WO2003061672A1

WO2003061672A1 - Method of treatment of gastrointestinal disease and polymeric composition for use therein

Info

Publication number: WO2003061672A1
Application number: PCT/AU2003/000039
Authority: WO
Inventors: Graham John Hamilton Melrose; Andrew James Huxham; Damon Matthew Goadby Tilbrook; Vincent Leonard Wycoco
Original assignee: Chemeq Ltd
Current assignee: Chemeq Ltd
Priority date: 2002-01-18
Filing date: 2003-01-17
Publication date: 2003-07-31
Anticipated expiration: 2004-07-18
Also published as: EP1474155A1; KR20040081135A; CN1617733A; CN1617733B; EP1474155A4; BR0306960A; MXPA04007005A; CA2472685C; CA2472685A1; KR100971520B1; NZ533811A

Abstract

A method of treatment of gastrointestinal disease by administering polymeric antimicrobial comprising a derivative of poly(2-propenal, 2-propenoic acid) formed by reaction between a poly(2-propenal, 2-propenoic acid) and an alcohol or phenol to form protected carbonyl groups. The invention also relates to composition for use in treatment of gastrointestinal disease.

Description

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

or this unit in its hydrated, hemi-acetal or acetal form, represented by the formulae:

CH₂>

CH \ (d)

RO

RO. /

\

CH

CH / \

CH

\

CH, (f)

CH₅ ^■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. WO88/04671. 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 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:

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 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.

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. 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 contents of which are herein incorporated by reference. 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 aldol-Michael self condensation form. Typical examples of these forms are shown in formula (a) to (f) below:

(I)

CH₂

CH

(a)

CH

/ \

RO RO

\ (d)

RO o RO.

\ /

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 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 Ci to Cι₀ 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 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/or 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 po!y(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 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 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.

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 poultry 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, 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 conforms or Enterobacteria. It is particularly useful in treatment of gastrointestinal diseases resulting from infection by E. coli such as enterotoxigenic E. coli and β-haemolytic E. coli. Colibacillosis is a devastating disease in the pig-rearing industry. The disease is generally associated with proliferation of β-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. 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 Helicobacter pylori 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. 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. 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 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 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 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 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 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 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 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, α-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 carboxylmethylcellulose, 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. 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 1 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. 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. 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 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 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 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 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 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 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 10⁷-10⁸ 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 10.5-11.0.

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 40° 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. 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 31 g 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

Co ony forming units/mL 1g poly(2-propenal, 2-propenoic acid) was dissolved in 200 mL of 0.1% w/w Na₂C0₃ 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 NaHC0₃ used as the diluent. The "aged" sample showed a surprising improvement in performance, as can be seen by reference to Table 2:

Table 2

* 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 was treated with cone. 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:

Table 3

* Colony forming units/mL Example 3

This example examines the product produced by reaction of the poly(2- propenal, 2-propenoic acid) with polyethylene glycol.

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 1 provides proton (¹H) and carbon (¹³C) NMR data. As can be seen from Table 1, Nuclear Magnetic Resonance spectroscopy of the residue showed peaks at δ 3.58 and 3.56 in the ¹H Nuclear Magnetic Resonance spectrum and δ 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.

Table 4

Data from the 600 MHz ¹H- and 125MHz ¹³C-Nuclear Magnetic Resonance Spectra in DgO with 1% w/w Na?CO₃ of the solid residue of Superactivated polymer after dialysis and concentration.

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 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

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. All samples were stored at 40° 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

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

* 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 M_w = ca. 10000 and M_n = 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). 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

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

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

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 : Table 11

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 12(b):

Table 12(a) Activity against Staphylococcus aureus Initial Count, 3 x 10⁶ cfu/mL; polymer 350 ppm.

Table 12(b) Activity against Pseudomonas aeruginosa Initial Count, 3.7 x 10⁶cfu/mL; polymer 350 ppm.

(b) 1200 g of poly(2-propenal, 2-propenoic acid) was dissolved in 7680 g of PEG200 at 60° C and then 96g Na₂C0₃ in 3024 g water was added. The solution was heated at 100°C for 6 hours. 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 μS. 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

* Colony forming units/mL

The data indicate the treatment programme maintained the microbial counts within the guidelines of AS/NZ Standard 3666.3 (lnt):1998 and below that in the 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. 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 7.0. 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. 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 ±3° 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 - 9.0.

Stir the solution for 45 minutes at 65 ±3° C.

NOTE: pH should be within the range 7-9. Temperature should be within the range 65 ±3° 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 superacfivated acetal derivative described in Example 10b. 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 during trial with normally activated antimicrobial of Example 10a

The treatment group had measurably greater weight gains at the end of the 14- day period in comparison to the control group. 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 Weight gains during trial with superacfivated acetal antimicrobial of Example 10b

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 (χ²; 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 superacfivated 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 superacfivated antimicrobial in their feed or water, APRALAN^® (Elanco) orally, autogenous vaccination, or neither, were challenged with the stresses 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 5 were assessed and are recorded in Table 15.

Table 15

Notes: 0 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 b 1 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. 0

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 5 challenged with β-haemolytic Escherichia coli after weaning: 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 1 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 μL of the diluted overnight culture. (One culture per tube). Mix well. 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 glutaraldehyde 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.

Negative controls were performed using un-inoculated 5 mL of diluent and subject to incubation, recovery and confirmation.

RESULTS: Results are tabulated below

Table 16

MKC of Selected Preservatives and Synergy Index

Note: all ratio values are expressed as ratio of polymeric antimicrobial: preservative

Legend:

1) Polymeric antimicrobial 0.025% w/w

2) Polymeric antimicrobial 0.2% w/w

3) Glutaraldehyde 0.025% w/w

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

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

Note: Synergy <1.0, Additive = 1.0, Antagonistic >1.0

Whereby SI=CD/A + CE/B

A= MKC polymeric antimicrobial

B= MKC Preservative

C= MKC Mix

D= Ratio of A to B

E= Ratio of B to A

CONCLUSION: It was shown that the acetal derivative of poly(2-propenal, 2-propenoic acid) was synergistic with Glutaraldehyde, EDTA, and Methyl Paraben, respectively versus A. niger, C. albicans, E. coli, P. aeruginosa, S. aureus.

Example 14

This example demonstrates the activity of the antimicrobial of the invention in combination with "Dettol" brand antimicrobial.

The sample was serially diluted 1 to 1 using sterile normal saline (5mL).

Each sample dilution was inoculated with 100μL of the diluted overnight culture. (One culture per tube). The samples were mixed well.

They were incubated at 37 ± 2oC for < 24 hours. (Aspergillus niger inoculated tubes were incubated at 28 ± 2°C for < 24 hours).

From each test tube 1mL was subcultured into 9mL recovery broth and vortexed well (NBT, or Sabouraud + 3% Tween 80 (SABT) for A. niger).

They were incubated at 37 ± 2oC for < 48 hours. (A. niger, was incubated at 28 ± 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 and/or "Dettol"

Legend

1) 0.1% w/w polymeric antimicrobial 2) 0.2% w/w polymeric antimicrobial 3) "Dettol" 4.8% w/v (Diluted 1 :20) 4) 0.1% w/w polymeric antimicrobial + "Dettol" (Diluted 1 :20) 5) 0.2% w/w polymeric antimicrobial + "Dettol" (Diluted 1:20)

Table 19 Synergy Index of polymeric antimicrobial and Dettol

Note: Antagonistic if SI >1 , Additive if Sl=1 , Synergistic if Sl<1 Sl= CD/A + CE/B

A= MKC polymeric antimicrobial (ppm) B= MKC Dettol (ppm)

C= MKC of polymeric antimicrobial / "Dettol" 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 "Dettol" 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. Example 15

Antiseptic Qualities of polv(2-propenal. 2-propenoic acid) fsuperactivated]

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. 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 asepfically 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 100μL of culture suspension. 1mL of sample was immediately transferred into the Deactivation/Recovery broth (Nutrient Broth plus 3% Tween 80) and then serially diluted. 100μL 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 minutes. 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. 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.

Table 20 Biocidal Activity of CHEMEQ^R™ polymeric antimicrobial on H. pylori (NCTC 11637)

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. Table 21 Biocidal Activity of Superactivated Polymer at 0.125% pH 7

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 CHEMEQ^R™ polymeric antimicrobial at 0.125% and pH 4.

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. 3μL 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:

Coding of treatments:

Group 1 : No treatment (negative control) Group 2: 0.1% w/v of superactivated polymeric antimicrobial according to Example 10b; 30 mg/kg/day.

Table 23 PCR results of Helicobacter SDD. using previously optimised genus specific primers where + represents a positive detection of Helicobacter spp. and - represents no detection.

In group 1 (no treatment) there were five positive PCR results to Helicobacter spp (1 - 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 (χ²: P<0.025) reduces the incidence of porcine Helicobacter spp in the gastric and oesophageal mucosa in weaner pigs. 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

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 7.0. 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 Dissolution

The resulting polymer was found to have an approximate solubility of 90 - 95% w/w in 1 % w/w sodium carbonate.

Example 20(b) 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 and place 64 kg polyethylene glycol 200 into the mixing vessel.

Commence stirring with the mechanical stirrer and heat the PEG200 to 65 ±3° 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.

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 - 9.0.

Stir the solution for 45 minutes at 65 ±3° 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. 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 μl) 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 μl 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. 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 ICgn of CHEMEQ^R™ polymeric antimicrobial against HT-29 human colon cancer cells.

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.

Claims

1. A method for treatment or prophylaxis of gastrointestinal disease in an animal (including human) comprising gastrointestinal administration to the animal of an effective amount of a polymer comprising a derivative of poly(2- propenal, 2-propenoic acid) formed by reaction between a poly(2-propenal, 2- propenoic acid) and an organic compound containing one or more hydroxyl groups to form protected carbonyl groups.

2. A method according to claim 1 wherein the polymer is orally administered.

3. A method according to claim 1 wherein the animal is suffering from at least one gastrointestinal disease selected from the group consisting of gastroenteritis, ulcer, diarrhoea and gastrointestinal cancer and dysentery- promoted insufficient weight gain.

4. A method according to claim 1 wherein the animal is suffering from at least one of diarrhoea, gastroenteritis and dysentery.

5. A method according to claim 1 wherein the animal is selected from the group consisting of dogs, pigs, sheep, horses, cattle, cats, poultry, ducks, turkeys and quail.

6. A method according to claim 1 wherein the animal is selected from ruminant animals and the polymer is rectally administered.

7. A method according to claim 1 wherein the animal is selected from poultry and pigs.

8. A method according to claim 1 wherein the animal is a partially grown pig-

9. A method for treatment or prophylaxis of porcine post weaning colibacillosis comprising orally administering to young pigs after weaning, an antimicrobially effective amount of the polymer of claim 1.

10. A method according to claim 1 wherein the antimicrobial of claim 1 is administered at a dose of from 0.05 to 5000 mg/kg/day.

11. A method according to claim 1 wherein the antimicrobial of claim 1 is administrated at a dose in the range of from 0.5 to 500 mg/kg/day.

12. A method according to claim 1 wherein the young pigs are administered a dose of the antimicrobial in the range of from 0.05 to 50 mg/kg/day.

13. A method according to claim 1 wherein the gastrointestinal disease results from one or more microbes selected from the group consisting of

Coliforms, Salmonella, P.aeruginosa, Helicobacter, Proteus, Enterobacteria, Yeasts, Protozoa, Clostridia, Shigella and Coccidia

14. 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 a polymer comprising 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.

15. A method according to claim 1 wherein the gastrointestinal disease results from at least one of enterotoxigenic E. coli and β-haemolytic E. coli.

16. A method of treatment or prevention of necrotic enteritis in poultry comprising administering to poultry an effective amount of the antimicrobial of claim 1.

17. A method according to claim 1 wherein the antimicrobial is administered in combination with a further chemotherapeutic adsorbed thereon to thereby reduce membrane penetration of the further chemotherapeutic.

18. A method of treatment or prevention of coccidiosis in poultry comprising administering to poultry an antimicrobially effective amount of antimicrobial of claim 1.

19. A method according to claim 1 wherein the derivative comprises a multiplicity of protected carbonyl groups selected from at least one of hemiacetal groups and acetal groups.

20. A method according to claim 20 wherein the protected carbonyl groups include acetal groups.

21. A method according to claim 1 wherein the alcohol is selected from alkanols, phenols, polyols and mixtures thereof.

22. A method according to claim 22 wherein the alcohol is selected from at least one polyol.

23. A method composition according to claim 23 wherein the polyol comprises a polyalkylene glycol.

24. A method according to claim 23 wherein the polyol comprises a polyethylene glycol.

25. A method according to claim 23 wherein the polyol is a polyethylene glycol of molecular weight of from 200 to 2000.

26. An antimicrobial for treating or preventing gastrointestinal disease in animals by gastrointestinal administration said antimicrobial composition comprising a derivative of poly(2-propenal, 2-propenoic acid) formed by reaction between a poly(2-propenal, 2-propenoic acid) and an organic compound containing one or more hydroxyl groups to form protected carbonyl groups and a pharmaceutically or veterinarially acceptable inert carrier for gastrointestinal administration to animals.

27. An antimicrobial for treating or preventing gastrointestinal disease according to claim 27 wherein the carrier for gastrointestinal administration is selected from the group consisting of water, controlled release polymers, olive oil, peanut oil, sesame oil, sunflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides, polyvinyl alcohol, partially hydrolysed polyvinylacetate and mixtures thereof.

28. An antimicrobial composition according to claim 27 in the form of a feed additive or drinking water additive comprising from 0.1 to 70% by weight of the antimicrobial according to claim 1.

29. An animal (including humans) feed or drinking water composition comprising a feed material or water and an antimicrobially effective amount of an antimicrobial according to claim 1.

30. An animal feed composition according to claim 30 wherein antimicrobial is present in an amount of from 0.001 to 25% by weight of the total feed or water composition.

31. An antimicrobial composition comprising an antimicrobial according to claim 1 and a further active agent selected from the group consisting of antimicrobials and chemotherapeutic agents.

32. A composition according to claim 31 wherein the further antimicrobial comprising (on a weight basis of the composition) at least one of

(a) a phenol in an amount of 0.1 to 10%;

(b) an isothiazolinone in an amount of 0.001 to 1%;

(c) alkyl parabens in an amount of 0.02 to 2% and

(d) lower alkanol in an amount of from,20 to 99.9%.