WO 2006/053383 PCT/AU2005/001746
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Bioactive Compositions
Field
[0001] The invention relates to bioactive compositions, and in particular relates
to bioactive compositions which are pH sensitive and need to act in or traverse
through a gastric environment, in order to provide benefits for a wide population
of individuals.
[0002] Examples of oral bioactive composilions include bovine colostrum (which
can be used to relieve the symptoms of gastrointestinal diseases), colostral IgG
fraction, hyperimmune colostrum, hyperimmune egg yolk material and other
materials described in International Patent Application PCT/AU03/00348
(published as WO 03/080082), which is incorporated by reference.
[0003] There are several challenges associated with the use of pH sensitive
bioactive compositions. The gastric environment varies greatly between
individuals and at different times during the day in the same individual. For
example a fasted gastric environment which may occur late in the night or
before breakfast may have a pH in the range 1.5 to 2, whereas a post-prandial
gastric environment may have a pH in the range 2 to 5 or even higher.
Furthermore, in individuals with arrested gastric secretion resulting from age or
medication, the gastric pH may be in the range 6 to 7. In addition, the resting
volume of gastric fluid in an individual can vary between 5 and 40 ml or even
more widely.
[0004] A number of strategies have been proposed to improve the efficacy of
pH sensitive bioactives.
[0005] The following discussion of documents, acts, materials, devices, articles
and the like is included in this specification solely for the purpose of providing a
context for the present invention. It is not suggested or represented that any or
all of these matters formed part of the prior art base or were common general
knowledge in the field relevant to the present invention before the priority date
of each claim of this application.

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[0006] US Patent No. 6569453 (Under and Dietrich) filed in 2001, describes an
administration form for acid - labile active compounds including a proton pump
inhibitor such as Omeprazole. The formulations have no enteric layers and are
suitable for oral administration - they may comprise a sterol such as cholesterol
in combination with a polymer such as polyvinyl acetate or PVP/vinyl acetate
co-polymer. The formulations may be made by dissolving sterol and polymer in
a suitable solvent, suspending the acid labile proton pump inhibitor there-in and
spray drying the resulting suspension.
[0007] Another strategy is to provide an acid labile active compound with an
enteric coating which is rapidly dissolved in the alkaline medium of the intestine
after gastric passage. Such a strategy is adopted in European Patent
Publication EP-A-0 005 129, EP-A-0 166 287, EP-A-0 174 726 and EP-A-0 268
956. In adopting this strategy the active ingredient must frequently be provided
in the form of its alkaline salt, or together with alkaline substances. The
substances of use in making enteric coatings are typically those having free
carboxyl groups, and in the presence of an alkaline moiety in the interior of the
dosage form, dissolution of the coating cap take place from the inside out. Free
carboxyl groups may promote the decomposition of the active compound. It
may therefore be necessary to provide an insulating intermediate layer between
the coating and the core material. Under and Dietrich address this problem by
performing a matrix encapsulation to provide protection against harsh acid
conditions. The active compound needs to be provided in dry form and comes
into contact with non-aqueous solvent. The above features increase cost and
solvent use and can lead to denaturing of many bioactive materials, particularly
denaturing of the tertiary structure of peptide based materials.
[0008] US Patent No. 6312712 (Whittle et al) filed in 2000, teaches that the
bioavailability of certain pharmaceutically active moieties can be increased by
administering said moieties in combination with a cyclodextrin. The use of this
method is limited to active moieties which undergo appropriate reactions with
the cyclodextrin for example to form inclusion complexes.

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[0009] US Patent No. 5998216 {O'Donnell) filed in 1996, describes stabilising
formulations for preserving the integrity of proteins present in a body fluid. An
important ingredient in the stabilising formulation is a water soluble, high-
potency buffering compound. The buffer capacity can be established by
measuring the pH change caused by the addition of increments of strong acid
or strong alkali to the buffer. High potency soluble buffering compounds include
TRIS, di-basic phosphate/mono-basic phosphate, sodium bicarbonate and
triethanolamine. O'Donnell refer to a range of body fluids including blood,
saliva, pleural fluid, gastric fluid, ascites fluid and synovial fluid.
[0010] Many of the buffers referred to by O'Donnell cannot be used over a long
course of treatment because of adverse side-effects.
[0011] US Patent No 5851579 (Wu et al) filed in 1997, describe an aqueous
enteric coating composition comprising an alkali soluble acrylic latex polymer
and an aqueous solution of ammonium or alkaline salts of cellulose polymers.
Enteric coating strategies such as these have been found to be problematic with
many peptide based bioactive materials.
[0012] US Patent No. 6468959 (Wunderlich et al) filed in 1994, describes a
dosage form for peptides such as insulin. The dosage form comprises a matrix
of gelatin or gelatin derivatives having distributed there-in the peptide
pharmaceutical. The gelatin derivatives carry a sufficient contrary net charge to
form a pseudo-coacervate. This is limited to bioactive materials which form
coacervate or pseudo-coacevates with gelatin, a criterion which tends to
exclude low molecular weight bioactive materials. Importantly, the coacervated
material needs to protect the bioactive from the adverse effects both of gastric
pH and gastric enzymes - this requires significant optimisation and testing, and
may not be achievable at all.
[0013] US Patent No. 5780434 (A. Fjellestad-Paulsen) filed in 1995, describes
a composition for oral administration of small and medium sized peptides,
particularly vasopressin, oxytocin and their analogues, said composition
comprising peptide, a protease inhibitor, and a carrier comprising a buffering

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agent buffering at a pH of about 5, wherein said mixture is in the form of
spheres smaller than 2 mm, said spheres; being coated with polymers having
dissociable carboxyl groups (enteric coating). Difficulties arise with enteric
coating as described previously. The patent provides no information about a
desirable gastric pH end value, or pH end value range.
[0014] US Patent No. 5525634 describes a matrix-drug combination, wherein
the matrix contains a saccharide-containing polymer. The polymer is resistant to
chemical and enzymatic degradation in the stomach and is susceptible to
enzymatic degradation in the colon by colonic bacteria. This patent provides no
teaching on pH modulation in the gastric environment.
[0015] International Publication WO 03/080082 of PCT/AU03/00348 (Rawlin
and Lichti) describes a method of improv.ng the viability of a labile bioactive
substance in a hostile environment, comprising forming a mixture of the
bioactive substance and mammalian colostrum. This patent provides no
teaching on the protective effect of any material other than colostrum.
[0016] Skim milk and/or egg yolk is known to be effective in preserving the
viability of spermatozoa in hostile environments such as freezing and thawing.
(Squire et al (2004) in Theriogenology Sep 15; 62(6):1056-65).
[0017] There is a need for a composition for pH sensitive bioactive materials
which provides good bioavailability to a wida range of individuals having diverse
gastric environments.
Summary
[0018] Through our studies of bioavailability of pH sensitive materials we have
developed an understanding of the requirements for such compositions. We
have found that part of the requirements for such a composition is to provide
good alkalising activity to acidic gastric liquors, however it is a significant
advantage if the composition does not cause neutral gastric environments to
become alkaline beyond pH 8.5, and preferably beyond pH 8. We believe the
latter constraint arises because gastric alkaline surges (even transient surges)

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can cause the digestive enzyme pepsin to be permanently inactivated (The
Pharmacological Basis of Therapeutics - Goodman and Gillman, 5th edition, p
960). The requirement to meet both of the above constraints (alkalising effect ai:
low pH, non-alkalising effect at neutral pH) does not arise in the formulation of
antacids, because antacids are only taken by people with acidic (pH less than
4) gastric liquors.
[0019] We have found that the optimal presentation of pH labile bioactives to a
gastric environment may involve maintaining the gastric pH between pH 4 and
5, rather than at higher pH values (eg pH 7 or 8) - this is because the rate of
gastric acid replenishment increases markedly at higher pH levels.
[0020] We have found that by using a carboxylic acid containing moiety and
alkali containing moiety and selecting quantities of each it is possible to protect
bioactive materials in individuals having a very acid gastric environment at the
time of administration whilst also avoiding gastric alkali surges in individuals
with less acidic or neutral gastric environment at the time of administration. In
this way the bioavailability of the bioactive; agent can be improved without the
risk of damaging the bioactive agent or inactivating pepsin.
[0021] Accordingly we provide in a first aspect of the invention a bioactive agent
composition comprising
(a) pH sensitive bioactive agent
(b) an edible carboxylic acid containing moiety and
(c) an edible alkalising moiety,
wherein the proportion of said moieties and active agent provide pH control
such that (i) when 400mg of said composition is added to 20 ml of 0.033 normal
hydrochloric acid and at a temperature of 37 +/- 3°C, the pH reaches a value in
the range 4 to 8, and (ii) when 400mg of said composition is added to 20 ml of
deionised water at pH 7 and at a temperature of 37 +/- 3°C, the pH reaches a
value less than 8.5.
[0022] In a further aspect the invention provides use of a pH sensitive bioactive
agent in preparation of a medicament for oral administration comprising forming

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a mixture of the pH sensitive bioactive agent with (a) an edible carboxylic acid
containing moiety and (b) an edible alkalising moiety, wherein the composition
is formulated to react so that (i) when 400mg of said composition is added to 20
ml of 0.033 normal hydrochloric acid and at a temperature of 37 +/- 3°C, the pH
reaches a value in the range 4 to 8, and (ii) when 400mg of said composition is
added to 20 ml of deionised water at pH 7 and at a temperature of 37 +/- 3°C,
the pH reaches a value less than 8.5.
[0023] In a further aspect the invention provides a unit dosage composition
comprising a pH sensitive bioactive agent and an edible carboxylic acid
containing moiety and an edible alkalising moiety wherein: (i) when said unit
dosage is added to 20 ml of 0.033 normal hydrochloric acid at a temperature of
37°C +/- 3°C the pH reaches a value in the; range 4 to 8; and (ii) when said unit
dosage is added to 20 ml of deionised water at pH7 and at a temperature of
37°C +/- 3°C the pH reached a value less than 8.5.
[0024] We have surprisingly found that the resulting compositions of the
invention provide a composition of the pH sensitive bioactive which is stable to
a wide population of individuals having a wide variety of gastric resting volumes
and gastric pH values, which in practice cannot be known prior to individual
dosing.
[0025] The term moiety where used herein refers to a chemical functional group
or segment of a molecule or entire molecule. This molecule may be a molecule
which may be a molecule of relatively low molecular weight or a macromolecule
such as a protein.
[0026] The term agent where used herein refers to a chemical entity such as a
molecule or substance.
[0027] Throughout the description and the claims of this specification the word
"comprise" and variations of the word, such as "comprising" and "comprises" is
not intended to exclude other additives, components, integers or steps.

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[0028] Where referred to herein in the specification and claims pH values
reported for addition of solid compositions to aqueous materials are determined
by adding the solid material in finely divided form to the aqueous composition
(at less than 10% w/v), stirring for 30 mimtes at 37°C and measuring pH using
a glass electrode calibrated for 37°C (This is explained in further detail in the
section of the Examples headed "pH protocol').
Detailed Description
[0029] In the preferred aspect of our invention the composition comprises a a
pH sensitive bioactive agent and further comprises an edible carboxylic acid
containing moiety and an edible alkaiising moiety, preferably so that (i) when
400mg of said formulation is added to 20 rn! of 0.033 normal hydrochloric acid,
the pH reaches a value in the range 4 to 8, and (ii) when 400mg of said
formulation is added to 20 ml of deionised water at pH 7, the pH reaches a
value less than 8.0.
[0030] The bioactive composition comprises a pH sensitive bioactive agent.
The bioactive agent is considered pH sensitive if it loses 50% or more activity
(preferably at least 75% activity) when 100 mg of the active is continually mixed
with 20 ml of 0.033 normal hydrochloric acid for 30 minutes.
[0031] The pH sensitive bioactive agent may be selected from the group
including vitamins, nutritional supplements, growth promoters, antineoplastic
agents, oral vaccines, inhalants, living microorganisms (for example protobiotics
such as Lactobacillus spp), peptides, polypeptides, nucleotides,
polynucleotides, nucleosides, proteins, giycoproteins, sugars and complex
carbohydrates, anti-infectants, antimicrobials, disinfectants, antiseptics,
antidepressants, psychoactive agents, genetically modified organisms and
infectious agents used as vectors for other bioactive substances eg bacterial
vectors (including E. coli, Salmonella, Vibrio, Lactobacilli, Bacillus,
Mycobacteria, Shigella), viral vectors (including Adenovirus, Poxvirus,
Bacculovirus, Herpesvirus, Enterovirus, Paramyxovirus and Orthomyxovirus),
plant vectors (including tobacco, potato and banana), yeast vectors,
immunoglobulins, affinity purified immunoglobulins including antibodies directed

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against diseases and disease causing agents (for example Helicobacter pylori,,
E. coli, Bacillus spp, pathogenic Yersinia spp., and allergens) and fragments,
derivatives and complexes containing any af the above.
[0032] The edible carboxylic acid containing moiety may comprise one or more
selected from the group consisting of: acetic acid; ascorbic acid; polyacid
moieties such as citric acidand tartaric acid; amino acids; peptide chains or
proteins; alginic acid; polyacrylic acid; polymethacrylic acid; and copolymers of
one or more monomers selected from the group of acrylic acid methacrylic
acids; and carboxylic acid containing cellulose derivatives. The edible carboxylic
acid may have nutritional value, for example a protein or protein fragment
derived from the processing of dairy fluids or vegetables or meats.
[0033] The edible alkalising moiety can be any moiety which at 400mg dose is
capable of elevating the pH of 20 m! of 0.033 normal hydrochloric acid to a final
pH of 4 or greater. Preferably the edible alkalising moiety is capable of elevating
the pH of 20ml 0.033 normal hydrochloric acid to a final pH of 5 or more, or
even 6 or more. This moiety may comprise an alkali agent such as selected
from an alkaline phosphate salt, an alkaline carbonate, an alkaline bicarbonate
salt, a hydroxy salt and mixtures of two or more thereof. The alkaline moiety
may comprise at least one of the salts selected from the group consisting of the
calcium and magnesium salts of one or more of carbonate, bicarbonate, silicate
salts and magnesium carbonate co-precipitate. The alkalising agent may be in
the form of other basic salts comprising nitrate, carbonate or gallate moieties.
The alkalising moiety may comprise a weak acid containing moiety which has
been reacted with an alkali such as an amine containing alkali or an alkali such
as at least one of potassium hydroxide, lithium hydroxide, sodium hydroxide,
aluminium hydroxide, calcium hydroxide, magnesium hydroxide, and aluminium
oxide.
[0034] It is possible that the edible carboxylic acid containing moiety (which
may be in protonated or de-protonated form) and the edible alkalising moiety
are one and the same that is part of the same substance. For example, if acid
groups in a protein or polycarboxylic acid are partially pre-reacted with sodium

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hydroxide or an amine-containing base then the resulting product will contain
both moieties.
[0035] The carboxylic acid containing moiety may be present in the bioactive
agent. For example, the bioactive agent may comprise macromolecules such as
proteins which have acid and/or alkali rroieties and may in some cases be
modified to provide the appropriate balance of the required moieties.
[0036] In one preferred embodiment, the bioactive agent comprises: a
hyperimmune fraction derived from bovine colostrum or avian egg yolk. The
hyperimmune fraction may for example be a hyperimmune material directed
against enteric bacteria, enteric viruses, anthrax, plague, oral bacteria,
respiratory bacteria, food borne bacteria.
[0037] In another preferred embodiment the bioactive agent comprises
hyperimmune colostrum harvested from dairy cows vaccinated with a vaccine
comprising one or more cell wall antigens reactive in a manner characteristic of
O group serotypes from enteric disease causing Gram negative bacteria. These
colostrum moieties and the associated vaccines, together with more preferred
embodiments, are described in our copend ng International Application PCT/AU
2004/00277 (published as WO 2004/073209), the contents of which are
incorporated by reference.
[0038] In another preferred embodiment the bioactive agent comprises
lactoferrin or lactoferracin.
[0039] In another preferred embodiment the formulation of this invention
comprises normal colostrum and hyperimmune colostrum harvested from cows
vaccinated with a vaccine comprising one or more cell wall antigens reactive in
a manner characteristic of O group serotypes from enteric disease causing
Gram negative bacteria, wherein the ratio of normal colostrum to hyperimmune
colostrum is greater than 1 to 1, preferably greater than 2 to 1, more preferably
greater than 3 to 1.

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[0040] In a particularly preferred embodiment the edible alkaline moiety is a
relatively insoluble alkali such as calcium or magnesium carbonate.
[0041] The unit dosage composition may be in the form of a tablet, capsule,
caplet, syrup or other suitable form. Preferably the unit dosage is in the form of
a tablet, capsule or caplet. Typically the unit dosage will contain in the range
from 50 to 700 mg of composition and mere preferably from 100 to 500 mg of
composition.
[0042] The carboxylic acid containing moiety preferably is a dairy derived
protein such as colostrum or milk or a fraction or concentrate or enzymic or non-
enzymic hydrolysate thereof and the edible alkalising moiety is calcium or
magnesium carbonate. The dairy derived protein and the calcium or magnesium
carbonate may be co-added as a mixed powder fill in a capsule or tablet, or
these materials may be co-suspended in an aqueous liquor which is
subsequently dried and processed to form a powder.
[0043] The dairy concentrate preferably contains greater than 80% protein.
' [0044] Preferably the weight ratio of dairy derived protein to calcium or
magnesium carbonate is greater than 2 to 1, more preferably greater than 3 to
1, still more preferably greater than 5 to 1.
[0045] The invention will now be described with reference to the following
i examples. It is to be understood that the examples are provided by way of
illustration of the invention and that they are in no way limiting to the scope of
the invention.
Examples
pH Protocol
[0046] Where referred to herein in the specification and claims pH values
reported for addition of solid compositions to aqueous materials are
determined by:

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(a) adding simulated gastric liquor ( unless otherwise specified 20 ml of
.033N hydrochloric acid of nominal pH 1.5) or distilled water (unless
otherwise specified 20 ml) to a 50 mi Falcon tube, and incubating in a 37
degrees centigrade water bath for 10 minutes,
(b) adding finely divided or ground up solid material (unless othrewise
specified 400 mg) to the Falcon tube and vortexing for 5 seconds,
(c) taping the Falcon tube to one extremity of a Ratek Rocking Platform
mixer (model ERPM4, sold by Ratek Instruments, Boronia, Victoria,
Australia), which rocks through 15 degrees of arc
and is 35 cm in length from one extremity to another, in such a manner
that the long axis of the tubes also rock through 15 degrees of arc,
(d) rocking the samples at 50 rpm for 30 minutes, with the rocking
platform and attached samples located inside a 37 degrees centigrade
incubator.
(e) after rocking and incubation measuring the pH of the contents of the
Falcon tube using a pH meter. The pH meter is calibrated between each
sample to be measured.
[0047] Example 1: Lactobacillus plantarum - Sample composition
Samples were made having the composition described in the following table -
the components were finely divided and mixed together. The bioactive was L.
plantarum (freeze-dried powder, see example 5).

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Table 1

Sample id Parts bioactive Parts carboxyiic moiety Parts alkalisingmoiety
Lp1 100 400 (colostrum) -
Lp2 100 378 (colostrum) 22 (calciumcarbonate)
Lp3 100 - 400 (calciumcarbonate)
Lp4 100 - 400 (sodiumcarbonate)
Lp6 100 320 (colost-um) 80 (sodiumcarbonate)
Lp7 100 320 (colostrum) 80 (calciumcarbonate)
Lp8 100 220 (citric acid) 180 (calciumcarbonate)
Lp9 100 270 (citric ecid) 130 (calciumcarbonate)

[0048] Example 2: Performance of samples described in example 1
400 milligrams of sample was added to 20 ml 0.033N HCI and to 20 ml distilled
water. Measurements were taken of the final pH (see section on pH protocol)
and of the viability of the Lplantarum after the treatment, expressed as cfu per
20ml (see example 6). The results are tabulated below (EXP5 means 100,000,
etc):
Table 2

Sample id PH(after acidtreatment) Viability(after acidtreatment) PH(after watertreatment) Viability(after watertreatment)
LP1 2.2 0 6.3 4.5EXP6
Lp2 3.7 1.6EXP5 6.9 5.2EXP6
Lp3 L6.6 4.4EXP6 8.8 3.1EXP6
Lp4 9.3 2.2EXP6 L9.6 2.1EXP6
Lp6 6.9 6.0EXP4 9.2 2.7EXP6
LP7 5.9 3.6EXP6 6.9 5.9EXP6
Lp8 4.75 6.4EXP6 5.98 5.7EXP6
Lp9 2.15 0 2.27 0

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[0049] Discussion:
Sample Lp1 is not a composition according to this invention - the pH after acid
treatment (representative of a resting gastric pH) was 2.2 (well below the limit oi
4 for the invention), and the Lplantamm was not viable.
[0050] Sample Lp2 is not a composition according to this invention - the pH
after acid treatment is 3.7 (marginally below the limit of 4 for the invention). The
Lplantarum viability is down by a factor of 40 on the water treatment.
[0051] Sample Lp3 is not a composition according to the invention - the pH
after acid treatment is above 4 however the pH after water treatment is above
8.5. Whilst the Lplantarum is viable, the final pH is above 8.5, and it would be
expected that the ingestion of this formulation, particularly on a prolonged
repetitive basis would promote gastric acid secretion or undesirable gastric
alkalinity in people with relatively neutral gastric environments.
[00523 Sample Lp4 is not a composition according to the invention - the final
pH after acid and water treatment is over 8.5 and it would be expected that that
the ingestion of this formulation, particularly on a prolonged repetitive basis
would promote gastric acid secretion or undesirable gastric alkalinity in people
with relatively neutral gastric environments.
[0053] Sample Lp6 is not a composition according to this invention - the final
pH after water treatment is over 8.5 and it would be expected that that the
ingestion of this formulation , particularly on a prolonged repetitive basis would
promote undesirable gastric alkalinity in people with relatively neutral gastric
environments.
[0054] Sample Lp7 is a composition according to the invention - the final pH
after acid treatment is over 4, and the final pH after water treatment is less than
8.5, and the Lplantarum is viable.

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[0055] Sample Lp8 is a composition according to this invention - the final pH
after acid treatment is over 4, and the final pH after water treatment is less than
8.5, and the Lplantarum is viable.
[0056] Sample Lp9 is not a composition according to the invention - the final
pH after acid treatment is less than 4 and the Lplantarum is not viable.
[0057] Example 3: Erythromycin, urease sample compositions
The bioactive materials in this example were erythromycin and Urease {see
example 7).
Table 3

Sampleid Parts bioactivematerial Parts component 1 Parts component 2
Er1 1.0 (erythro) 320 (colostrum, carboxylmoiety) 80 (calciumcarbonate, alkalisingmoiety)
Er2 1.0 {erythro) 400 (sucrose, blankmoiety) -
Ur1 168 (urease) 320 (colostrum,carboxylmoiety) 80 (calciumcarbonate, alkalisingmoiety)
Ur2 168 (urease) 400 (sucrose.blankmoiety) -
[0058] Example 4: Performance of samples described in Example 3
Table 4

Sample id PH(after acidtreatment) Relative activity(after acidtreatment) PH(after watertreatment Relative activity(after watertreatment)
Er1 5.8 65% 7.0 95%
Er2 1.7 <5% 6.8 100%
Ur1 6.0 75% 76.9 105%
Ur2 1.8 <5% 6.9 100%
[0059] Discussion:
Sample Er1 is a composition according to the invention - the pH after acid
treatment is over 4 and the pH after water treatment is less than 8.5. The
relative activity of erythromycin is substantial after both treatments.

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[0060] Sample Er2 is not a composition according to the invention - there are
no carboxyl components, no alkalising components, and low relative activity of
bioactive after acid treatment. The activity of bioactive in this sample after water
treatment has been used to set the 100% level.
[0061] Sample Ur1 is a composition according to the invention - the pH after
acid treatment is over 4 and the pH after water treatment is less than 8.5. The
relative activity of urease is substantial after both treatments.
[0062] Sample Ur2 is a not a composition according to the invention - there are
no carboxyl components, no alkalising components, and low relative abundance
of bioactive after acid treatment. The activity of bioactive in this sample after
water treatment has been used to set the 100% level.
[0063] Example 5: Preparation of L.piantarum freeze-dried powder
Freeze-drying media (FDM) was prepared as follows (method derived from
Conrad PB et al. (2000) in Stabilisation and preservation of Lactobacillus
acidophilus in saccharide matrices. Cryobiclogy 41,17-24): Trehalose dihydrate
(Sigma) and sodium tetraborate decahydrate (Sigma) were dissolved in sterile
0.6mM potassium phosphate pH 7.2 at 40%w/v and 5.7%w/v respectively. The
pH was adjusted to 6.5 with solid citric acid (Sigma) and then to 8.5 with
ammonium hydroxide (Sigma 29.5% solution).
[0064] L.piantarum used in this study was taken from the culture collection of
the Microbial Research Unit Royal Childrens Hospital, Parkville, Victoria,
Australia, and typed by 16S sequencing (sequencing of DNA from ribosomal
subunits). The strain was grown as a lawn culture (20 plates) on MRS agar at
37deg C in an incubator with a 5% CO2: air mix for 48 hrs. The culture was
scraped off the 20 plates and combined in 20 mis saline 0.85%. The saline
liquor was centrifuged for 15 min at room temperature, and the pellet was
resuspended in 5mls saline. To this 5ml aliquot was added 5ml FDM, with
vortexing, and the combined liquor was freeze-dried in a Dynavac "Mini Ultra
Cold" freeze-dryer. Max vacuum from pump = O.imbar; vacuum (steady state)

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of freeze-drying chamber = 0.38mbar; working temp = -100deg C; volume
(condenser capacity) = 1.7 L The freeze-drying process was run over-night
(18hrs) until the vacuum approached 0.38mbar. The freeze-dried material was
ground into a fine powder and added to other sample components.
[0065] Example 6: Determination of L.plantarum viability.
After acid or water treatment in the 50 ml Falcon tube (20ml liquor volume), 250
microlitres of liquor was taken (duplicates) and the liquor quenched by the
addition of 62 microlitres of chilled 200mM Tris-HCI pH 8.0. L plantarum
survival was assayed using a viable plate count technique. Ten-fold dilutions of
the incubation mixture were prepared in MRS broth immediately after
quenching. 100 microlitres of each dilution were then spread onto duplicate
plates. Plates were incubated for 48 hrs and the number of colonies per plate
counted. The number of colony forming units in the incubation mixture was then
calculated by multiplying the number cf colony forming units of diluted
suspension by the dilution factor.
[0066] Example 7: Preparation /Acquisition of Other Components
Urease was Sigma Jack Bean Urease Type III (Cat No U-1500). This freeze-
dried powder had a quoted activity of 16 units per mg (one unit liberates 1.0
micro-moles of ammonia from urea per min at pH 7.0 and 25 deg C).
Erythromycin powder was from Boehringer Mannheim.
[0067] Colostrum
The following diagram shows the principles used to take colostrum and convert
it to a processed form.

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[0068] The raw colostrum is collected from dairy cows most preferably at the
first milking after calving. The colostrum is stored at 4°C on farm and then
transported either for longer term storage at -20°C or sent directly to wet
manufacturing.
[0069] The raw colostrum is warmed to approximately 37°C and then skimmed
with a rotary milk separator to remove fat. The resultant liquid may be
pasteurised or microfiltered with a 7-10 micron ceramic filter system (to remove
bacteria and debris. The liquid is then Ultrafiltered (for example in a Abcor 10m2
Ultrafiltration plant) to remove a majority of the water, lactose and electrolytes
leaving a high protein concentrate. The resultant high protein concentrate is
further processed preferably by lyophilization (freeze-drying) or spray-drying.
[0070] The above method yield a processed bovine colostrum powder with the
specifications as below. This product as defined below is listed as a substance
suitable for inclusion in therapeutic goods by the Therapeutic Goods Authority
of Australia.

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[0071] Definition: Bovine colostrum powder is derived from the first milking of
Australian or New Zealand* cows (Bos taurus) following concentration and
lyophilisation.
Appearance: Free-flowing, paie yellow powder.
Properties: Soluble in water. Mild odour of milk when contacted with moisture.
Moisture Range 2 to 5 % m/m AS 2300.1.1(1988)
Fat Range 1 to 4 % m/m AS 2300.1.3 (1988)
Ash (@550°C) Not more than 8 % m/m AS 2300.1.5 (1988)
Total Nitrogen (TN) For information** AS 2300.1.2 (1991)
Non-protein nitrogen(NPN) For information** AS 2300.1.2.2 (1988)
True protein Not less than 60 % m/m (rM-NPN)%x 6.38
Protein Not less than 60 % m/m AS 2300.1.2 (1991)
Lactose (monohydrate) Not more than 15 % m/m
UV assay following enzymatic hydrolysis and oxidation
(Boehringer Mannheim)
Total immunogiobulins Not less than 20 % m/m
Radial immunodiffusion assay
Microbiai limits Complies with TGA guidelines
Residues: Heavy metals Agricultural and Veterinary chemicals
[0072] Subject to ANZFA Food Standards Code for dairy products. Where
there is no applicable Food Standard, the BP test for heavy metals applies
(2 ppm calculated as lead) and also the BP requirements for pesticide
residues.
[0073] * These countries being BSE-free. Colostrum powder from other
countries will require pre-clearance from the TGA
** Used to calculate the value for true protein
[0074] 'AS' refers to document of the Australian Standards Organisation series
of 'Australian Standards' - in this case referring to standardised methods of
quality and component testing for dairy products.

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[0075] Example 8: Measurement of activity for Erythromycin
Erythromycin activity was assayed usirg a bacillus subtilis disc diffusion
susceptibility test (Barry AL and Thornsberry C ,1991, Susceptibility tests:
diffusion test procedures. In Balos A, Hauser WJ, Herman KL, Isenberg HD,
and Shadomy HJ, Manual of Clinical Microbiology 5th Edition, American Society
for Microbiology, Washington pp1117-1125). An inoculum of B.subtilis (ATCC
6633) was prepared by picking at least 2 colonies from an overnight culture
grown on horse blood agar (HBA) and inoculating 2ml of saline to reach a
turbidity equivalent to a 0.5 McFarlane standard. HBA plates for the assay were
then inoculated by streaking a sterile swab, dipped into the standardised
solution, evenly in three directions over the entire surface of the plate to obtain
a uniform inoculum. Plates were then allowed to dry for 3 to 5 minutes before
the discs were applied.
[0076] An aliquot of erythromycin liquor (after acid or water treatment) was
serially diluted 1:2 with MilliQ water, resulting in six dilutions for each. 20 micro-
litres of each dilution was then loaded into duplicate blank susceptibility discs
(Oxoid, Hampshire, England). These discs; were allowed to dry for at least 30
minutes before being placed onto duplicate plates. Each plate contained six
evenly placed discs corresponding to the six dilutions of a single treatment.
Sample Er2 (reacted with water according to the pH protocol) was used as a
control to obtain a standard curve. Plates were incubated for 16-18 hours at 37
degC.
[0077] After 16-18 hours incubation the susceptibility of B. subtilis to
erythromycin was determined by measuring the diameter of the zones of
inhibition which appear around the discs. These zones result from the diffusion
of the antibiotic from the disc into the surrounding agar. A standard curve was
generated using the diameters of zones resulting from the serially diluted
erythromycin control. Diameters from the test samples were then used to obtain
thepercentage of erythromycin activity remaining compared with the untreated
control.

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[0078] Example 9: Measurement of activity for Urease
250 micro-litre aliquots were taken after sample treatment and the aliquots were
quenched by the addition of 0.25 volumes of chilled 160mM Na2CO3 . Aliquots
were then centrifuged for 3 minutes at 20,000g and stored on ice. Supernatants
were assayed for urease activity.
[0079] Urease activity was assayed using a coupled enzyme assay for
increased sensitivity (modified from Kaltwasser and Schlegel, 1966, NADH-
dependent coupled enzyme assay for urease and other ammonia-producing
systems. Analytical Biochem, 16:132-138). In this reaction, the urease enzyme
catalyses the hydrolysis of urea:
Urea + H2O + 2H+ => 2NH4+ + CO2
which is measured by coupling ammonia production to a glutamic
dehydrogenase reaction:
2NH4+ + 2 α-ketoglutarate + 2 NADH => 2 glutamate + 2NAD+ + 2H2O
the reaction is followed by the oxidation of NADH to NAD.
[0080] The final assay volume of 1ml contained final concentrations of 1.6mM
a-ketoglutarate {Boehringer Mannheim Cat No.127 205), 1.5mM NADH (Sigma
p-NADH Cat No. N-8129), 15 units/ml of L-giutamic dehydrogenase (Sigma Cat
No. G-4387), 10mM Urea (Boehringer Mannheim Cat No. 100 164) and 1mM
sodium sulphide (Sigma Cat No S-4766) in 50mM Tris-HCI buffer (pH 8.0).
These reagents were mixed in 1 cm path length polystyrene cuvettes (Sarstedt
Cat No 67.742) and then allowed to equilibrate for several minutes to room
temperature in a Beckman DU70 recording spectrophotometer. The
spectrophotometer was zeroed using the above mixture, before the addition of
the sample.
[0081] A ten-microlitre sample of the supernatant of each incubation mixture
was added to the assay mixture to start the assay. The reaction rate was
recorded every 10 seconds at 340nm for up to 4 min at RT. Reaction rate was
calculated from the linear portion of curve (generally after first 1-2 mins) and

WO 2006/053383 PCT/AU2005/001746
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urease activity was then noted as jimole of urea hydrolysed per min per mg of
protein. Sample Ur2 was used as the control.
[0082] Example 10 - Bioactives Erythromycin, Lactoferrin: sample
composition
Table 5

Sample ID Parts Bioactive Parts Carboxylicmoiety Parts Alkalisingmoiety
S1 1(Ery) 320 (Colostrum) 62 (NaOH)
S2 1(Ery) 320 (Colostrum) 31 (NaOH)
S3 1(Ery) 320 (Colostrum) 15.5 (NaOH)
S4 1(Ery) 320 (Colostrum) 7.8 (NaOH)
S5 1 (Ery) 320 (Colostrum) 111 (Na2HPO4) dihydrate
S6 1 (Ery) 320 (Colostrum) 55.5 (Na2HPO4) dihydrate
S7 1(Ery) 320 (Colostrum) 27.5 (Na2HPO4) dihydrate
S8 40 (lactoferrin) 320 (Colostrum) 40 (CaCO3)
S9 20 (lactoferrin) 320 (Colostrum) 60 (CaCO3)
S10 10 (lactoferrin) 320 (Colostrum) 70 (CaCO3)
S11 10 (lactoferrin) 320 (Colostrum) 80 (CaCO3)
S12 10 (lactoferrin) 320 (Colostrum) 90 (CaCO3)
S13 10 (lactoferrin) 320 (Colosxum) 100(CaCO3)
S14 1(Ery) 8 Citric aciddihydrate 390 (Na2HPO4) dihydrate
S15 1 (Ery) 250 Citric aciddihydrate 150 (NaOH dry)

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[0083] Example 11 - Performance of samples described in Example 10
Table 6
Sample ID pH (after acid treatment) pH (after water treatment)
S1 11.5 11.7
S2 8.4 11.7
S3 3.3 11.4
S4 2.5 10.5
S5 5.5 7.6
S6 3.2 7.8
S7 2.5 7.0
S8 3.8 6.9
S9 4.2 7.0
S10 4.6 7.0
S11 4.9 7.0
S12 5.2 7.1
S13 5.4 7.1
S14 6.6 8.2
S15 5.6 6.7
[0084] In the above example S5, S9, S10, S11, S12, S13, S14 and S15 are
compositions according to the invention.

WO 2006/053383 PCT/AU2005/001746
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Claims
1. A bioactive composition comprising:
(a) pH sensitive bioactive agent
(b) an edible carboxylic acid containing moiety and
(c) an edible alkalising moiety,
wherein the proportion of said moieties and active agent provide pH controi
such that (i) when 400mg of said composition is added to 20 ml of 0.033 normal
hydrochloric acid and at a temperature of 37 +/- 3°C, the pH reaches a value in
the range 4 to 8, and (ii) when 400mg of said composition is added to 20 ml of
deionised water at pH 7 and at a temperature of 37 +/- 3°C, the pH reaches a
value less than 8.5.
2. A bioactive composition according to claim 1 wherein when the 400mg of
said composition is added to 20 ml of deionised water at pH 7, the pH reaches a
value is less than 8.0.
3. A bioactive composition according to any one of the previous claims
comprising the bioactive agent and at least one pH regulating agent component
comprising at least one of said alkalising moiety and said carboxylic acid
containing moiety wherein the other of said alkalising moiety and said carboxylic
acid containing moiety is present in at least one of said bioactive agent and said
pH regulating agent component.
4. A bioactive composition according to any one of the previous claims
comprising a bioactive agent, and a pH regulating agent comprising both said
edible carboxylic acid containing moiety and said edible alkalising moiety.
5. A bioactive composition according [o claim 1 wherein the composition
comprises the bioactive agent, an alkalising agent comprising the alkalising
moiety and an agent comprising the carboxylic acid moiety.

WO 2006/053383 PCT/AU2005/001746
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6. A bioactive composition according to any one of the previous claims
wherein the bioactive agent is selected from the group consisting of vitamins.,
nutritional supplements, growth promoters, antineoplastic agents, oral vaccines,
inhalants, living microorganisms, peotides, polypeptides, nucleotides,
polynucleotides, nucleosides, proteins, glycoproteins, sugars and complex
carbohydrates, anti-infectants, antimicrobials, disinfectants, antiseptics,
antidepressants, psychoactive agents, genetically modified organisms and
infectious agents used as vectors for other bioactive substances eg bacterial
vectors (including E. coli, Salmonella, Vibrio, Lactobacilli, Bacillus,
Mycobacteria, Shigella), viral vectors (including Adenovirus, Poxvirus,
Bacculovirus, Herpesvirus, Enterovirus, Paramyxovirus and Orthomyxovirus),
plant vectors (including tobacco, potato and banana), yeast vectors,
immunoglobulins, affinity purified immunoglobulins including antibodies directed
against diseases and disease causing agents (for example Helicobacter pylori,
E. coli, Bacillus spp, pathogenic Yersinia spp., and allergens) and fragments,
derivatives and complexes containing any of the above.
7. A bioactive composition according to any one claims 1 to 6 wherein the
bioactive agent is selected from the group consisting of growth promoters, oral
vaccines, probiotic microrganisms, antimicrobials, bacterial vectors,
immunoglobulins, antibodies and antibody fragments.
8. A bioactive composition according to any one of the previous claims
comprising a pH regulating agent comprising said carboxylic acid containing
moiety comprising at least one substance selected from the group consisting of
acetic acid, polyacid moieties, ammo acids, or peptide chains, proteins, or
alginic acid, polyacrylic acid, polymethacrylic acid, copolymers of one or both of
acrylic and methacrylic acids and carboxyl containing cellulose derivatives.
9. A bioactive composition according to any one of the previous claims
comprising a pH regulating agent having a carboxylic acid containing moiety
comprising at least one of colostrum, citric acid and tartaric acid.

WO 2006/053383 PCT/AU2005/001746
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10. A bioactive composition according to claim 8 wherein the acidic agent
comprising the carboxylic acid moiety is bovine colostrum.
11. A composition according to any of the previous claims wherein the
400mg of said composition contains sufficient edible alkalising moiety to elevate
the pH of 20ml 0.033 normal hydrochloric acid to a final pH of 4 or more, more
preferably to a final pH of 5 or more.
,12. A composition according to any of the previous claims wherein the
composition comprises a pH regulating agent comprising an alkalising moiety
selected from the group consisting of alkaline phosphate salts, alkaline
carbonate salts, alkaline bicarbonate salts, hydroxy salts and mixtures of two or
more thereof.
13. A bioactive composition according to any one of the previous claims
comprising an edible alkalising agent comprising the alkalising moiety and
selected from at least one of calcium carbonate, magnesium carbonate,
magnesium bicarbonate, silicate salts, and basic salts comprising nitrate,
carbonate or gallate moieties.
14. A bioactive composition according to any one of the previous claims
wherein the alkalising moiety comprises a weak acid containing moiety which
has been reacted with an alkali selected from the group consisting of amine
containing alkali, potassium hydroxide, lithium hydroxide, aluminium hydroxide,
calcium oxide or hydroxide, magnesium oxide or hydroxide and aluminium
oxide or hydroxide.
15. A bioactive composition according to any one of the previous claims
wherein the bioactive agent comprises at least one substance selected from the
group consisting of antibodies derived from hyperimmune bovine colostrum or
hyperimmune avian egg yolk.
16. A bioactive composition according to claim 15 wherein the antibodies are
directed against at feast one antigen selected from the group selected from

WO 2006/053383 PCT/AU2005/001746
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enteric bacteria, enteric viruses, .anthrax, plague, oral bacteria,- respiratory
bacteria and foodbome bacteria.
17. A composition according to any one of the previous ciaims wherein the '
bioactive agent comprises the hyperimmune fraction of colostrum harvested
from dairy cows vaccinated with a vaccine comprising one or more" cell wall
antigens reactive in a manner characteristic-of O group serotypes from enteric
disease causing gram negative bacteria.
18. A bioactive composition according to any one of the previous claims
wherein the oioactive agent comprises iactoferrin or lactoferracin.
19. A bioactive composition according to anyone of the previous claims
comprising as the bioactive agent the riyperimmune fraction of colostrum
harvested from cows vaccinated with a vaccine comprising one or more cell wall
antigens reactive in a manner characterisec of O group serotypes from enteric -
disease causing Gram negative bacteria, and a pH regulating agent comprising
colostrum wherein the ratio of normal colostrum to hypenmmune colostrum is
greater than 1 to 1.
20. A bioactive composition according to claim 19 wherein the ratio is greater
than 3 to 1.
21. A bioactive composition according to any one of the previous claims
wherein the composition comprises an alkalising agent comprising the edible
alkalising moiety selected from calcium and magnesium carbonate.
22. A composition according to claim 1 wherein the composition comprises
an agent comprising a carboxylic acid moiety selected from the group consisting
of colostrum, milk or a fraction, concentrate or hydrolysate thereof'and'the'
edible alkalising moiety is at least one of calcium carbonate and magnesium
carbonate.

WO 2006/053383 PCT/AU2005/001746
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23. A composition according to claim 22 wherein the weight ratio of dairy
derived protein to calcium or magnesium carbonate is greater than 2 to 1.
24. A bioactive composition according to claim 23 wherein the ratio is greater
than 4 to 1.
25. A unit dosage composition comprising a-pH sensitive bioactive agent an
edible carboxyiic acid containing moiety and an edible-alkalising moiety
wherein: (i) when said unit dosage is added to 20 mi of 0.033 normal
hydrochloric acid at a temperature of 37°C +/- 3°C the pH reaches a value in the
range 4 to 8; and (ii) when said unit dosage is added to 20 m! of deionised
water at pH 7 and at a temperature of 37°C +/- 3°C the pH reached a value less
than 8.5.
26. A unit dosage composition acceding to claim 25 wherein the
composition is as defined according to any one of claims 1 to 24.
27. Use of a pH sensitive bioactive agent in preparation of a medicament for
oral administration comprising forming a mixture of the pH sensitive bioactive
agent with (a) an edible carboxyiic acid containing moiety and (b) an edible
alkalising moiety, wherein the composition is formulated to react so that (i) when
400mg of said composition is added to 20 ml of 0.033 normal hydrochloric acid
and at a temperature of 37°C +/- 3°C, the pH reaches a value in the range 4 to
8, and (ii) when 400mg of said composition is added to 20 ml of deionised water
at pH 7 and at a temperature of 37°C +/- 3°C, the pH reaches a value less than
8.5.

This invention relates to a bioactive composition comprising: (d) pH sensitive bioactive agent (e) an edible carboxylic acid containing moiety and (f) an edible alkalising moiety, wherein the proportion of said moieties and active agent provide pH control such that (i) when 400mg of said composition is added to 20 ml of 0.033 normal hydrochloric acid and at a temperature of 37 +/- 3°C, the pH reaches a value in the range 4 to 8, and (ii) when 400mg of said composition is added to 20 ml of deionised water at pH 7 and at a temperature of 37 +/- 3°C, the pH reaches a value less than 8.5.