* 2.0 5.0 1.0 Sample 2 Lactoferrin 0.625 ?2.5 ?2.5 Selectivity* ?4.0 1.0 Sample 3 ?2.5 ?2.5 ?2.5 Lactoperoxidase Selectivity* 1.0 Sample 4 Cationic ?2.5 ?2.5 ?2.5 fraction Selectivity* 1.0 MIC values @ 48 hrs (mg/ml) Organism Streptococcus Streptococcus Streptococcus mitis mutans salivarius Type Commensal Pathogen Commensal Sample 1 Activated 0.625 0.002 0.156 cationic fraction Selectivity* 1.0 78.0-300** 1.0 Sample 2 Lactoferrin 1.25 ?2.5 2.5 Selectivity* ?2.0 1.0 Sample 3 ?2.5 ?2.5 ?2.5 Lactoperoxidase Selectivity* 1.0 Sample 4 Cationic ?2.5 ?2.5 ?2.5 fraction Selectivity* 1.0 *1.0 = no selectivity **relative to S. salivarius and S. mitis

(44) Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof.