Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF TREATMENT OF GASTROINTESTINAL DISEASE AND
POLYMERIC COMPOSITION FOR USE THEREIN
FIELD OF THE INVENTION
The present invention relates to treatment or prophylaxis of gastrointestinal
disease and promotion of animal growth and to antimicrobial compositions for
use in such treatments.
BACKGROUND ART
Antimicrobials are compounds which kill microorganisms, such as bacteria.
Antibiotics are a subset of antimicrobials that are (usually) derived from
other
microorganisms and work by interfering with specific mechanisms within the
target microorganism. Antibiotics were first used in the 1940s and 1950s and
their use has increased ever since. The development of antibiotic resistance
has become a serious and potentially life threatening event worldwide. Some
strains of Staphylococcus have shown resistance to almost all antibiotics and
have had fatal infection occurring in hospitals. Other drug resistant
organisms
include pneumococci that cause pneumonia and cryptosporidium and E.coli
which cause diarrhoea.
The use of antibiotics in animal feed is widely considered to be responsible
for
the accelerated development of resistance and as a result many countries
control their use. This has lead to problems in animal farming resulting in
difficulty in controlling disease and obtaining optimum growth rates. This is
a
particular problem in farming of pigs and poultry. For example,
gastrointestinal
diseases such as colibacillosis in pigs and coccidiosis in poultry can have a
devastating effect.
Melrose et al in (International Patent Publication No. 96/38186) were the
first to
describe the preparation of acrolein polymers for use in treatment of
gastrointestinal disease.
These polymers have a repeating unit of formula I
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2
H
~ HZ~c/ ~I)
OH
or this unit in its hydrated, hemi-acetal or acetal form, represented by the
formulae:
CH2
~
CH
i H (a)
H ~
RO OR
CH2
`
\ CH \CH,-' CH2
(b)
CH IH
/ \ / OR
RO O
CH2 CH2 O CH~
CH CH CH
(c)
I I L!H
CH CH RO \ O \ O n `OR
CH2 \
H
CH
RH 0 (d)
RO /
~CH
I
CH
CH 2
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3
CH=CH2
I (e)
CHOO
~o~
CH2 (f)
I H2 CH
wherein R is hydrogen and n is an integer of one or more, have been
demonstrated previously. Prior to this the Biocidal properties of acrolein
polymers in antiseptic applications was described by Melrose et al in
International Application No. W088/04671. German Patent Application
P4404404 and equivalents such as EP667358 and AU 11686/95 (now lapsed)
discloses a process in which acrolein is polymerised in an aqueous sodium
hydroxide medium. The disclosure states that the resulting ' polyacrolein is
soluble in polyhydric alcohol at 40 to 50 C to form a solution of the
polyacrolein
in a polyhydric alcohol. As explained below the assignee of this German
application subsequently found such polymers to be problematic and have low
solubility in aqueous media.
European publication No. 792895 Werle et al. (corresponding to US 6060571)
relates to acrolein releasing polymers prepared by copolymerisation of
acrolein
monomer and a polyhydric alcohol. Werle et al. observes that the polyacroleins
20' described in German Application No. P4404404 are problematic in that the
yield
is less than desired and the polymers are virtually insoluble in water.
European
Application 792895 teaches that these problems are overcome by forming an
acrolein releasing polymer by copolymerisation of acrolein monomer and a
polyhydric alcohol monomer. The proposed structure of the copolymer is as
follows:
O O O~
,,,,,,,/ ,.,,,,,,,, O
n y
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4
While free acrolein acts as an antimicrobial it is irritating to the eyes,
lungs,
tissues and skin. There is a need in a range of applications particularly in
gastrointestinal treatments for antimicrobials which are stable, highly water
soluble, and safe to use. There is a further need for an effective
antimicrobial
for treatment or prevention of gastrointestinal disease, which can reduce the
pressure for development of resistance in antibiotics.
The discussion of the background to the invention herein is included to
explain
the context of the invention. This is not to be taken as an admission that any
of
the material referred to was published, known or part of the common general
knowledge as at the priority date of any of the claims.
Summary of the invention
We have now found the activity and stability of poly(2-propenal, 2-propenoic
acid) polymers in treatment or prophylaxis of gastrointestinal disease is
substantially increased if they are reacted with an alcohol or polyol to form
protected carbonyl groups such as acetal and/or hemiacetal derivatives.
Surprisingly we have found that the activity of the derivative in treatment or
prophylaxis of gastrointestinal disease is substantially increased
notwithstanding that the free acrolein content may be extremely low or
negligible in the polymer. The solubility of the polymer in water is also very
high.
The invention provides a method of treatment or prophylaxis of
gastrointestinal
disease in an animal (including humans) comprising administering to the animal
an effective amount of a derivative of poly(2-propenal, 2-propanoic acid)
formed
by reaction between poly(2-propenal, 2-propenoic acid) and an organic
compound containing one or more hydroxyl groups such as an alcohol
preferably selected from alkanols, phenols, polyols and mixtures thereof, to
form protected carbonyl groups.
In a further aspect the invention provides an antimicrobial for treatment of
gastrointestinal disease comprising a derivative of poly(2-propenal, 2-
propenoic
acid) formed by reaction between poly(2-propenal, 2-propenoic acid) and an
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organic compound containing one or more hydroxyl groups such as an alcohol
preferably selected from alkanols, phenols, polyols and mixtures thereof, to
form protected carbonyl groups.
5 The term polyol as used herein means a molecule containing at least two
hydroxyl groups.
The derivatives formed are typically selected from hemiacetal and acetal
derivatives. Without wishing to be bound by theory we believe that the
reaction
of the poly(2-propenal, 2-propenoic acid) with the alcohol forms hemiacetal
and/or acetal groups from at least a proportion of the pendent aldehyde groups
thereby stabilising the carbonyl groups of the polymers against alkaline
degradation by the Cannizzaro reaction. The formation of acetal groups has
been found to significantly reduce or eliminate the release of free acrolein
while
surprisingly increasing the activity of the resulting derivative.
In yet another embodiment the invention provides the use of the above
described antimicrobial for preparation of a medicament for treatment or
prophylaxis of gastrointestinal disease.
Throughout the description and claims of this specification, the word
"comprise"
and variations of the word such as "comprising" and "comprises", is not
intended to exclude other additives or components or integers.
Detailed Description Of The Invention
The antimicrobial of the invention may be prepared by heating poly(2-propenal,
2-propenoic acid) in the presence of the alcohol, preferably a polyol such as
polyethylene glycol. Water is invariably present in the alcohols and it will
be
understood that the presence of at least some water assists in the
nucleophilic
reaction resulting in hemiacetal or acetal formation.
The solution is generally heated at a temperature in the range of from 40 C to
150 C, more preferably 40 to 115 C and most preferably 70 to 115 C.
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The antimicrobial of the invention is prepared from poly (2-propenal, 2-
propenoic acid) polymers. Such polymers and their preparation are described
in International Patent Publication No. WO 96/38186 (PCT/AU96/00328). The
poly (2-propenal, 2-propenoic acid) polymers are preferably prepared by
polymerisation of acrolein preferably in aqueous solution by anionic
polymerisation, followed by autoxidation. The polymers contain the repeating
unit of formula I and at least one (and typically a mixture) of the hydrated,
hemiacetal and acetal forms.
The hydrated, hemiacetal and acetal forms formed by polymerisation of acrolein
are known to arise from the various carbon-carbon and carbon-oxygen
polymerisation mechanisms of acrolein. For example the hydrated form is
typically the hydrated diol form, the hemiacetal or acetal form may be formed
from the condensation of the diol form with the aldehyde or diol form, the
tetrahydropyran or fused tetrahydropyran form may be formed from
condensation of. the diol form and the aidol-Michaei self condensation form.
Typical examples of these forms are shown in formula (a) to (f) balow:
H
I
CH2 C
O H
(j)
CH2
i (a)
/CH`
RO RO
CH2
CH CH,--' CH2
I I (b)
CH CH
\ OR
RO/ \O/
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CH2 CH2 CH2,,,
CH CH CH
(c)
CH CH ~CH
RO O \ O n \OR
CH2
CH
RO \ (d)
O
RO /
~CH
I
CH
CH 2
CH=CH2
I (e)
/
\p~CH O
~O\
CH2 (f)
I H2 CH
wherein R is hydrogen and n is an integer of one or more. The proportion of
repeating unit of formula I is typically less than 20% and frequently from 5
to
15%. Notwithstanding the relatively low proportion of these units we have
found
that they have a significant effect on the stability of the polymer.
The poly(2-propenal, 2-propenoic acid) will generally contain no more than 10%
on a molar basis of monomer units from monomers other than acrolein and is
most preferably an acrolein homopolymer (before autoxidation). Where used
other monomers may be selected from the group consisting of acrylic acid and
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8
vinyl pyrrolidone. The 2-propenoic acid groups are typically present in an
amount of from 0.1 to 5 moles of carboxyl groups per kilogram. The poly(2-
propenal, 2-propenoic acid) polymers typically have a number average
molecular weight of over 1000 and most preferably over 2000. Typically the
molecular weight is less than 10,000.
The antimicrobial of the invention is a derivative of poly(2-propenal, 2-
propenoic
acid) prepared by reaction with an alcohol or phenol to form protected
carbonyl
groups. The protected carbonyl groups are formed from the 2-propenal groups,
which react with the alcohol to form hemiacetal and acetal groups. The alcohol
is preferably a polyol by which is meant that it preferably contains at least
two
hydroxyl groups. Alkanols such as C, to CIo may be used. Where the alcohol
is a polyol the reaction may produce acetals or hemiacetals formed by reaction
of one or more than one alcohol group. Furthermore when two alcohol groups
react it is possible for them to react at the same carbonyl or different
carbonyl
groups within the polymer.
Referring to the above formula I and hemiacetal and acetal forms the invention
produces derivatives in which there are fewer units of formula I and forms a
group wherein one or more groups R are derived from an alcohol, or when the
alcohol is a polyol, more than two groups R may together form a bridging group
such as a cyclic acetal group.
The propensity for polyols to give rise to internal cyclic groups will depend
on
the spacing and configuration of the polyol. The preferred alcohols are
polyalkylene glycols and more preferred alcohols are polyethylene glycols.
The molecular weight of the polyalkylene glycols is preferably from 200 to
2000
and more preferably from 200 to 1000.
Preferably, the alcohol such as polyethylene glycol is present during the
preparation of the antimicrobial polymers in an amount of between 50 and 99%
by weight. Relatively dilute compositions of the acrolein polymer are
particularly
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preferred where the alcohol is a polyol as the incidence of intermolecular
cross-
linking is reduced by dilution.
More preferably, polyethylene glycol is present during preparation of the
polymers in the amount of between 64 and 95% by weight.
Base or alkali is preferably added to the polymers followed by a drift to
acidic
pH before and%r during heating, as neutralization of the acid groups of the
polymer occurs, thereby enhancing the antimicrobial activity of the polymers.
Preferably, the addition of the base or alkali initially brings the pH of the
poly(2-
propenal, 2-propenoic acid) polymers to between 7 and 9. Still more
preferably,
the initial pH on addition of the base is about 8. The base is preferably an
alkali
metal hydroxide, carbonate, bicarbonate or mixture thereof.
In a still further form of the invention, the release of free acrolein monomer
is
inhibited, from continuous release, whereby the polymers are less likely to
present a source of tissue or dermal irritation.
We have found that the antimicrobial of the invention has significantly
improved
activity in controlling gastrointestinal disease when compared with the poly(2-
propenal,2-propenoic) from which it is prepared. The superactivated derivative
of the present invention may be used to treat a wide range of animals
(including
humans) and a wide range of microbial infections.
The antimicrobial of the invention may be used in treatment of
gastrointestinal
disease in humans, however it is particularly preferred that it be used in
treatment of other animals particularly animals selected from the group
consisting of dogs, pigs, sheep, horses, goats, cattle, cats,. poultry, ducks,
turkeys and quail.
The antimicrobial of the invention may be formulated for oral or rectal
administration. Rectal administration may be particularly useful in ruminant
animals. Oral formulations for ruminant animals may also be prepared using
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enteric coatings to provide optimal activity in the later part of the
gastrointestinal
tract.
The antimicrobial of the invention is particularly useful in treatment and
5 prophylaxis of gastrointestinal ulcers, diarrhoea and gastrointestinal
cancers.
The antimicrobial of the invention may also be used to improve the rate of
weight gain in farm animals by improving the feed to weight conversion in
animals.
10 We have found that the antimicrobial of the invention may be used as a
growth
promotant and that the polymer may be used in place of the presently used
antibiotics. Drug resistance in pathogenic bacteria is a problem of major
clinical
importance in human medicine. This problem is exacerbated by the use of
important antibiotics in animal feed to provide weight gain in farm animals
particularly pouitry and pigs. Indeed, in some European countries the use of
conventional antibiotics in animal feeds has been banned. The antimicrobial of
the invention may be used in treatment of animals to significantly extend the
useful life of conventional antibiotics in human treatment.
We have found the antimicrobial of the invention to be effective against a
wide
range of microbes including protozoa, yeasts, Gram positive bacteria and Gram
negative bacteria. The polymers of the invention contain multiple structures
of
diverse configurations and can find. a fit with the proteins found in the cell
wall of
target organisms, this speeds up the inactivation of the protein and the
destruction of the cell. Of the Gram negative bacteria the antimicrobial of
the
invention has been found to be particularly useful in providing broad spectrum
activity against coliforms or Enterobacteria. It is particularly useful in
treatment
of gastrointestinal diseases resulting from infection by E. coli such as
enterotoxigenic E. coli and P-haemolytic E. coli. Colibacillosis is a
devastating
disease in the pig-rearing industry. The disease is generally associated with
proliferation of P-haemolytic E. coli. in the small intestine after weaning
and
gives rise to high mortality rates and.morbidity rates in young weaner
piglets.
Infected-weaner piglets fail to make normal weight gains.
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Coccidiosis is a protozoal disease of animals particularly poultry and if left
uncontrolled has a devastating effect. We have found that the antimicrobial of
the invention may be used in the treatment or prevention of coccidiosis in
birds
particularly in poultry. In chickens typical clinical signs of coccidiosis
include
lack of thriving, rapid loss of weight, diarrhoea and dysentery. The most
serious
effects take place in the intestine where the protozoa tend to invade the
mucosa
and cause epithelial damage, lesions and haemorrhage. Vaccines have been
used in an attempt to prevent coccidiosis but have side effects including the
tendency to reduce weight and feed efficiency.
The antimicrobial of the invention may be used in combination with other drugs
known to have activity against coccidiosis. Such drugs include nitro-
carbanilide,
quinoline, pyridon, guanidine, quinoxaline, toltrazural, toluamide,
potentiated
sulfa drugs and ionophore with carbanilide.
Clostridia are Gram positive bacteria responsible for serious disease in a
range
of animals. For example, necrotic enteritis is a disease known to affect
commercial poultry. Clostridia bacterial produce exotoxins which are some of
the most toxic of all known toxins. Necrotic enteritis particularly effects
broilers
of between 14 and 42 days of age. The condition causes pronounced apathy,
diarrhoea and can cause death within hours.
Upper gastrointestinal disease including chronic gastritis, gastric ulcer and
duodenal ulcer are significant human health problems. Helicobacter is
understood to be responsible for the development of ulcers and the
development of gastrointestinal cancers particularly adenocarcinoma of the
stomach. We have found the antimicrobial of the invention to be particularly
useful against Helicobacter including H. pylori in gastrointestinal disease in
animals, particularly humans.
The infection of the stomach with Helicobacterpylori is one of the most
frequent
infectious diseases in the world. About 50% of the population are infected
with
H. pylori. In developing countries it has been estimated that more than 80% of
the population is already infected with H.pylori during childhood.
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Helicobacter pylori is a Gram-negative, microaerophilic, spiral-shaped,
bacilli
that is motile by way of flagella at one end of the cell. The standard
treatments
of H. pylori infections are the so-called triple antibiotic therapies all of
which
include either metronidazole or clarithromycin. Unfortunately strains of H.
pylori
have emerged, which are resistant to both these antibiotics.
H. pylori live in the stomach at the interface between the surface of gastric
epithelial cells and the overlying mucus gel layer. H. pylori can additionally
be
found on top of the gastric epithelium in the duodenum and oesophagus. Other
animal species have their own unique Helicobacter species present in their
gastrointestinal tracts, which have similar properties to H. pylori. In
addition to
its association with gastrointestinal cancers; H. pylori has been directly
linked in
humans to gastritis and peptic ulcer formation.
Helicobacter species in general, and H. pylori in particular, survive the
extreme
conditions of the stomach by secreting urease, which hydrolyses urea to give
ammonia and bicarbonate ion, thus raising the pH of the immediate
surroundings of the bacilli. This local alteration of the conditions protects
the
bacteria from the bactericidal effect of the gastric acid. The preferred
position
underneath the stomach's protective mucus layer is also a survival advantage
and its motility allows it to burrow through the layer to attain this
position.
The epithelial cells lining the stomach are naturally difficult to penetrate;
this is
part of their function to protect the rest of the body from gastric acid and
digestive juices. This difficulty in penetration also makes it difficult for
the body's
natural defences to pass through the stomach wall and reach the site of H.
pylori infection. This has two consequences; the body sends more nutrients to
the site to aid the white cells, T-cells, and other defence mechanisms,
concomitantly supplying the bacilli; and the defence cells eventually die,
releasing their cargo of superoxide ion and other lethal chemicals, damaging
the surrounding epithelial cells.
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It is apparently this activity that leads to gastritis, which can easily
progress to
peptic ulcers. Should the insult continue, the possibility of the appearance
of
gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT)
lymphoma is greatly increased. Gastric adenocarcinoma begins in the mucosa
and the first stage of development, intestinal metaplasia, is a response of
the
stomach to rid itself of the H. pylori infection. Also, studies performed in
UCL
Medical School have shown that the MALT lymphoma requires help from H.
pylori specific T-cells to grow. Treatment of H. pylori infection has been
shown
to be extremely effective in curing MALT lymphoma. The World Health
Organisation has labelled the pathogen a Group I carcinogen.
Accordingly the present invention also provides for a method for the treatment
or prophylaxis of diseases of the gastrointestinal tract caused by
Helicobacter
infection comprising the gastrointestinal administration of a therapeutic
amount
of an agent wherein the agent comprises a derivative of poly(2-propenal, 2-
propenoic acid) formed by the reaction between a poly(2-propenal, 2-propenoic
acid) and an organic compound containing hydroxyl groups selected from
alkanols, phenols, polyols and mixtures thereof, to form protected carbonyl
groups. The term polyol where used herein means a compound containing at
least two hydroxyl groups. The derivatives formed are typically selected from
hemiacetal and acetal derivatives
Hence, the use of the method of the present invention provides an alternative
to
the use of surgery, radiation therapy or traditional chemotherapy in the
treatment of gastrointestinal cancers.
The invention further provides a method of treatment for gastrointestinal
infection by a species of Helicobacter bacteria such as gastritis, gastric
ulcer,
duodenal ulcer, gastric malignant lymphoma or gastric cancer, comprising the
gastrointestinal administration of a therapeutic amount of an agent wherein
the
agent comprises a derivative of poly(2-propenal, 2-propenoic acid) formed by
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the reaction between a poly(2-propenal, 2-propenoic acid) and an organic
compound containing one or more hydroxyl groups to form protected carbonyl
groups.
The present invention provides an alternative to standard treatments of
Helicobacter infections, which, in general, comprise the so-called triple
antibiotic
therapies all of which include either metronidazole or clarithromycin. Strains
of
H. pylori have emerged which are resistant to both these antibiotics and we
have shown that the method of the present invention can effectively treat such
antibiotic resistant bacteria.
The agent which is a product of the reaction between poly(2-propenal, 2-
propenoic acid) and an organic compound containing one or more hydroxyl
groups has been shown to be more effective in the treatment of Helicobacter
infections than the corresponding non-superactivated poly(2-propenal, 2-
propenoic acid) groups.
The invention further provides the use of a derivative of poly(2-propenal, 2-
propenoic acid) in manufacture of a medicament for treatment or prophylaxis of
a disease caused by Helicobacter infection.
The method of the present invention may be used in treatment or prophylaxis of
gastrointestinal cancers. These may include, for example, cancers of the
oesophagus, stomach, intestine and colon. An example of such a type of
cancer is the human colon cancer cell line HT-29.
When the antimicrobial of the invention is incorporated into an animal feed or
water this may be done in the usual manner. In a preferred embodiment the
antimicrobial of the invention is incorporated in a premix. The premix will
preferably include the antimicrobial, a physiologically acceptable carrier and
optionally a feedstuff. The premix is generally in a relatively concentrated
form
and is adapted to be diluted with other material such as one or more of the
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other carriers, vitamins and mineral supplements and feedstuff to form the
final
animal feed. The premix preferably includes the antimicrobial in a
concentration
in the range of from 0.1 to 70% by weight, preferably 0.5 to 50% by weight.
The
optimum concentration will depend on whether the treatment is preventative,
for
5 control or remedial and whether the antimicrobial of the invention is the
only
active or whether it is used in concomitant therapy with other materials or
antimicrobials.
In a preferred embodiment the concentrated composition of the antimicrobial is
10 in a controlled-release form. The controlled release form will include the
antimicrobial and a polymeric material for providing controlled release of the
antimicrobial from the controlled-release system and is particularly useful in
compositions for addition to solid feed material. As a result of the
controlled
release formulation the release of the antimicrobial may be delayed so as to
15 occur mainly in the duodenum. A controlled release polymer may also
minimise
rejection of the composition due to taste or be used for rectal suppositories.
An antimicrobial composition in accordance with the invention may be in the
form of pellets, pills or like solid composition. The pellets containing the
antimicrobial of the invention may be prepared by the steps of:
(i) dissolving said antimicrobial in an aqueous alkaline or basic
solution;
(ii) neutralising said solution with acid;
(iii) adding to said neutralised solution insoluble, cross-linked,
absorbent polymers of acrylic acid and/or copolymers of
acrylamide and acrylic acid, to form wet swollen pellets; and
(iv) optionally, wholly or partially drying said wet swollen pellets.
The so-formed wet, swollen pellets may be used either wet, partially dried or
wholly dried, as an additive to, for example, animal feed. This system is
further
designed so that the carboxyl-containing groups of the outer polymeric matrix
cause the Subject Polymers to remain essentially contained within the matrix
when in the acidic environment of the stomach. However, in the alkaline
environment of the duodenum, the carboxyl groups of the matrix become
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ionised and mutually-repelling, and the pellet rapidly swells to allow the
Subject
Polymers, aided by repulsion among their own ionic groups, to be excluded by a
diffusion process, approximately matching the speed of passage of feed
through the duodenum.
In this invention, the term, "controlled release system" is used in the same
context as that in, and includes the same range of examples as quoted in
"Controlled Drug Delivery" (Robinson & Lee, 1987). Many other pH-sensitive
controlled-release systems which are known in the art (Robinson and Lee,
1987) may be substituted for the polymer of acrylic acid or copolymer of
acrylamide and acrylic acid. For example, soluble and anionic, or insoluble
cross-linked and anionic, cellulosic systems; or soluble and anionic, or
insoluble
,
cross-linked and anionic polymers derived from any generic acrylic acid
polymer
and/or its derivatives. Such cross-linked and insoluble polymers are preferred
since they swell and also are less likely to be metabolised.
It is preferred that the controlled release system comprises a pH-sensitive,
cross-linked, water-absorbent pellet, which when wet is a gel.
The invention also provides an animal feed composition comprising the
antimicrobial of the invention and a feedstuff. The antimicrobial is
preferably
present in an amount of from 0.0001 to 25% of the total feed composition and
preferably from 0.0001 to 5% of the total feed composition.
In another preferred embodiment, the antimicrobial of the invention may be
formulated for addition to the drinking water of animals.
The antimicrobial of the invention is preferably administered in amounts of
from
0.05 to 5000 mg/kg of bodyweight/day more preferably from 0.05 to 50
mg/kg/day.
Examples of suitable inert carriers for use in compositions for administration
of
the antimicrobial of the invention include water, olive oil, peanut oil,
sesame oil,
sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin,
ethylene
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glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol,
glycerol, fatty alcohols, triglycerides, polyvinyl alcohol, partially
hydrolysed
polyvinyl acetate and mixtures thereof.
Solid forms for oral or rectal administration may contain pharmaceutically or
veterinarally acceptable binders, sweeteners, disintegrating agents, diluents,
flavourings, coating agents, preservatives, lubricants and/or time delay
agents.
Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth,
sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable
sweeteners include sucrose, lactose, glucose or flavonoide glycosides such as
neohesperidine dihydrochalcone. Suitable- disintegrating agents include corn
starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic
acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose,
kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate.
Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry,
orange or raspberry flavourings. Suitable coating agents include polymers or
copolymers of acrylic acid and/or methacrylic acid and/or their esters, and/or
their amides, waxes, fatty alcohols, zein, shellac or gluten. Suitable
preservatives include sodium benzoate, vitamin E, a-tocopherol, ascorbic acid,
methyl parabens, propyl parabens or sodium bisulphite. Suitable lubricants
include magnesium stearate, stearic acid, sodium oleate, sodium chloride or
talc. Suitable time delay agents include glyceryl monostearate or glyceryl
distearate.
Suspensions for oral or rectal administration may further comprise dispersing
agents and/or suspending agents. Suitable suspending agents include sodium
carboxyimethylcellulose, methylcellulose, hydroxypropylmethylcellulose, poly-
vinyl-pyrrolidone, sodium alginate or cetyl alcohol. Suitable dispersing
agents
include lecithin, polyoxyethylene esters or fatty acids such as stearic acid,
polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate,
polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the
like.
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The composition of the antimicrobial may further comprise one or more
emulsifying agents. Suitable emulsifying agents include dispersing agents as
exemplified above or natural gums such as gum acacia or gum tragacanth.
Compositions for administration in the method of the invention may be prepared
by means known in the art for the preparation of compositions (such as in the
art of veterinary and pharmaceutical compositions) including blending,
grinding,
homogenising, suspending, dissolving, emulsifying, dispersing and where
appropriate, mixing of the Subject Polymers together with selected excipients,
diluents, carriers and adjuvants.
For oral administration, the pharmaceutical or veterinary composition may be
in
the form of tablets, lozenges, pills, troches, capsules, elixirs, powders,
including
lyophilised powders, solutions, granules, suspensions, emulsions, syrups and
tinctures. Slow-release, or delayed-release, forms may also be prepared, for
example in the form of coated particles, multi-layer tablets or microgranules.
It is generally preferred that poly(2-propenal, 2-propenoic acid) is prepared
from
poly(2-propenal) by oxidation of the solid in air. The poly(2-propenal)
polymer
may be initially heated, predominantly in the dry state, to between 80 and
110 C. More preferably, the polymer is initially heated to about 85 C. The
poly(2-propenal, 2-propenoic acid) is preferably heated in the alcohol for a
period in the range of from 1 hour to 1400 hours and more preferably from I
hour to 60 hours.
In accordance with the present invention there is further provided a
preservative
compound or composition comprising the antimicrobial of the invention.
In accordance with the present invention there is yet still further provided a
disinfectant or antiseptic compound or composition comprising the
antimicrobial
of the invention.
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In accordance with a further aspect of the invention we provide a composition
for treatment of gastrointestinal disease comprising an antimicrobial polymer
as
hereinbefore described and a further chemotherapeutic agent wherein the
further chemotherapeutic agent is adsorbed onto the antimicrobial.
The adsorption will typically reduce membrane penetration of the further
chemotherapeutic. The suitable chemotherapeutics for use in this embodiment
are those which exhibit a significant reduction in membrane penetration when
admixed with the polymeric antimicrobial. Preferably the penetration is
inhibited
by a factor of at least 50%.
The useful chemotherapeutic agents for use in this aspect of the invention
include antibiotics for treatment of gastrointestinal disease and anticancer
agents for treatment of gastrointestinal cancers.
The use of chemotherapeutics in combination with the polymeric antimicrobial
reduces membrane penetration of the chemotherapeutic thereby reducing
systemic side effects and providing more targeted therapy. In many cases
odour is also reduced.
Examples of chemotherapeutics for treatment of gastrointestinal disease
include antibiotics and anticancer agents.
Examples of antibiotics which may be used in combination with the
antimicrobial
polymer include tetracyclines, penicillins, aminoglycosides, sulfa drugs,
cephalosporins and nitrofurans.
The antibiotics may be conventional antibiotics used to treat infections of
the
gastrointestinal tract.
Examples of anticancer agents which may be used in combination with the
polymeric antimicrobial of the invention are alkylating agents,
antimetabolites,
anticancer antibiotics, plant alkaloids, hormones and other anticancer agents,
particularly anticancer agents containing carbon, hydrogen and oxygen only.
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The compositions of the invention may also comprise one or more further
antimicrobials such as those selected from the group of a phenol (preferably
in
an amount of 0.1 to 10% by weight), an isothiazolinone (preferably in an
amount
5 of fromk 0.001 to 1%), an alkyl parabens (preferably in an amount of from
0.02
to 2%) and a lower alcohol (preferably in an amount of from 20 to 99%) wherein
the amounts are on the basis of weight by weight of the composition.
The derivative of poly(2-propenal, 2-propenoic acid used in the method of the
10 invention has been found to have significantly increased stability compared
with
of poly(2-propenal, 2-propenoic acid) polymers. Since the prior art recorded
some instability of poly(2-propenal, 2-propenoic acid), as evidenced by loss
of
antimicrobial activity of its compositions, we conducted "accelerated ageing"
at
elevated temperature, ie. at 40 C. However, to our greatest surprise, the
15 elevated temperature of "ageing" poly(2-propenal, 2-propenoic acid) in
aqueous
or in aqueous-polyethylene glycol solutions at 40 C, not only slowed the
decrease in antimicrobial activity-but in fact, actually increased
antimicrobial
activity of the poly(2-propenal, 2-propenoic acid), see Example 2(a) and (b).
This finding is totally contradictory and unexpected in view of the prior art
which
20 predicts that the rise in temperature should lead to "accelerated ageing",
ie.
accelerated loss of antimicrobial activity.
Herein, the process of providing increased antimicrobial activity by the
formation
of a new configuration of the subject polymers including poly(2-propenal, 2-
propenoic acid), is referred to as "super-activation" and the polymers
referred to
as "super-activated polymers".
Even more surprising, in view of prior art, the inventors have found
superactivation in aqueous polyethylene glycol solution is promoted by basic,
followed by acidic conditions. Also, super-activation is promoted by heat and
moisture.
Super-activation is facilitated by the presence of polyethylene glycols or
polyols
or alkanols, we believe, since the presence of the polyethylene glycol or
polyol
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or alkanol protects, and stabilises the carbonyl groups of the polymers, by
formation of acetals, from alkaline degradation by the Cannizzaro reaction.
An added advantage of super-activation is that it reduces or eliminates,
contaminant acrolein which is a source of tissue and dermal irritation.
It is emphasised that super-activation is quite distinct and additional to any
increase of antimicrobial activity which may result, merely from more polymer
being available in any aqueous test-medium as the result of increased
hydrophilicity of the polymer such as was demonstrated in lapsed Australian
Patent Application AU-A-11686/95 (hereinafter "11686/95"). The inventors have
repeated exactly the method described in 11686/95 and then, following, found
that subsequent super-activation of the partially soluble polymer
demonstratively gave rise to additional, substantial antimicrobial activity.
It
should be noted that even super-activation did not render the polymer from
11686/95 completely soluble-in contrast to super-activation beginning with
polymer firstly heated between to 80-85 C.
The optimum time to achieve super-activation of solutions of poly(2-propenal,
2-
propenoic acid) depends inversely upon the temperature. It will be apparent
that even ageing at room temperature may be used for superactivation,
especially when facilitated in the presence of hydroxylic solvent and/or base
followed by acidity, but obviously, this may be impractical due to the longer
time
periods required.
The inventors have found polymers super-activated as described herein,
suitable for gastrointestinal therapy, preservatives in water-based products
or
processes, and active ingredients in disinfectants or antiseptics having the
advantage of enhanced antimicrobial activity. Furthermore, the inventors found
that the antimicrobial activity of such disinfectants or antiseptics was
increased
by increase in their pH, for example above pH 6.
A common feature of the invention is the attachment of a group capable of
hydrophobic interaction, to the antimicrobial of the invention, by way of
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hemiacetal/acetal formation or by way of adsorption, in order to enhance
antimicrobial activity.
The invention will now be described with reference to several Examples, which
should not be construed as limiting the scope thereof.
BIOCIDAL TEST
Dissolve sample with 1% by weight aqueous sodium bicarbonate to obtain the
required concentration (unless specified to the contrary, 0.125% by weight of
polymer). Weigh 19.9g of diluted sample into a sterile jar and inoculate with
0.1
mL of 107-10$ cfu of Ps.aeruginosa and mix. At specified time-intervals,
transfer
1 mL of inoculated sample to 9 mL of Letheen broth and vortex. Plate out
serial
1 in 10 dilutions. Pour with trypticase soy agar. Incubate 3 days at 37 C.
Example 1
The example describes a method of preparing poly(2-propenal, 2-propenoic
acid) by oxidation of a solid acrolein polymer in air. This poly(2-propenal, 2-
propenoic acid) is the preferred method of preparing a starting material for
use
in the method of the invention. Water (720 mL at ambient temperature, about
20 C) and acrolein (60g ; freshly distilled, plus hydroquinone added to 0.25%
w/w) were placed in an open beaker, within a fume cupboard, and very
vigorously stirred, mechanically. Then, 0.2 M aqueous sodium hydroxide (21.4
mL) was added to bring the pH to 10.5-11Ø
The solution immediately turned a yellow typical of the hydroquinone anion and
within a minute, the colour had disappeared and the clear solution became
milky.
About 1 minute later, precipitation of a white flocculent polymer began, and
appeared complete within 15-30 minutes. The precipitate was filtered and
washed with water (250 mL), dried at room temperature upon filter papers for 2
days (yield 25g), then spread as a thin layer in glass petri dishes and heated
at
C/8 hours. This heating was continued at the following schedules : 50 C/15
hours ; 65 C/4 hours ; 75 C/18 hours ; 84 C/24 hours.
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It is envisaged that this method may be scaled-up,to include, eg the stepwise
addition of acrolein, in a closed vessel, and followed by more rapid drying
(compare example 10).
Typically, a solution of the resulting poly(2-propenal, 2-propenoic acid) was
prepared by adding 2g of the subject polymer, with stirring over 15-30
minutes,
to a 1% w/w aqueous sodium carbonate solution (100 mL), and then diluted as
required. Such solutions were perfectly clear-in contrast to attempted
dissolutions, using alternatively, polymer derived from Example 5 of 11686/95.
Example 2
This example describes acetal formation from poly(2-propenal, 2-propenoic
acid).
(a) 5g of poly(2-propenal, 2-propenoic acid) was dissolved in 64g
polyethylene glycol ("PEG") 200 and combined with 31g of a 0.71 % w/w
solution of sodium carbonate. A portion of the solution (apparent pH=5.8) was
retained at room temperature while
(b) the remainder of the sample from part (a) was heated at 60 C for periods
of 12 or 25 days.
Samples from (a) and (b) were diluted with 1% w/w sodium bicarbonate and
submitted for biocidal testing at polymer concentrations of 0.125% w/w.
Surprisingly, the samples which had undergone "accelerated ageing" showed
improved antimicrobial activity, as can be seen by reference to Table 1:
Table 1
Cfu/mL * (Pseudomonas aeruginosa)
Sample 0 min 10 min 15 min 30 min 60 min
25 days at room
temperature 7.8x106 4.1 x 106 6.1 x105 9.8x104 <10
12 days at 60 C 7.7x10 1.4x10 9.8x10 <10 <10
25 days at 60 C 1.OX10 1.3X10b 6.6X10 <10 <10
* Colony forming units/mL
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1 g poly(2-propenal, 2-propenoic acid) was dissolved in 200 mL of 0.1% w/w
Na2CO3 and allowed to stand overnight. Sodium lauryl sulphate was introduced
at a level of 0. 05% w/w and the solution was acidified with HCI to pH 5.9.
Portions were stored at both room temperature and 60 C. Biocidal Tests were
carried out on 0.125% w/w polymer solutions, with 1% w/w NaHCO3 used as
the diluent. The "aged" sample showed a surprising improvement in
performance, as can be seen by reference to Table 2:
Table 2
Cfu/mL * (Pseudomonas aeruginosa)
Sample 0 min 10 min 15 min 30 min
days at room
temperature (RT) 9.0x106 5.1x 105 6.8x102 <10
7 days at 60 C +
13 days at RT 9.0x106 1.2x102 <10 <10
* Colony forming units/mL
(c) A 5% w/w solution of super-activated polymer was prepared as in
example (2a) but replacing PEG200 with PEG1000. A portion of this solution
15 was treated with conc. NaOH to pH 8.1. Samples were heated at 60 C and
submitted for biocidal testing. The sample exposed to more basic conditions,
unexpectedly gave superior biocidal performance, as can be seen by reference
to Table 3:
20 Table 3
Cfu/mL * (Pseudomonas aeruginosa)
Sample 0 min 5 min 10 min 15 min 30 min
pH 5.8, 12 days 60 C 3.8x10 2.7x10 1.5xlOb 3.3x10 <10
pH 8.1, 7 days 60 C 90X10 - 10 <10 <10
pH 8.1, 17 days 60 C 8.3x10 3.3x10 1.3x10 <10 <10
* Colony forming units/mL
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Example 3
This example examines the product produced by reaction of the poly(2-
propenal, 2-propenoic acid) with polyethylene glycol.
5 The presence of acetals in the polymers of Example 2(b) may be determined by
examining the solid residue left after dialysis and concentration of the
polymer
solution using proton (1H) and carbon (13C) NMR spectroscopy. Dialysis
removes all material of molecular weight less than a 1000. Table I provides
proton (1H) and carbon (13C) NMR data. As can be seen from Table 1, Nuclear
10 Magnetic Resonance spectroscopy of the residue showed peaks at 8 3.58 and
,
3.56 in the 'H Nuclear Magnetic Resonance spectrum and 8 71.62, 69.48 and
60.25 in the 13C Nuclear Magnetic Resonance' spectrum. These peaks are
indicative of the attachment of polyethylene glycol units as acetals.
15 Table 4
Data from the 600 MHz 1H- and 125MHz 13C-Nuclear Magnetic Resonance
Spectra in D20 with 1% w/w Na2C03 of the solid residue of Superactivated
polymer after dialysis and concentration.
Region carbonyl alkenyl methine methylen acetals alkoxy- yls PEG Alkyls
'H 8 9.5-8.5 7.3-6.0 6.0-5.5 5.5-5.0 5.0-4.0 4.0-3.0 3.58, 3.56 3.0-0.5
71.62,
13C S 185-175 140-105 105-95 80-55 69.48, 55-15
60.25
Example 4
(a) 5% w/w solutions of polymers of a range of degrees of super-activation,
apparent pH 5.7, were prepared similarity to Example 2(a), but varying the
percentage of PEG 200.
Samples were heated at 60 C and stabilities were monitored over time.
Physical stability was considered to have failed with the occurrence of
precipitation or gelling. UV measurements were made on a 0.01% w/w polymer
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concentration in 1% w/w sodium carbonate solution. A decrease of the ratio of
absorption at 268 nm : 230 nm is considered synonymous with a decrease in
chemical stability. Results are shown in Table 5:
Table 5
Composition A B C D
PEG 200 (% by weight) 0 50 64 95
Physical Stability
Time A B C D
4 days 60 C Fail Pass Pass Pass
11 days 60 C Fail Fail Pass Pass
Chemical Stability Ratio = 260-270 peak absorbance
228-235 peak absorbance
Time A B C D
0 days 60 C 1.38 1.41 1.43 1.46
4 days 60 C 0.98 1.04 1.21 1.27
11 days 60 C - 0.97 1.03 1.09
18 days 60 C - 0.89 0.92 1.04
25 days 60 C - - 0.84 1.04
Both physical and UV spectral results demonstrate the positive effect of PEG
on
stability; higher PEG content results in greater physical and chemical
stabilities.
(b) The following solutions A and B were prepared by dissolving 4g of poly(2-
propenal, 2-propenoic acid) in 196g 1% w/w sodium bicarbonate and adjusting
the pH to 7 (A) and 5.5 (B) with dilute HCI. Solution C was prepared by
dissolving 50 g of poly(2-propenal, 2-propenoic acid) in PEG 200 (640 g) at 65
to -70 C. Then a solution of 4 g sodium carbonate in water (306 g) was added,
the apparent pH being 7, and then 5.5 at the end of the treatment period of 31
days.
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All samples were stored at 400 C. At various time intervals samples containing
equivalent to 0.125% w/w polymer were submitted for biocidal testing. Results
are shown in Table 6:
Table 6
Time (days) Time for complete kill (minutes) <10 cfu/mL
at 40 C (Pseudomonas aeruginosa)
Solution A Solution B Solution C
0 30 30 30
7 30 60 -
14 - - 10
31 60 60 10
Example 5
1 g of poly(2-propenal, 2-propenoic acid) was heated in either a dry or a
humid,
enclosed chamber, both at 60 C, for 3 days. Solutions of the dry polymer and
the humidified polymer, respectively were prepared at 0.125% w/w (with
correction for moisture content) and submitted for evaluation by the Biocidal
Test:
Table 7
Cfu/mL * (Pseudomonas aeruginosa)
0 min 5 min 10 min 15 min 30 min 60 min
4
1.2x10 <10
Polymer (dry) 4.9x10 - 7.6x10 5.9x10
Polymer
(humidified) 1.1x10' 6x106 3.4x103 3.7x103 <10 -
* Colony forming units/mL
The polymers exhibited carbonyl and/or carboxyl absorption in the I.R. between
1700 - 1730 cm"1, carbonyl groups (e.g. with Schiffs reagent) and have MW =
ca. 10000 and Mõ = ca.5000; titration shows carboxyl groups ca. 5 mole %.
These parameters are similar (but not the same) as those of poly(2-propenal, 2-
propenoic acid).
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Example 6
In duplicate experiments, a sample of polymer was prepared and then dissolved
in ethane-diol, exactly as described in Example 5 of 11686/95. Half of this
material was further heated at 80 C for 24 hours (following which, solubility
in
aqueous media remained incomplete). The samples were compared for
antimicrobial activity, using the standard Biocidal Test. Both of the samples
treated by heating, ie. super-activation showed a clear enhancement of
antimicrobial activity, as shown in Table 8:
Table 8
Cfu/mL * (Pseudomonas aeruginosa)
Treatment of solution Initial Count 5 min 10 min 15 min 30 min
(1) None 4.6x10 5.7x10 2.9x10 <10 <10
(2) None 4.6x10 4.2x10 1.5x10 10 <10
(1) 24 hours 80 C 4.6x10 3.7x10 <10 <10 <10
(2) 24 hours 80 C 4.6x10 8.Ox10 <10 <10 <10
* Colony forming units/mL
Example 7
50 g of poly(2-propenal, 2-propenoic acid) was dissolved in PEG200 (640 g) at
between 65 to 70 C. Then, an aqueous solution of sodium carbonate (4 g) in
water (306 g) was added. The sample was divided and either stood at room
temperature or heated at 80 C for 24 hours. The acrolein content of the
solution
was determined over time, by reverse phase HPLC and results are shown in
Table 9:
Table 9
Days stored at 20 C Acrolein Content (ppm)
Superactivated Not Superactived
0 274 144
7 - 126
16 34 103
13 80
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Example 8
Solutions of poly(2-propenal, 2-propenoic acid) were prepared as in Example 7
and treated at temperatures of 40, 60, 80, 100 and 115 C for varying time
periods. Samples were subjected to the standard Biocidal Test to confirm the
increased kill rate and results are shown in Table 10:
Table 10
Super-activation Optimum Time Range Total Kill Time
Temperature( C) (Hours) (minutes)
Room Temperature >1400 <10
40 1400 <10
60 120-170 <10
80 16-24 <10
100 4-7 <10
115 1-3 <10
The amount of time required for super-activation is seen to be inversely
proportional to temperature. All solutions of polymers derived from the
superactivation process were completely miscible, in all proportions, with
aqueous solvents.
Example 9
(a) 540 g of poly(2-propenal, 2-propenoic acid) was dissolved in 2304 g
PEG200 at 65 C, prior to mixing with 43.2 g of sodium carbonate in 712 g of
water. Then, the solution was heated to 100 C for 4 hours, and 36g sodium
lauryl sulphate, 7 g ECOTERIC T20 (non-ionic detergent) and 2 g lemon
fragrance were added. The formulation, pH6, was diluted 1:30 with hard water
and challenged against Staphylococcus aureus (a Gram-positive bacterium, of
particular significance regarding infections in hospitals) and Salmonella
choleraesuis (a Gram-negative bacterium, of particular significance regarding
infections in food preparation areas), respectively using the Association of
Agricultural Chemists Official Methods of Analysis (1995) 991.47, 991.48,
(Hard
Surface Carrier Test Method). Results are shown in Table 11:
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Table 11
Micro-organism Positive Tubes Result
S aureus 2/60 Pass
S. choleraesuis 1/60 Pass
Adjustment of this formulation to higher pHs, increases the antimicrobial
activity,
as monitored by the Biocidal Test. Results are shown in Tables 12(a) and
5 12(b):
Table 12(a)
Activity against Staphylococcus aureus
Initial Count, 3 x 106 cfu/mL; polymer 350 ppm.
10 20 30 45 60
pH minutes minutes minutes minutes minutes
cfu/mL cfu/mL cfu/mL cfu/mL cfu/mL
5.6 2.8x10 4.4x10 2.3x10 20 <10
7.2 2.7x10 <10 <10 <10 <10
8.9 3.2x10 <10 <10 <10 <10
10.5 1.1x10 <10 <10 <10 <10
Table 12(b)
Activity against Pseudomonas aeruginosa
Initial Count, 3.7 x 106 cfu/mL; polymer 350 ppm.
10 20 30 45 60
pH minutes minutes minutes minutes minutes
cfu/mL cfu/mL cfu/mL cfu/mL cfu/mL
5.6 2.9x10 8.6x10 02 40 <10
7.2 5.8x10 9.1x10 4.3x10 <10 <10
8.9 9.5x10 8.2x10 4.6x10 <10 <10
10.5 1.1x10 3.0x10 <10 <10 <10
(b) 1200 g of poly(2-propenal, 2-propenoic acid) was dissolved in 7680 g of
PEG200 at 60 C and then 96g Na2CO3 in 3024 g water was added. The
solution was heated at 100 C for 6 hours.
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The formulation was added to the basin of an induced draft cooling tower, to a
concentration of 300ppm (30ppm polymer) 3 times/week. Dosing was carried
out at evening to allow contact times of 8-12 hours before operation
recommenced; residual concentration was expected to be halved every 3-6
hours of operation. Recirculation water had on average, temperature 27 C, pH
8.5, conductivity 3000 S. Microbial counts were determined and compared to
an adjacent, identical, tower which was dosed with a biodispersant, daily.
Results are shown in Table 13:
Table 13
Cfu/mL*
Treatment Time Treated Tower Control Tower
(days)
1 2.4 x 10 1.1 x 10
2 2.0x10 1 x10
3 3.3 x 10 -
4 2.5 x 10 -
14 6.1 x10 2.6x10
5.1 x 104 1.1 x 10
16 104 4.9x10
~ Colony forming units/mL
The data indicate the treatment programme maintained the microbial counts
within the guidelines of AS/NZ Standard 3666.3 (Int):1998 and below that in
the
15 adjacent tower, containing biodispersant (which was found to be unusually
inadequate during the demanding conditions of the very hot, summer period of
the test).
Example 10
10(a) Comparative Example
This example demonstrates a method of preparing an acrolein polymer in which
the method of the invention is not used.
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1.0 0.8% w/w Sodium Hydroxide
Place 9.90 kg of deionised water in a 10L stainless steel vat and add
0.08 kg sodium hydroxide to the water and stir until dissolved.
2.0 Polymerisation
Place 100.1 kg of deionised water in a 200L stainless steel vat and add
4.99 kg of the 0.8% w/w sodium hydroxide solution to the 200L vat.
Equilibrate the solution to 15 - 20 C. Simultaneously add 20 kg acrolein
monomer and the remaining 0.8% w/w sodium hydroxide solution to the
200L vat at a rate over 1 hour such that the pH remains at 10.5 -11.0,
and the temperature does not rise above 30 C. Continue the
polymerisation for a further 90 minutes.
3.0 Washing
Filter/centrifuge the polymerisation mixture and wash the polymer with
deionised water until the pH of the wash water is less than 7Ø The
approximate yield is 8 kg.
4.0 Drying
Dry the polymer in air, then heat in an oven using the following program.
Step Time Temperature
1 2 hrs 25 C
2 1 hr 40 C
3 1 hr 70 C
4 1 hr 75 C
5 2 hrs 85 C
5.0 Dissolution
Place 400L of water in a 500L vat and add 4 kg of sodium carbonate and
stir until dissolved. Slowly add 8 kg of dry, heated polymer and stir for
thirty minutes.
The resulting polymer was found to have an approximate solubility of 90
- 95% w/w in 1% w/w sodium carbonate.
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Example 10(b)
This example describes a method of preparing an acrolein polymer in which the
polymer of comparative example is super activated by the method of the
invention.
1.0 Base Manufacture
Dissolve 0.4 kg sodium carbonate in 30.6 kg water in a suitable container,
and place 64 kg polyethylene glycol 200 into the mixing vessel.
Commence stirring with the mechanical stirrer and heat the PEG200 to
65 t3 C. Add 5 kg of the dry acrolein polymer from Example 10a to the
PEG200 and stir until a uniform mixture is obtained.
NOTE: The solid may not completely dissolve at this stage.
Slowly add the sodium carbonate solution to the glycol mixture at a rate
that ensures the pH of the solution remains in the range 3.5 - 9Ø
Stir the solution for 45 minutes at 65 30 C.
NOTE: pH should be within the range 7-9. Temperature should be within
the range 65 t3 C.
2.0 Superactivation
Cover the mixing vessel and heat to 100 C for four (4) hours. The
resulting polymer was found to have an approximate solubility 99.5 -
100% w/w in water.
Example 11
This example examines the antimicrobial activity of the dry, normally
activated
poly(2-propenal, 2-propenoic acid) polymer of Example 10a and the
antimicrobial activity of the superactivated acetal derivative described in
Example 10b.
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Chickens treated each of the antimicrobials were compared with a control group
according to the following procedure:
In each trial 20 Cob chickens (Line 53), day old were purchased from a
commercial hatchery. They were weighed, sexed and randomly assigned into
adjacent pens in a room of an isolated animal house. There was an even
distribution of male and female chickens. Water and feed were available ad
libitum. The diet was a commercial crumble (Chick Starter, Milne Feeds: 18%
crude protein) with a coccidiostat present (125 ppm Dinitolmide).
Ten chickens were administered the formulation of 0.1% w/w of normally
activated antimicrobial from Example 10a in the water for 14 days through
static
drinkers; dose rate of 30 mg/kg/day. The other ten chickens were the Control
Group.
Both groups of chickens were weighed on days 0, 4, 7, 11 and 14. At the
completion of the trial all chickens were euthanised, and the treated chickens
were autopsied post mortem. A thorough gross examination of the thoracic and
abdominal cavities was performed.
RESULTS:
Table 14a
Weight gained durinqtrial with normally activated antimicrobial of Example 10a
Day Control Group Treatment Group Differences between
(Average Weight in g) (Average Weight in g) groups (%)
0 42.5 42.5 0
4 65.0 72.5 11.5
7 98.5 98.0 -0.5
11 145.5 148.5 2.4
14 185.5 200.5 8.1
The treatment group had measurably greater weight gains at the end of the 14-
day period in comparison to the control group.
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At the completion of the trial, at post-mortem, no clinical or pathological
signs of
toxicity were evident at this gross examination in the treated group of
chickens.
Table 14b
5 Weight gains during trial with superactivated acetal antimicrobial of
Example
10b
Day Control Group Treatment Group Differences between
(Average Weight in g) (Average Weight in g) groups (%)
0 42.5 42.5 0
4 62.5 67.5 8
7 97.5 103 6
11 130 145 11.5
14 178 219 23
At post mortem at the completion of the trial, on the remaining chickens, no
clinical or pathological signs were evident at gross examination in both
groups.
CONCLUSION:
There was a significant difference in weight gains in the treatment group in
comparison to the control group (x2; P<0.015). The treatment group was 23%
heavier than the control group at the completion of the trial.
The significant improvement in weight gain of the superactivated acetal
derivative relative to the control, and in the next example, when compared
with
the normally activated poly(2-propenal, 2-propenoic acid) demonstrates the
significant improvement in enteric antimicrobial activity of the acetal
derivative.
Example 12
This example evaluates the polymeric antimicrobial of Example 10b under field
conditions for the control of porcine post weaning colibacillosis (PWC).
METHOD:
146 Young pigs [weaners], either receiving various formulations of
superactivated antimicrobial in their feed or water, APRALAN (Elanco) orally,
autogenous vaccination, or neither, were challenged with the stresses
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associated with weaning on a large commercial piggery that had a long history
of problems with PWC.
Their responses in the development of diarrhoea, weight gains, and mortality
were assessed and are recorded in Table 15.
Table 15
Superactivated Mortality Diarrhoea days Mean Faecal
polymeric Rates [Days loose: Score4
antimicrobial [% died]2 score 1-2]3
group number'
Superactivated 1 3.33 1.79 0.14
polymeric [F;P<0.0001] [F;P<0.0001]
antimicrobial 2 0 1.17 0.11
[F;P<0.0001] [F;P<0.0001]
Untreated control 3 16.67 3.77 0.41
group [F;P<0.05] [F;P<0.0001] [F;P<0.0001]
Apralan group 4 13.33 3.89 0.38
[F; P<0.05] [F; P<0.0001 ] [F; P<0.0001 ]
Vaccinated Group 5 6.67 3.10 0.30
[F; P<0.0001 ] [F; P<0.0001 ]
Notes:
1. Coding of treatments:
i. Group 1= 0.1 % w/w superactivated polymeric antimicrobial in feed
ii. Group 2 = 0.02% w/v superactivated polymeric antimicrobial in water
2. Percentage of group that died from PWC during the trial
3. The mean number of days for each pig in the group when a faecal score of
lb I or 2 was recorded; faecal score is a measure of the intensity of
diarrhoea.
4. The sum of the faecal scores divided by the number of observed samples
per pig.
5. F = Fisher's Exact Test.
CONCLUSION:
This field trial highlighted the following points that positively reflected on
the
efficacy of the acetal derivative of poly(2-propenal, 2-propenoic acid)
(superactivated antimicrobial) for use in piglets in commercial piggery, when
challenged with (3-haemolytic Escherichia coli after weaning:
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1. Mortality:
Lower mortality rates for either groups treated with superactivated
polymeric antimicrobial than either the untreated, vaccinated or Apralan
groups.
2. Diarrhoea days:
Significantly lower diarrhoea days in either of the superactivated
antimicrobial treated groups [F; P<0.0001] than in either of the untreated,
vaccinated or Apralan groups.
3. Faecal scores:
Significantly lower mean faecal scores in either of the superactivated
antimicrobial treated groups [F; P<0.0001] than in either of the untreated,
vaccinated or Apralan groups.
Example 13
This example examines the effect of using certain additives with the
antimicrobial of the invention.
METHOD:
Overnight broths of challenge cultures were prepared and the Total Viable
Count (TVC) of the overnight cultures was estimated.
The samples were serially diluted 1 to I using sterile normal saline (5mL).
Sterile Hard Water (SHW) was used as diluent when testing samples containing
EDTA.
1 part of each overnight culture was diluted in 9 parts diluent. The resulting
diluted suspensions were used as the inocula.
Inoculate each sample dilution with 100 L of the diluted overnight culture.
(One culture per tube). Mix well.
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The tubes were incubated for <_ 24 hours at 37 C (A. niger was incubated at
28 C for <_ 24 hours).
From each test tube, 1 mL was subcultured into 9 mL of recovery broth and
mixed well (Recovery Broths: Nutrient Broth + 3% Tween 80 (NBT) for
polymeric antimicrobial and EDTA; Nutrient Broth + 3% Tween 80 + 0.1%
ammonia (NBTA) for glutaraidehyde and, Letheen Broth (LB) for methyl
parabens).
The samples incubated at 37 C for a further <_ 48 hours. (28 C for <_ 5 days
for
A niger).
Each tube was examined for growth. All tubes were then subcultured onto
selective agar and incubated for <_ 24 hours at 37 C. (28 C for <_ 5 days
for A.
niger). Growth on selective agar was taken to confirm growth of the test
organism.
Positive controls were performed using 5mL of diluent inoculated with culture
and subject to incubation, recovery and confirmation.
'20
Negative controls were performed using un-inoculated 5 mL of diluent and
subject to incubation, recovery and confirmation.
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SY
O ~ O
O r
o LO LO LO
O N N N
lf~ ~- Cp .. ',Ã cn M
--~ - - -
O
CD O ~ O O
ON LO N LO
o
O ~
O N ~
N U-) Cfl N
O LC)
O t.0
Gp p ~ Cfl LO
O U') iO
o t1) N N
N N O `-
-- -- -- -
O O O O
1` O O O O O
c- p r p
~ n A n
o o
o o
o
0 o N ~i
N
- - - - ---
LO~
Cfl U N
E
.Q C O
O N
=L _ , ;.._._ .- _
LO ._._-
E Lc~
X cl~ M (~
>+
~ - - - - - - --'-
O Q M N N ~ r
~ 0 A ( r- c"i c") r~
o o ---
O CO N O
~ '~ - - -
t6
v ttf tn O
N
G4 M
N
cu Q~
- ;, -
~ ~ X ~ .-.
O 0 O O O O O O O O O O O 0 O
~ E r-r- rT- rrrrrrrr- ~- T- r
w cu (~ O X X X X X X X X X X X X X X X
N co 04==MONONtUOCD0 d'OOLnCO M
p,= c~MMNMM
~ > y
CO
? p f6 .~ .~ Q U ~ Rl
U uLj r~ C/j
w zl
LO
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Legend:
1) Polymeric antimicrobial 0.025% w/w
2) Polymeric antimicrobial 0.2% w/w
3) Glutaraldehyde 0.025% w/w
5 4) Polymeric antimicrobial 0.025% w/w + Glutaraldehyde 0.025%
w/w
5) Polymeric antimicrobial 0.2% w/w + Glutaraldehyde 0.025% w/w
6) Polymeric antimicrobial 0.2% w/w
7) EDTA 0.1 % w/w
10 8) Polymeric antimicrobial 0.2% w/w + EDTA 0.1 % w/w
9) Polymeric antimicrobial 0.2% w/w (in 80% w/w glycerol)
10) Methyl Paraben 1% w/w (in 80% w/w glycerol)
11) Polymeric antimicrobial 0.2% w/w (in 80% w/w glycerol) + Methyl
Paraben 1% w/w (in 80% w/w glycerol).
Table 17
Synergy Index with polymeric antimicrobial
Culture Glutaraidehyde EDTA Methyl paraben
A.niger <0.6 <0.5 0.2
C.albicans 0.3 0.1 0.5
E.coli 0.5 <0.3 0.3
P.aeruginosa 0.6 0.4 0.2
S.aureus 0.3 <0.7 0.2
Note: Synergy <1.0, Additive = 1.0, Antagonistic >1.0
Whereby S1=CD/A + CE/B
A= MKC polymeric antimicrobial
B= MKC Preservative
C=MKCMix
D= Ratio of A to B
E= Ratio of B to A
CONCLUSION:
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41
It was shown that the acetal derivative of poly(2-propenal, 2-propenoic acid)
was synergistic with Glutaraidehyde, EDTA, and Methyl Paraben, respectively
versus A. niger, C. aibicans, E. coli, P. aeruginosa, S. aureus.
Example 14
This example demonstTates the activity of the' antimicrobial of the invEntion
in
combination with "Dettol" brand antimicrobial.
The sample was seriaUy diluted 1 to I using sterile normal saline (5mL).
Each sample dilution was inoculated with 100 L of the diluted ovemight
culture.
(One culture per tube). The samples were mixed well.
They were incubated at 37 f 2oC for s 24 hours. (Aspergillus niger inoculated
tubes were incubated at 28 t 2 C for 5 24 hours).
From each 'test tube 1 mL was subcultured into 9mL recovery broth and
TM
vortexed well (NBT, or Sabouraud + 3% Tween 80 (SABT) for A. niger).
They were incubated at 37 2oC for < 48 hours. (A. nioer, was incubated at 28
t 2 C for a further 5 days).
The recovery broths were examined for turbidity (Growth) and streaked onto
selective agars to confirm growth.
Table 18
MKC Results in ppm of superactivated polymeric antimicrobial andlor "DettolA
Culture Inoculum Polymeric 1 2 3 4 5
Approx Antimicrobiai
cfu/mL MKC ren e( m
A. niger 4.1x10 2000 - 500 WA 1000 300 WA 125:150
C. aib ns 5.2x10 100 - 250 WA 500 75 WA 62.5:75
. coli 7.6x10 125 - 31 125 A 75 31:75 A
P.aeruginosa 1. 0 250 - 62.5 A 250 600 A 125:150
S. aureus 2.0x10 31-7 N/A 15 75 N/A 15:19
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Legend
1) 0.1 % w/w polymeric antimicrobial
2) 0.2% w/w polymeric antimicrobial
3) "DettoP" 4.8% w/v (Diluted 1:20)
4) 0.1 % w/w polymeric antimicrobial +"DettoP' (Diluted 1:20)
5) 0.2% w/w polymeric antimicrobial +"Dettol" (Diluted 1:20)
Table 19
Svnergy Index of polymeric antimicrobial and Dettol
Culture Synergy Index (SI)
A. niger 0.3
C. albicans 0.6
E. coli 0.6
P. aeruginosa 0.4
S. aureus 0.6
Note: Antagonistic if SI >1, Additive if Sl=1, Synergistic if SI<1
S1= CD/A + CE/B
A= MKC polymeric antimicrobial (ppm)
B= MKC Dettol (ppm)
C= MKC of polymeric antimicrobial /"DettoP' Mix (ppm)
D;= Ratio of polymeric antimicrobial in relation to Dettol
E= Ratio of "Dettol" in relation to polymeric antimicrobial
Note: The active antimicrobial in "Dettol" is chloroxylenol
CONCLUSION:
It was shown that the polymeric antimicrobial of the invention was synergistic
with "DettoP" against E. coli, S. aureus, P. aeruginosa, C. albicans and A.
niger:
This shows that "Dettol" and the polymeric antimicrobial when used together as
a mixed solution, will work substantially better than when used on their own.
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Example 15
Antiseptic Qualities of poly(2-propenal, 2-propenoic acid) [superactivatedj
The antimicrobial, poly(2-propenal, 2-propenoic acid) [superactivated], was
trailed for antiseptic qualities.
The number of bacteria present on the hands of subjects was determined
before and after application of antimicrobial followed by donning of surgical
gloves. The antiseptic effect of poly(2-propenal, 2-propenoic acid)
[superactivated] was compared to the commonly used surgical antiseptic, 4%
Chlorhexidine Surgical Scrub (manufactured by Orion Laboratories, in Perth,
Western Australia).
= 3% w/w aqueous solutions of poly(2-propenal, 2-propenoic acid)
[superactivated] reduced baseline counts of bacterial populations on
gloved hands after 3 hours.
= 2% w/w aqueous solution - of poly(2-propenal, 2-propenoic acid)
[superactivated] with 70% ethanol showed a sustained reduction in
baseline bacterial counts after 3 hours on gloved hands, as did 4%
chlorhexidine.
= 3.2% w/w aqueous solution of poly(2-propenal, 2-propenoic acid)
[superactivated] with 3.1% sodium lauryl sulphate, followed by 4% w/w
aqueous solution of poly(2-propenal, 2-propenoic acid) [superactivated]
in 70% ethanol, applied to hands prior to donning surgical gloves, gave a
significant reduction in the baseline bacterial counts after 3 hours.
The results indicate that poly(2-propenal, 2-propenoic acid) [superactivated]
possesses good residual antimicrobial activity that is required for sustained
control of bacterial numbers in surgical asepsis. The inclusion of 70% ethanol
to the formulation aids the initial rapid decrease in bacterial numbers.
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Example 16
The Biocidal Activity of 0.125% w/w and 0.05% w/w Superactivated Polymer
against the reference strain H. pylori NCTC 11637 at pH 7 and pH 4.
The efficacy in vitro of the Superactivated Polymer was first established
against
the H. pylori reference strain, H. pylori NCTC11637. As variables, two
concentrations were chosen; one was a 40-fold dilution of the 5% solution of
the
Superactivated Polymer prepared in Example 2 giving a 0.125% w/w
concentration of the Superactivated Polymer, mimicking the dilution in the
stomach; the other was a 100-fold dilution giving 0.05% w/w concentration of
Superactivated Polymer. Two pHs were chosen, pH 7 as the baseline and pH 4
to mimic conditions in the stomach.
Cultures H. pylori NCTC11637 were grown microaerophilically on selective agar
plates at 37 2 C until sufficient growth was observed. Growth was
aseptically
removed from the plates and prepared as a standardized turbid suspension of
10% T, as displayed on a Vitek colorimeter, diluting with sterile normal
saline.
19.9g of sample was weighed out and inoculated with 100 L of culture
suspension. 1 mL of sample was immediately transferred into the
Deactivation/Recovery broth (Nutrient Broth plus 3% Tween 80) and then
serially diluted. 100 L aliquots were placed onto selective agar plates and
spread using a sterile disposable spreader. The transfer steps were repeated
at
time intervals of 5, 10, 15 and 20 miriutes. All plates were incubated
microaerophilically at 37 2 C until sufficient growth was achieved
(approximately 5 to 7 days). All colonies were counted and the population
decline over time was determined. The test was repeated using sterile normal
saline as the sample to determine the natural die-off rate at atmospheric
conditions.
Legend:
Culture 1. Superactivated Polymer, pH 7, 0.125% w/w
Culture 2. Superactivated Polymer, pH 7, 0.05% w/w.
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Culture 3. Superactivated Polymer, pH 4, 0.125% w/w.
Culture 4. Superactivated Polymer, pH 4, 0.05% w/w.
Culture 5. Sterile Normal Saline, pH 7.
Culture 6. Sterile Normal Saline, pH 4.
5
Table 20
Biocidal Activity of CHEMEQRT"" polymeric antimicrobial on H. pylorl
(NCTC 11637)
Culture T= 0 min T = 5 min T= 10 min T= 15 min T = 20 min
1 1.0x105 8.6x103 1.0x102 <1.0x102 <1.0x102
2 1.6x105 2.0x103 4.0 x 102 1.0x102 <1.0x102
3 1.0x105 3.2x104 1.1x104 4.0x102 <1.0 x102
4 3.0x104 1.7x104 1.1x104 8.2x103 3.6x103
5 2.0x105 1.0x105 9.2x105 8.0x104 _47.9
6 2.0x104 1.4x104 8.0x103 6.0x103 6.0x103
NOTE: Counts in Colony Forming Units (cfu) per mL of Deactivation Broth
The results against the reference strain (Table 20) show that the
Superactivated
Polymer was effective at pH 7 at both 0.125% w/w and 0.05% w/w, and was
also effective at pH 4 and 0.125% w/w.
Example 17
Further to the analysis of Example 16, three additional strains of H. pylori
at pH
7 and pH 4 were examined: H. pylori 01/303, which is resistant to
clarithromycin
and metronidazole; H. pylori SS1, a clinical strain isolated in Sydney with a
high
colonising ability of interest for possible animal models and H. pylori ATCC
700392, a strain whose genome has been sequenced and comes from the UK.'
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Table 21
Biocidal Activity of Superactivated Polymer at 0.125% pH 7
Culture T = 0 min T = 5 min T = 10 min T= 15 min T = 20 min
Control
2.Ox105 1.0 x 105 9.2 x 105 8.0 x 104 7.9 x 104
H. pylori 11637
H. pylori 11637 1.Ox105 8.6x103 1.Ox102 <1.Ox102 <1.Ox102
H. pylori 01/303 3.6 x 105 2.8 x 103 <1.0 x 102 <1.0 x 102 <1.0 x 102
H. pylori SSI 2.8 x 105 1.4 x 103 2.0 x 102 <1.0 x 102 <1.0 x 102
H. pylori 700392 2.0 x 106 1.3 x 106 1.8 x 105 8.8 x 103 <1.0 x 102
When treated with the Superactivated Polymer at 0.125% w/w at pH 7, all
strains were rapidly killed, with the antibiotic resistant strain being
particularly
vulnerable with death coming at less than 10 minutes (Table 21). The control
strain was untreated.
Example 18
The method of Example 3 was repeated in testing the Biocidal Activity of
0.125% w/w Superactivated polymer (pH 4) against all strains of H. pylori.
Table 22
Biocidal Activity of CHEMEQRTM polymeric antimicrobial at 0.125% and pH 4.
Culture T= 0 min T = 5 min T = 10 min T= 15 min T= 20 min
Control
2.0x104 1.4x104 8.0x103 6.0x103 6.0x103
H. pylori 11637
H. pylori 11637 1.O x 105 3.2 x 104 1.1 x 104 4.0 x 102 <1.0 x 102
H. pylori 01 /303 4.2 x 106 1.1 x 106 5.0 x 104 2.0 x 104 2.0 x 102
H.pyloriSS1 4.9x106 1.0x106 1.Ox105 2.8x104 2.0x102
H. pylori 700392 5.5 x 106 1.9 x 106 1.2 x 105 3.6 x 104 <1.0 x 102
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Testing the Superactivated Polymer at the stomach mimicking pH of 4 and at
0.125% w/w resulted in all strains being killed within 20 minutes (Table 3).
This
result is significant because this time frame for killing H. pylori is less
than the
time for passage through the stomach (40 min -1 hour). This demonstrates the
effectiveness of the Superactivated Polymer at a pH, a concentration, and in a
time frame consistent with treating an H. pylori infection in the, stomach.
The
control strain was untreated.
Example 19
This example demonstrates the enteric antimicrobial activity of the acetal
derivative of poly(2-propenal, 2-propenoic acid) prepared according to the
procedure of Example 10b.
MATERIAL AND METHODS:
Sixteen weaner pigs (age: 18 days 2 days and weight: 5.5 kg 1.0 kg) were
purchased from a commercial piggery. They were randomly assigned into 2
groups of 8 pigs (equal distribution of sexes) and housed in an
environmentally
controlled isolation animal house.
Water and feed were available ad libitum upon entry to the animal house, and
the diet was a commercial antimicrobial-free weaner pellet [19% crude
protein].
All the weaner pigs were euthanised with an intravenous injection of sodium
barbiturate, and then necropsied. DNA from Gastric and Oesophageal regions
of the stomach of twenty-four weaner pigs were extracted at necropsy using a
Qiagen Dneasy tissue kit according to provided instructions. 3iaL of the
extracted DNA was used to test for the presence of Helicobacter spp. in the
biopsy tissue samples. Polymerase Chain Reaction (PCR) was conducted
twice on each sample with seven control DNA samples being included in each
PCR run. No discrepancies were found between the PCRs conducted.
RESULTS:
Codinq of treatments:
Group 1: No treatment (negative control)
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Group 2: 0.1% w/v of superactivated polymeric antimicrobial according to
Example 10b; 30 mg/kg/day.
Table 23
PCR results of Helicobacter spp. using previously optimised genus specific
primers where + represents a positive detection of Helicobacter spp. and -
represents no detection.
Group Number Gastric Region Oesophageal Region
1 - -
1 - +
1 - +
1 + -
1 - -
1 - +
1 - -
1 - +
2 - -
2 - -
2 - -
2 - -
2 - -
2 - -
2 - -
2 - -
In group 1(no treatment) there were five positive PCR results to Helicobacter
spp (I - Gastric, 4 - Oesophageal), while in group 2 (0.1% w/v of polymeric
antimicrobial) there were no positive PCR results.
CONCLUSION:
The acetal derivative of poly(2-propenal, 2-propenoic acid) at 0.1% w/v
significantly (x2: P<0.025) reduces the incidence of porcine Helicobacter spp
in
the gastric and oesophageal mucosa in weaner pigs.
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Example 20
Comparative Example 20(a)
This example demonstrates a method of preparing non-superactivated poly(2-
propenal, 2-propenoic acid).
0.8% w/w Sodium Hydroxide
Place 9.90 kg of deionised water in a 10L stainless steel vat and add
0.08 kg sodium hydroxide to the water and stir until dissolved.
Polymerisation
Place 100.1 kg of deionised water in a 200L stainless steel vat and add
4.99 kg of the 0.8% w/w sodium hydroxide solution to the 200L vat.
Equilibrate the solution to 15 - 20 C. Simultaneously add 20 kg acrolein
monomer and the remaining 0.8% w/w sodium hydroxide solution to the
200L vat at a rate over 1 hour such that the pH remains at 10.5 -11.0,
and the temperature does not rise above 30 C. Continue the
polymerisation for a further 90 minutes.
Washing
Filter/centrifuge the polymerisation mixture and wash the polymer with
deionised water until the pH of the wash water is less than 7Ø The
approximate yield is 8 kg.
Drying
Dry the polymer in air, then heat in an oven using the following program.
Step Time Temperature
1 2 hrs 25 C
2 1 hr 40 C
3 1 hr 70 C
4 1 hr 75 C
5 2 hrs 85 C
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Dissolution
Place 400L of water in a 500L vat and add 4 kg of sodium carbonate and
stir until dissolved. Slowly add 8 kg of dry, heated polymer and stir for
thirty minutes.
5
The resulting polymer was found to have an approximate solubility of 90
- 95% w/w in 1% w/w sodium carbonate.
Example 20(b)
10 This example describes a method of preparing an acrolein polymer in which
the
polymer of comparative example 20(a) is super activated
Base Manufacture
Dissolve 0.4 kg sodium carbonate in 30.6 kg water in a suitable container
15 and place 64 kg polyethylene glycol 200 into the mixing vessel.
Commence stirring with the mechanical stirrer and heat the PEG200 to
65 t3 C. Add 5 kg of the dry acrolein polymer from Example 20(a) to
the PEG-200 and stir until a uniform mixture is obtained.
20 NOTE: The solid may not completely dissolve at this stage.
Slowly add the sodium carbonate solution to the glycol mixture at a rate
that ensures the pH of the solution remains in the range 3.5 - 9Ø
Stir the solution for 45 minutes at 65 t3 C.
NOTE: pH should be within the range 7-9. Temperature should be within
the range 65 t3 C.
Superactivation
Cover the mixing vessel and heat to 100 C for four (4) hours. The
resulting Superactivated Polymer was found to be miscible with water in
all proportions.
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Example 21
In Example 14 of PCT/AU9600328, it was demonstrated that poly(2-propenal,2-
propenoic acid) polymer in 0.5% w/w sodium carbonate solution possessed
anticancer activity against the Ehrlich ascites cell line in a mouse model.
The anticancer activity of poly(2-propenal,2-propenoic acid) polymer [Example
20(a)] against that of the Superactivated Polymer [Example 20(b)]. An in vitro
model of a gastrointestinal cancer was carried out on the human colon cancer
cell line, HT-29. Poly(2-propenal, 2-propenoic acid) was used in a 5% w/w
concentrate. The test used incubates the cancer cells with varying
concentrations of polymer to give a plot from which an IC50 can be
established.
Methodology
HT-29 cells (human colon cancer cells) were seeded (in 100 ial) into the wells
of
96-well culture plates and incubated overnight at 37 C in a humidified 5% C02,
95% air atmosphere. The polymer [poly(2-propenal,2-propenoic acid) polymer
in Comparative Example 20(a) and the Superactivated Polymer in Example
20(b)] was dissolved in water and then diluted in medium to 10 concentrations
spanning a 4-log range. 100 pl of each solution was then added to each of 5
wells. The plates were incubated for a further 72 hr after which viable cells
were
measured using the sulforhodamine B assay (Skehan et al., (1990) J. Nat.
Cancer Inst. 82: 1107-1112.; Monks et al., (1991) J. Nat. Cancer Inst. 83: 757-
766.). The cells were then fixed with 10% cold trichloroacetic acid for 1 hr
at 4 C
and the plates rinsed with distilled water, left to air dry and then stained
with
0.4% sulforhodamine B (Aldrich) in 1% acetic acid (v/v) for 30 min. Unbound
dye is then removed by washing twice with distilled water and finally with 1%
acetic acid. Protein-bound dye is then solubilized in 10 mM unbuffered Tris
base and the absorbance read at 550 nm using an automatic plate reader. The
mean absorbance for each drug dose is expressed as a percentage of the
control untreated well absorbance.
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The results of the test are shown in Table 24.
The poly(2-propenal,2-propenoic acid) polymer of Comparative Example 20(a)
gave an average IC50 over two tests of 0.030%; the poly(2-propenal',2-
propenoic
acid) polymer being assigned the value of 100%. This translates to 0.0015%
w/w of active polymer.
The Superactivated Polymer in Example 20(b) gave an average IC50 over four
tests of 0.025%. This translates to 0.00125% w/w of the Superactivated
Polymer and indicates that the Superactivated Polymer has potent anticancer
activity.
Table 24
IC50 of CHEMEQRTM polymeric antimicrobial against HT-29 human colon cancer
cells.
Compound Drug Average Average IC50
exposure IC50 (%) ICso (as % w/w of
tested
(hrs) (% w/w) active polymer)
Comparative
72 0.037, 0.023 0.030 0.0015
Example 20(a)
Example 20(b) 72 0.017, 0.034, 0.025 0.00125
0.025, 0.023
IC50 is the concentration required to inhibit cell growth by 50%.
Finally, it is understood that various other modifications and/or alterations
may be made without departing from the spirit of the present invention as
outlined herein.
