JP4732691B2 - Method for treating gastrointestinal diseases and polymer composition for use therein - Google Patents

Description

  The present invention relates to the treatment or prevention of gastrointestinal diseases and the promotion of animal growth and antimicrobial compositions for use in such treatment.

  Antibacterial agents are compounds that kill microorganisms such as bacteria. Antibiotics are usually part of antimicrobial agents obtained from other microorganisms and act by interfering with specific mechanisms in the target microorganism. Antibiotics were first used in the 1940s and 1950s, and their use has increased since then. The development of antibiotic resistance has become a serious and potentially life-threatening event worldwide. Some strains of staphylococci are resistant to almost all antibiotics and have caused fatal infections that occur in hospitals. Other drug-resistant microorganisms include pneumococci that cause pneumonia and Cryptosporidium and E. coli that cause diarrhea.

  The use of antibiotics in animal feed is widely believed to be responsible for promoting resistance development, and as a result, many countries limit their use. This causes problems in animal farm management that pose difficulties in controlling disease and obtaining optimal growth rates. This is a particular problem in pig and poultry farming. For example, gastrointestinal diseases such as colibacillosis in pigs and coccidiosis in poultry have devastating effects.

で示される繰り返しユニット、または式：

［式中、Ｒは水素であり、ｎは１以上の整数である］
で示されるその水和ヘミアセタールもしくはアセタール体を有することがこれまでに記載されている。これに先立って、消毒処理におけるアクロレインポリマーの殺生物特性が、Melroseら（International Application No. WO88/04671）によって記載された。ドイツ特許出願P4404404およびその対応出願EP667358およびAU 11686/95(現在、失効)は、水性水酸化ナトリウム媒体中でアクロレインが重合されるプロセスを開示している。この開示は、得られるポリアクロレインが、40〜50℃にて多価アルコールに可溶性であり、多価アルコール中でポリアクロレインの溶液を形成することを述べている。以下に説明するように、ドイツ出願の譲受人は、続いて、このようなポリマーが問題があり、水性媒体には溶解度が低いことを見出した。 Melrose et al. (International Patent Publication No. 96/38186) first described the production of acrolein polymers for use in the treatment of gastrointestinal diseases.
These polymers have the formula (I):

Repeating unit indicated by or formula:

[Wherein R is hydrogen and n is an integer of 1 or more]
It has been described so far to have its hydrated hemiacetal or acetal form as shown. Prior to this, the biocidal properties of acrolein polymers in disinfection were described by Melrose et al. (International Application No. WO88 / 04671). German patent application P4404404 and its corresponding application EP667358 and AU 11686/95 (currently expired) disclose a process in which acrolein is polymerized in aqueous sodium hydroxide medium. This disclosure states that the resulting polyacrolein is soluble in polyhydric alcohol at 40-50 ° C. and forms a solution of polyacrolein in the polyhydric alcohol. As explained below, the assignee of the German application subsequently found that such polymers are problematic and have low solubility in aqueous media.

European Publication No. 792895 Werle et al. (Corresponding to US 6060571) relates to an acrolein releasing polymer produced by copolymerization of an acrolein monomer and a polyhydric alcohol. Werle et al. Notice that the polyacrolein described in German application No. P4404404 has problems in that the yield is lower than desired and the polymer is substantially insoluble in water. European application 792895 teaches that these problems are overcome by forming an acrolein releasing polymer by copolymerization of an acrolein monomer and a polyhydric alcohol monomer. The structure of the proposed copolymer is as follows:

Free acrolein acts as an antibacterial agent, but is irritating to the eyes, lungs, tissues and skin. In particular, for a series of applications in gastrointestinal treatment, there is a need for antibacterial agents that are stable, highly water soluble, and highly safe in use. There is a further need for effective antibacterial agents for the treatment or prevention of gastrointestinal diseases that can reduce the pressure on resistance development in antibiotics.
The discussion of the background of the invention is included to illustrate the context of the invention. This should not be taken as an acknowledgment that for any of the claims, any of the substances referred to were published, publicly known, or part of the common general knowledge on the priority date.

Summary of invention

  We react poly (2-propenal, 2-propenoic acid) polymers with alcohols or polyols to form protected carbonyl groups such as acetals and / or hemiacetal derivatives to form poly (2 in the treatment or prevention of gastrointestinal diseases. It has been found that the activity and stability of the 2-propenal, 2-propenoic acid) polymer is substantially increased. Surprisingly, we have substantially increased the activity of these derivatives in the treatment or prevention of gastrointestinal disease despite the very low or negligible amount of free acrolein content in the polymer I found out. The solubility of the polymer in water is also very high.

  The present invention relates to an organic compound containing one or more hydroxyl groups such as poly (2-propenal, 2-propenoic acid) and alkanols, phenols, polyols and mixtures thereof preferably for forming protected carbonyl groups. A method of treating or preventing a gastrointestinal disease in an animal comprising administering to the animal (including human) an effective amount of a poly (2-propenal, 2-propenoic acid) derivative formed by a reaction between .

  In another embodiment, the present invention relates to poly (2-propenal, 2-propenoic acid) to form a protected carbonyl group and one or more alcohols, preferably alcohols selected from alkanols, phenols, polyols and mixtures thereof. Provided is an antibacterial agent for the treatment of gastrointestinal diseases comprising an effective amount of a poly (2-propenal, 2-propenoic acid) derivative formed by a reaction between an organic compound containing a hydroxyl group.

The term “polyol” as used herein means a molecule having at least two hydroxyl groups.
The formed derivative is typically selected from hemiacetal and acetal derivatives. Although not theoretically elucidated, we have found that the reaction of poly (2-propenal, 2-propenoic acid) with alcohol removes hemiacetal and / or acetal groups from at least some pendent aldehyde groups. By forming, it is thought that the carbonyl group of the polymer is stabilized against alkali decomposition by the Cannizzaro reaction. The formation of acetal groups has surprisingly been found to significantly reduce or eliminate the release of free acrolein while increasing the activity of the resulting derivative.

In yet another aspect, the present invention provides the use of the antibacterial agent for the manufacture of a medicament for the treatment or prevention of gastrointestinal diseases.
Throughout the detailed description and claims of this specification, variations of this word, such as the word “comprising” and “including”, exclude other additives or ingredients or integers. Is not intended.

Detailed Description of the Invention

The antibacterial agent of the present invention can be produced by heating poly (2-propenal, 2-propenoic acid) in the presence of an alcohol, preferably a polyol such as polyethylene glycol. It is understood that moisture is always present in the alcohol, and the presence of at least some moisture assists the nucleophilic reaction in which hemiacetal or acetal is formed.
The solution is generally heated in the temperature range of 40 ° C to 150 ° C, preferably 40 to 115 ° C, and most preferably 70 to 115 ° C.
The antibacterial agent of the present invention is produced from a poly (2-propenal, 2-propenoic acid) polymer. Such polymers and their production are described in WO 96/38186 (PCT / AU96 / 00328), which is incorporated herein by reference in its entirety. The poly (2-propenal, 2-propenoic acid) polymer is preferably prepared by polymerization of acrolein, preferably in an aqueous solution by anionic polymerization followed by anionic polymerization. The polymer includes repeating units of formula (I) and includes at least one (typically a mixture) of hydrated hemiacetal and acetal bodies.

［式中、Ｒは水素であり、ｎは１以上の整数である］。式(Ｉ)で示される繰り返しユニットの割合は、代表的には、２０％以下であり、５〜１５％であることが多い。これらのユニットの割合が相対的に低いにもかかわらず、我々は、それらがポリマーの安定性において重大な効果をもつことを見出した。 Hydrated hemiacetals and acetals formed by polymerization of acrolein are known to arise from the various carbon-carbon and carbon-oxygen polymerization mechanisms of acrolein. For example, typically, a hydrate is a hydrated diol, a hemiacetal or acetal is formed from the condensation of a diol and an aldehyde or diol, and a tetrahydropyran or fused tetrahydropyran is a diol. And the aldol-Michael self-condensate. Representative examples of these features are shown in the following formulas (a) to (f):

[Wherein R is hydrogen and n is an integer of 1 or more]. The ratio of the repeating unit represented by the formula (I) is typically 20% or less and often 5 to 15%. Despite the relatively low proportion of these units, we have found that they have a significant effect on polymer stability.

  Poly (2-propenal, 2-propenoic acid) generally includes 10% or less on a molar basis of monomer units from monomers other than acrolein, with acrolein homopolymer (before auto-oxidation) being most preferred. When used, the other monomer is selected from the group consisting of acrylic acid and vinyl pyrrolidone. The 2-propenoic acid group is typically present in an amount of 0.1 to 5 moles of carboxyl groups / kg. The poly (2-propenal, 2-propenoic acid) polymer typically has a number average molecular weight of 1000 or more, more preferably 2000 or more. Typically, the molecular weight is 10,000 or less.

The antimicrobial agent of the present invention is a derivative of poly (2-propenal, 2-propenoic acid) produced by reaction with an alcohol or phenol to form a protected carbonyl group. Protected carbonyl groups are formed from 2-propenal groups and react with alcohols to form hemiacetal and acetal groups. The alcohol is preferably a polyol, which means that it preferably contains at least two hydroxyl groups. It can be used alkanols such as C ₁ -C _10. When the alcohol is a polyol, the reaction creates an acetal or hemiacetal formed by the reaction of one or more alcohol groups. Furthermore, when two alcohol groups react, they can react at the same carbonyl or different carbonyl groups in the polymer.

With respect to formula (I) above and hemiacetals and acetals, the present invention produces derivatives containing fewer units of formula (I) to form a group in which one or more R groups are derivatives from alcohols; Alternatively, when the alcohol is a polyol, two or more R groups together form a crosslinking group such as a cyclic acetal group.
The tendency of polyols to generate internal cyclic groups depends on the spacing and configuration of the polyol. A preferred alcohol is polyalkylene glycol, and a more preferred alcohol is polyethylene glycol.
The molecular weight of the polyalkylene glycol is 200 to 2000, more preferably 200 to 1000.

During the preparation of the antibacterial polymer, it is preferred that an alcohol such as polyethylene glycol is present in an amount of 50 to 99% by weight. A relatively dilute composition of an acrolein polymer is particularly preferred when the alcohol is a polyol because the occurrence of intermolecular crosslinking is reduced by dilution.
More preferably, polyethylene glycol is present in the amount of 64 to 95% by weight during the production of the polymer.

It is preferred to add a base or alkali to the polymer prior to and / or during heating and then to an acidic pH since neutralization of the acidic groups of the polymer enhances the antimicrobial activity of the polymer. It is preferred that the addition of base or alkali first brings the pH of the poly (2-propenal, 2-propenoic acid) polymer to 7-9. Even more preferably, the initial pH by addition of base is about 8. The base is preferably an alkali metal hydroxide, carbonate, bicarbonate or a mixture thereof.
In yet another aspect of the present invention, the release of free acrolein monomer is blocked and is not released continuously, thereby reducing the likelihood that the polymer provides a source of tissue or skin irritation.

We have found that the antibacterial agent of the present invention has significantly improved activity in the control of gastrointestinal diseases compared to poly (2-propenal, 2-propenoic acid) as the production source. The superactivated derivatives of the present invention can be used to treat a wide range of animals (including humans) and a wide range of microbial infections.
The antibacterial agent of the present invention can be used for the treatment of human gastrointestinal diseases, but animals selected from other animals, particularly dogs, pigs, sheep, horses, goats, cattle, cats, poultry, ducks, turkeys and quails Particularly preferred for use in the treatment of
The antimicrobial agent of the present invention can be formulated for oral or rectal administration. Rectal administration is particularly useful in ruminants. Oral formulations for ruminants can also be prepared with enteric coatings to provide optimal activity in the posterior part of the gastrointestinal tract.

The antimicrobial agents of the present invention are particularly useful in the treatment and prevention of gastrointestinal ulcers, diarrhea and gastrointestinal cancer. Using the antibacterial agent of the present invention, the rate of weight gain of livestock can be improved by improving the feed for converting the weight of the animal.
We have found that the antibacterial agents of the present invention can be used as growth promoters and that they can be used in place of currently used antibiotics. Drug resistance in pathogens is a problem of great clinical importance in human medicine. This problem is exacerbated by the use of important antibiotics in animal feed to increase the weight of livestock, particularly poultry and pigs. In fact, some European countries prohibit the use of conventional antibiotics in animal feed. The antibacterial agent of the present invention can be used in the treatment of animals to greatly extend the useful life of conventional antibiotics in human treatment.

  We have found that the antimicrobial agents of the invention are effective against a wide range of microorganisms such as protozoa, gram positive and gram negative bacteria. The polymers of the present invention encompass a large number of structures with various configurations and can find compatibility with proteins found in the cell walls of target organisms, which can lead to protein inactivation and cell destruction. Promote. Of the gram-negative bacteria, the antibacterial agents of the present invention have been found to be particularly useful in providing a wide range of activity against E. coli or enteric bacteria. The polymers of the present invention are particularly useful in the treatment of gastrointestinal diseases due to E. coli infection, such as enterotoxigenic E. coli and β-hemolytic E. coli. E. coli is a devastating disease in the pig farming industry. This disease is generally associated with the growth of β-hemolytic E. coli in the small intestine after weaning, resulting in high mortality and morbidity in young weaned piglets. Infected, weaned piglets do not gain normal weight gain.

Coccidiosis is a protozoan disease in animals, particularly poultry, and has a devastating effect if left untreated. We have found that the antibacterial agents of the present invention can be used in the treatment or prevention of coccidiosis in birds, particularly poultry. In chickens, typical clinical signs of coccidiosis are no growth, weight loss, diarrhea and dysentery. The most severe effects occur in the intestines where protozoa invade the mucosa causing epithelial damage, lesions and bleeding.
Vaccines are being used to prevent coccidiosis, but have side effects such as weight loss and a tendency to decrease food efficiency.
The antibacterial agent of the present invention may be used in combination with other drugs known to have activity against coccidiosis. Such drugs include ionophores including nitrocarbanilide, quinoline, pyridone, guanidine, quinoxaline, toltrazal, toluamide, enhanced sulfa drugs and carbanilide.

Clostridium bacteria are Gram-positive bacteria that cause severe disease in various animals. For example, necrotizing enterocolitis is a disease that is known to affect poultry farming. Clostridium bacteria produce exotoxins, some of the most toxic of all known toxins. Necrotizing enterocolitis occurs particularly in 14-42 day old broilers. The disease causes significant lethargic symptoms, diarrhea, and death within hours.
Upper gastrointestinal tract diseases such as chronic gastritis, gastric ulcer and duodenal ulcer are serious human health problems. Helicobacter is understood to be responsible for the development of ulcers and gastrointestinal cancers, particularly gastric adenocarcinoma. We have found that the antibacterial agents of the present invention are particularly useful against Helicobacter, such as Helicobacter pylori, in animals, particularly human gastrointestinal diseases. Gastric infection with Helicobacter pylori is one of the most frequent infectious diseases in the world. About 50% of the population is infected with Helicobacter pylori. In developing countries, it is estimated that more than 80% of the population is already infected with Helicobacter pylori in childhood.

Helicobacter pylori is a gram-negative microaerobic spiral gonococci that moves with flagella on one end of the cell. The standard treatment for Helicobacter pylori infection is the so-called triple antibiotic therapy, all of which contains either metronidazole or clarithromycin. Unfortunately, strains of Helicobacter pylori that are resistant to both antibiotics have emerged.
Helicobacter pylori survives in the stomach at the interface between the surface of gastric epithelial cells and the mucosal gel layer that covers it. Helicobacter pylori can further be found in the upper part of the gastric epithelium in the duodenum and esophagus. Other animal species have their own Helicobacter species present in their gastrointestinal tract, and their characteristics are similar to Helicobacter pylori. In addition to its association with gastrointestinal cancer, Helicobacter pylori is directly linked to the formation of gastritis and peptic ulcers in humans.

In general, Helicobacter species, in particular Helicobacter pylori, survive under harsh conditions in the stomach by secreting urease, which raises the pH in the vicinity of Neisseria gonorrhoeae by hydrolyzing urea into ammonia and bicarbonate ions. This local environmental change protects the bacteria from the bactericidal effects of gastric acid. The preferred location under the protective mucosal layer of the stomach is also a survival advantage and its motility allows it to lie through the layer to achieve this location.
Gastric lining epithelial cells are difficult to penetrate naturally; this is an integral part of their function to protect the rest of the body from gastric acid and digestive fluids . This penetration difficulty also makes it difficult for the body's natural defenses to reach the site of Helicobacter pylori infection through the stomach wall. This has two consequences; the body sends more nutrients to this site to assist leukocytes, T cells and other defense mechanisms, but is inadvertently supplied to gonorrhea; , The protective cells eventually die, releasing a load of superoxide ions and other lethal chemical species, damaging surrounding epithelial cells.

It is clear that this activity leads to gastritis and can easily progress to peptic ulcers. As the injury continues, the likelihood of developing gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma is greatly increased. Gastric adenocarcinoma begins in the mucosa and intestinal metaplasia, the first stage of progression, is the gastric response to remove the Helicobacter pylori infection itself. Experiments conducted at the UCL Medical School also show that MALT lymphoma requires the development of help from Helicobacter pylori specific T cells. Treatment of Helicobacter pylori infection has proven very effective in the treatment of MALT lymphoma. The World Health Organization classifies this pathogen as a Group I carcinogen.
Thus, the present invention also provides a therapeutic amount of an agent (wherein the agent is a poly (2-propenal, 2-propenoic acid) and an alkanol, phenol, polyol and mixtures thereof to form a protected carbonyl group. Treatment of gastrointestinal diseases caused by Helicobacter infection, including gastrointestinal administration of poly (2-propenal, derivatives of 2-propenoic acid) formed by reaction with an organic compound containing a selected hydroxyl group or Provide prevention methods. As used herein, the term “polyol” means a compound having at least two hydroxyl groups. The formed derivative is typically selected from hemiacetal and acetal derivatives.

Thus, the use of the method of the present invention provides an alternative to the use of surgery, radiation therapy or conventional chemotherapy in the treatment of gastrointestinal cancer.
Furthermore, the present invention relates to poly (2-propenal, 2) formed by a reaction between poly (2-propenal, 2-propenoic acid) for forming a protected carbonyl group and an organic compound having one or more hydroxyl groups. A method for the treatment of gastrointestinal infections by a Helicobacter bacterium such as gastritis, gastric ulcer, duodenal ulcer, gastric malignant lymphoma or gastric cancer, comprising the gastrointestinal administration of a therapeutic amount of an agent comprising a derivative of propenoic acid
The present invention generally provides an alternative to standard treatment of Helicobacter infection, including so-called triple antibiotic therapy, all containing either metronidazole or clarithromycin. A strain of Helicobacter pylori that is resistant to both of these antibiotics has emerged and we have demonstrated that the method of the present invention can effectively utilize such antibiotic-resistant bacteria. Yes.

Agents that are products of reactions between poly (2-propenal, 2-propenoic acid) and organic compounds having one or more hydroxyl groups to form protected carbonyl groups are useful in the treatment of Helicobacter infection. It has proved to be more effective than the non-superactivated poly (2-propenal, 2-propenoic acid) group.
The present invention further provides the use of a derivative of poly (2-propenal, 2-propenoic acid) in the manufacture of a medicament for the treatment or prevention of diseases caused by Helicobacter infection.
The method of the present invention can be used for the treatment or prevention of gastrointestinal cancer. This cancer includes, for example, esophageal, stomach, intestinal and colon cancer. An example of this type of cancer is the human colon cancer cell line HT-9.

The antibacterial agent of the present invention is blended in animal feed or water, and this can be done by conventional methods. In a preferred embodiment, the antibacterial agent of the present invention is blended in a premix. The premix preferably includes an antimicrobial agent, a physiologically acceptable carrier and optionally feed. The premix is generally at a relatively high concentration and is suitable for dilution with other substances such as one or more other carriers, vitamins and mineral supplements and feeds into the final animal feed. It is preferred that the premix comprises an antimicrobial agent at a concentration of 0.1 to 70% by weight, preferably 0.5 to 50% by weight. The optimal concentration is whether the treatment is prophylactic or therapeutic and whether the antibacterial agent of the present invention is the only active ingredient or whether it is used in combination therapy with other substances or antibacterial agents, It depends on.
In a preferred embodiment, the high concentration composition of antimicrobial agent is a controlled release formulation. Controlled release formulations comprise a polymeric material for providing controlled release of antimicrobial agents from a controlled release system and are particularly useful in compositions for addition to chow material. As a result of the controlled release formulation, antimicrobial agents can be delayed so that they are released primarily in the duodenum. Controlled release polymers can minimize composition rejection by flavor or can be used as rectal suppositories.

The antibacterial agent composition of the present invention may be a preparation of a solid composition such as a pellet or pill. Pellets containing the antimicrobial agent of the present invention can be produced by the following steps:
(i) dissolving the antimicrobial agent in an aqueous alkali or basic solution;
(ii) neutralizing the solution with acid;
(iii) adding an insoluble crosslinked adsorbent polymer of acrylamide and acrylic acid to the neutralized solution to form wet swellable pellets;
(iv) If necessary, dry or partially dry the wet swellable pellets.

  The wet swellable pellets thus formed can be used, for example, as an additive to animal feeds in either a wet, partially dry or fully dry state. In addition, the system is designed such that in the acidic environment of the stomach, the carboxyl-containing groups of the outer polymer matrix leave the polymer of interest essentially contained within the matrix. However, in the alkaline environment of the duodenum, the carboxyl groups of the matrix become ionized and repel each other, promoted by the repulsion between the ionic groups, the pellet swells rapidly, and the passage of feed through the duodenum The polymer of interest will be released by the diffusion process, while roughly matching the rate.

In the present invention, the term “controlled release system” is used in the same context, including the same range of examples as cited in 鼎 ontrolled Drug Delivery Robinson & Lee, 1987). Many other pH-sensitive controlled release systems known to those skilled in the art (Robinson and Lee, 1987) can be substituted with polymers of acrylic acid or copolymers of acrylamide and acrylic acid. For example, soluble and anionic or insoluble crosslinkable and anionic cellulosic systems; or soluble and anionic or insoluble crosslinkable and anionic derived from any common acrylic acid polymer and / or derivative thereof Polymer etc. Such crosslinkable and insoluble polymers are preferred because they swell and are less likely to be metabolized.
It is preferred that the controlled release system, which gels when wet, comprises pH sensitive, crosslinkable, water absorbing pellets.
The present invention also provides an animal feed composition comprising the antibacterial agent of the present invention and a feed. The antibacterial agent is preferably present in an amount of 0.0001-25%, preferably 0.0001-5% of the total feed composition.
In other preferred embodiments, the antimicrobial agents of the invention may be added to animal drinking water.
The antibacterial agent of the present invention is preferably administered in an amount of 0.05 to 5000 mg / kg body weight / day, more preferably 0.05 to 50 mg / kg body weight / day.

Suitable inert carriers for use in the composition for administration of the antimicrobial agent of the present invention include water, olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, peanut oil, coconut oil, liquid paraffin, ethylene glycol , Propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohol, triglyceride, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate and mixtures thereof.
Solids for oral or rectal administration are pharmaceutically or veterinary acceptable binders, sweeteners, disintegrants, diluents, flavoring agents, coating agents, preservatives, lubricants and / or time delays. An agent may be included. Suitable binders include acacia gum, gelatin, corn starch, tragacanth gum, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include flavonoids such as sucrose, lactose, glucose or neohesperidin dihydrochalcone. Suitable disintegrants include corn starch, methyl cellulose, polyvinyl pyrrolidone, xanthine gum, bentonite, arginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavoring agents include peppermint oil, winter green oil, cherry, orange or raspberry flavoring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their ethers and / or their amides, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, α-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

The suspension for oral or rectal administration may further comprise a dispersing agent and / or a suspending agent. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, sodium alginate or cetyl alcohol. Suitable dispersants include lecithin, polyoxyethylene esters, or stearic acid, mono or dioleic acid, stearic acid or polyoxyethylene sorbitol, or stearic acid, mono or dioleic acid, stearic acid or polyoxyethylene sorbitan And fatty acids.
The antimicrobial composition may further comprise one or more emulsifiers. Suitable emulsifiers include the previously exemplified dispersants or natural rubbers such as acacia gum or tragacanth rubber.
The composition for administration in the method of the present invention includes mixing, grinding, homogenization, suspension, dissolution, emulsification, dispersion, and mixing of the main polymer with selected excipients, diluents, carriers and adjuvants as necessary. Can be prepared by means known in the art for the manufacture of such compositions (such as the veterinary and pharmaceutical compositions industry).

For oral administration, pharmaceutical and veterinary compositions comprise tablets, lozenges, pills, troches, capsules, elixirs, lyophilized powders, solutions, granules, suspensions, emulsifiers, Syrup and tincture dosage forms may also be used. For example, it may be manufactured in sustained or delayed release dosage forms such as coated particles, multilayer tablets or granules.
In general, it is preferred that poly (2-propenal, 2-propenoic acid) is produced from poly (2-propenal) by oxidation of a solid in the air. First, the poly (2-propenal) polymer is heated at 80-110 ° C. primarily in the dry state. More preferably, the polymer is first heated to about 85 ° C. In the alcohol, poly (2-propenal, 2-propenoic acid) is preferably heated in the range of 1 to 1400 hours, more preferably 1 to 60 hours.

The present invention further provides a preservative compound or composition comprising the antimicrobial agent of the present invention.
The present invention further provides a bactericidal and disinfecting compound or composition comprising the antimicrobial agent of the present invention.
Another aspect of the present invention provides a composition for the treatment of gastrointestinal diseases comprising the antimicrobial polymer described above and an additional chemotherapeutic agent, wherein the additional chemotherapeutic agent is adsorbed to the antimicrobial agent.
Adsorption typically reduces the membrane penetration of additional chemotherapeutic agents. Suitable chemotherapeutic agents for use in this embodiment are agents that exhibit a significant decrease in transmembrane when mixed with polymeric antimicrobial agents. It is preferred that penetration is inhibited at a rate of at least 50%.
Useful chemotherapeutic agents for use in this aspect of the invention include antibiotics for treating gastrointestinal diseases and anticancer agents for treating gastrointestinal cancer.
The use of chemotherapeutic agents in combination with polymeric antibacterial agents reduces chemotherapeutic agent transmembrane activity, thereby reducing systemic side effects and providing more targeted treatment. In many cases, odor is also reduced.

Examples of antibiotics that can be used in combination with antibacterial polymers include tetracycline, penicillin, aminoglycosides, sulfa drugs, cephalosporin and nitrofuran.
Antibiotics are conventional antibiotics used to treat gastrointestinal infections.
Examples of anticancer agents that can be used in combination with the polymeric antimicrobial agents of the present invention include alkylating agents, antimetabolites, anticancer antibiotics, plant alkaloids, hormones and other anticancer agents, particularly carbon, hydrogen And an anticancer agent containing only oxygen.
The composition of the present invention comprises phenol (preferably in an amount of 0.1 to 10% by weight), isothiazolinone (preferably in an amount of 0.001 to 1%), alkylparaben (preferably in an amount of 0.02 to 2%). And may include one or more additional antimicrobial agents, such as antimicrobial agents selected from lower alcohols (amounts of 20-99% are preferred), where the amounts are based on the weight of the composition.

  The derivatives of poly (2-propenal, 2-propenoic acid) used in the method of the present invention may have significantly enhanced stability compared to the stability of poly (2-propenal, 2-propenoic acid) polymer. It was found. Since the prior art describes some instability as demonstrated by a loss of antibacterial activity of poly (2-propenal, 2-propenoic acid) compositions, we increased the temperature (ie, 40 ° C.) “Acceleration of aging” was performed. However, very surprisingly, heating poly (2-propenal, 2-propenoic acid) in an aqueous solution or in an aqueous polyethylene glycol solution at an elevated temperature, ie 40 ° C., delayed the antimicrobial activity. In addition, the antibacterial activity of poly (2-propenal, 2-propenoic acid) was increased; see Example 2 (a) and (b). This finding is contradictory and unexpected as a whole, given the prior art that predicts that an increase in temperature should result in "promoting aging", i.e., loss of antimicrobial activity.

As used herein, a method that provides enhanced antibacterial activity by forming a novel configuration of a subject polymer comprising poly (2-propenal, 2-propenoic acid) is referred to as “superactivation” and the polypeptide is designated as “ Referred to as "super-activated polymer".
In view of the prior art, more surprisingly, the inventors have found that superactivation in aqueous polyethylene glycol solutions is promoted by basic and then acidic conditions. Superactivation is also promoted by heat and moisture.
The formation of acetals in the presence of polyethylene glycol or polyol or alkanol protects and stabilizes the carbonyl group of the polymer from alkaline decomposition by the Cannizzaro reaction, which is believed to promote superactivation.
A further advantage of superactivation is the reduction or elimination of contaminating acrolein that is responsible for tissue and skin irritation.

Superactivation is available in aqueous test media as a result of increased hydrophilicity of the polymer as demonstrated in expired Australian patent application AU-A-11686 / 95 (hereinafter “11686/95”). Emphasize that it is completely different from any increase in antibacterial activity obtained only from certain mower polymers. We have found that the method described in 11686/95 is repeated exactly, and then the subsequent superactivation of the partially soluble polymer explicitly causes a particular substantial antimicrobial activity. It should be noted that even the superactivation did not render the 11686/95 polymer completely soluble, in contrast to the superactivation starting with heating initially between 80-85 ° C. It is.
Conversely, the optimum time to reach superactivation of a solution of poly (2-propenal, 2-propenoic acid) depends on the temperature. Even aging at room temperature can be used for superactivation, especially when promoted by the presence of hydroxyl solvents and / or bases, then acidity, but this is evident because it requires longer times It is clear that it is not practical.

The inventors have developed a polymer superactivation as described herein suitable for active ingredients in gastrointestinal treatments, aqueous products or preservatives in the process and fungicides or disinfectants having the advantage of enhanced antimicrobial activity. I found it. Furthermore, the inventors have found that the antibacterial activity of such bactericides or disinfectants increases with increasing their pH, for example pH 6 or higher.
A general feature of the present invention is to attach groups that can exhibit hydrophobic interactions to the antimicrobial agent of the present invention, either by hemiacetal / acetal formation or by adsorption, to enhance antimicrobial activity.
The invention will be described with reference to several examples, which should not be construed as limiting the scope of the invention.

Biocidal test Samples are dissolved in 1 wt% aqueous sodium bicarbonate to obtain the required concentration (0.125 wt% polymer unless otherwise specified). Weigh 19.9 g of the diluted sample into a sterile wide-mouth bottle, inoculate 0.1 mL of 10 ⁷ -10 ⁸ cfu Pseudomonas aeruginosa (Pseudomonas aeruginosa) and mix. At specified time intervals, transfer 1 mL of inoculated sample to 9 mL of resane broth and mix by vortexing. Remove cereal 1 out of 10 dilutions. Pour into trypticase soy agar. Incubate at 37 ° C for 3 days.

Example 1
This example describes a process for producing 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 for preparing starting materials for use in the process of the present invention. In a fume cupboard, add water (720 mL, ambient temperature, about 20 ° C) and acrolein (60 g; freshly distilled, add hydroquinone to 0.25 w / w%) in an open beaker and vigorously mechanically stir To do. Then 0.2 M aqueous sodium hydroxide (21.4 mL) is added to bring the pH to 10.5-11.
The solution quickly changes to the yellow color typical of hydroquinone anions, and the color disappears within 1 minute, and the clear solution becomes milky.

After about 1 minute, it can be seen that the white fluffy polymer begins to precipitate and is completed within 15-30 minutes. The precipitate is filtered, washed with water (250 mL), dried on filter paper for 2 days at room temperature (yield 25 g), then spread into a thin layer in a glass petri dish and heated at 40 ° C. for 8 hours. This heating continues with the following setup: 50 ° C / 15 hours; 65 ° C / 4 hours; 75 ° C / 18 hours; 84 ° C / 24 hours.
For example, it is anticipated that this process can be scaled up such that acrolein is added stepwise in a closed container and then dried more rapidly (Comparative Example 10).
Typically, the poly (2-propenal, 2-propenoic acid) of the resulting poly (2-propenal, 2-propenoic acid) is added to a 1 w / w% aqueous sodium carbonate solution (100 mL) with stirring for 15-30 minutes. Prepare the solution and dilute if necessary. Such a solution is completely clear as opposed to the attempted dissolution using the polymer from Example 5 of 11686/95 as an alternative.

Example 2
This example describes the formation of acetals from poly (2-propenal, 2-propenoic acid).
(a) 5 g of poly (2-propenal, 2-propenoic acid) is dissolved in 64 g of polyethylene glycol (“PEG”) 200 and combined with 31 g of 0.71 w / w% sodium carbonate solution. Keep a part of the solution (apparent pH = 5.8) at room temperature,
(b) Heat the remaining sample at 60 ° C. for 12 or 25 days.
Samples from (a) and (b) are diluted with 1 w / w% sodium bicarbonate and subjected to biocidal testing at a polymer concentration of 0.125 w / w%. Surprisingly, as shown in Table 1, the samples that had undergone “accelerated aging” showed improved antimicrobial activity.

1 g of poly (2-propenal, 2-propenoic acid) is dissolved in 200 mL of 0.1 w / w% Na ₂ CO ₃ and left overnight. Sodium lauryl sulfate is added at a concentration of 0.05 w / w%, and HCl is added to the solution to make it pH 5.9. Store the solution at both room temperature and 60 ° C. Biocidal testing is performed with 0.125 w / w% polymer solution using 1 w / w% NaHCO ₃ as diluent. As shown in Table 2, the “aged” sample shows a surprising improvement in performance.

(c) A 5 w / w% solution of the superactivated polymer is produced in the same manner as in Example 2 (a) except that PEG1000 is used instead of PEG200. A portion of this solution is treated with concentrated NaOH to pH 8.1. Samples are heated to 60 ° C. and subjected to biocidal testing. As shown in Table 3, the samples exposed to more basic conditions unexpectedly showed excellent biocidal performance.

Example 3
This example examines the product produced by the reaction of poly (2-propenal, 2-propenoic acid) and polyethylene glycol.
Determine the presence of acetal in the polymer of Example 2 (b) by examining the solid residue left after dialysis and concentration of the polymer solution using proton (1H) and carbon (13C) NMR spectroscopy. Can do. Dialysis removes all substances with a molecular weight of 1000 or less. Table 4 provides proton (1H) and carbon (13C) data. As shown in Table 4, nuclear magnetic resonance spectroscopy of the residue shows peaks at δ 3.58 and 3.56 for the 1H nuclear magnetic resonance spectrum and at δ 71.62, 69.48 and 60.25 for the 13C nuclear magnetic resonance spectrum. These peaks indicate the binding of polyethylene glycol units as acetals.

物理的および紫外線スペクトルの両方の結果は、安定性におけるPEGの正の効果を実証する：高PEGを含むと物理的および化学的安定性が同大する。 Example 4
(a) A series of 5 w / w% solutions of polymer with a degree of superactivation at an apparent pH of 5.7 are prepared as in Example 2 (a) except that the percentage of PEG 200 is changed.
Samples are heated to 60 ° C and monitored for stability over time. Physical stability regards the occurrence of precipitation or gelation as its reduction. UV measurement is performed on a polymer with a concentration of 0.01 w / w% in a 1 w / w% sodium carbonate solution. Attenuation of the absorbance ratio at 268 nm: 230 nm is considered synonymous with a decrease in chemical stability. The results are shown in Table 5.

Both physical and ultraviolet spectrum results demonstrate the positive effect of PEG on stability: physical and chemical stability are the same when high PEG is included.

(b) by dissolving 4 g of poly (2-propenal, 2-propenoic acid) in 196 g of 1 w / w% sodium bicarbonate and adjusting the pH to 7 (A) and 5.5 (B) with dilute HCl, The following solutions A and B are prepared. Solution C is prepared by dissolving 50 g of poly (2-propenal, 2-propenoic acid) in PEG 200 (640 g) at 65-70 ° C. Then 4 g of an aqueous solution of sodium carbonate (306 g) is added to bring the apparent pH to 7, then to pH 5.5 at the end of the 31 day treatment period.
Store all solutions at 40 ° C. At various time intervals, samples containing the equivalent of 0.125 w / w% polymer are subjected to biocidal testing. The results are shown in Table 6.

ポリマーは、I.R.1700−1730 cm-1においてカルボニルおよび／またはカルボキシル吸収を示した。カルボニル基（たとえば、シッフ試薬など）、およびMw＝約10000 and Mn＝約5000;滴定は、カルボキシル基が約5 mole %であることを示す。これらのパラメーターは、ポリ(２−プロペナール，２−プロペン酸)のものと類似している（しかし、同じではない）。 Example 5
1 g of poly (2-propenal, 2-propenoic acid) is heated for 3 days at 60 ° C. in a dry or humidified closed chamber. Dry and humidified polymer solutions are each prepared at 0.125 w / w% (with water content correction) and subjected to evaluation by biocidal testing.

The polymer showed carbonyl and / or carboxyl absorption at IR1700-1730 cm-1. Carbonyl groups (such as Schiff's reagent), and Mw = about 10000 and Mn = about 5000; titration shows that the carboxyl group is about 5 mole%. These parameters are similar (but not the same) to those of poly (2-propenal, 2-propenoic acid).

Example 6
In duplicate experiments, a polymer sample is prepared exactly as in Example 5 of 11686/95 and then dissolved in ethanediol. Half of this material is further heated at 80 ° C. for 24 hours, after which the standard biocidal test is used to compare the antimicrobial activity of the samples. As shown in Table 8, both samples treated by heating (ie, superactivated) clearly increased antimicrobial activity.

Example 7
50 g of poly (2-propenal, 2-propenoic acid) is dissolved in PEG200 (640 g) at 60-70 ° C. An aqueous solution of sodium carbonate (4 g) in water (306 g) is then added. Divide the sample and let stand at room temperature or heat at 80 ° C. for 24 hours. The acrolein content of the solution was determined over time by reverse phase HPLC and the results are shown in Table 9.

超活性化に必要な時間は、温度に反比例するのが認められる。超活性化プロセスから誘導されたすべての溶液は、すべての比率において水性溶媒と完全に混和する。 Example 8
Analogously to Example 7, solutions of poly (2-propenal, 2-propenoic acid) are prepared and treated at 40, 60, 80, 100 and 115 ° C. for various times. Samples were subjected to standard biocidal testing to confirm increased kill rates and the results are shown in Table 10.

It can be seen that the time required for superactivation is inversely proportional to temperature. All solutions derived from the superactivation process are fully miscible with the aqueous solvent in all ratios.

Example 9
(a) 540 g of poly (2-propenal, 2-propenoic acid) is dissolved in 2304 g of PEG200 at 65 ° C. and then mixed with 43.2 g of sodium carbonate in 712 g of water. The solution is then heated to 100 ° C. for 4 hours, and 36 g sodium lauryl sulfate, 7 g ECOTERIC (nonionic surfactant), 2 g lemon flavor are added. Dilute pH 6 formulation with hard water at 1:30, Staphylococcus aureus (Staphylococcus aureus) (Gram positive bacteria, especially important for nosocomial infections) and Salmonella cholera swiss (S. cholera) (Gram negative bacteria, (Especially important for infections in the cooking area) are challenged using the Agricultural Chemical Society official analysis method (1995) 991.47, 991.48, (painted surface carrier test method), respectively. The results are shown in Table 11.

Adjusting the formulation to a high pH enhances antibacterial activity as monitored by biocidal testing. The results are shown in Tables 12 (a) and 12 (b).

データは、処置プログラムが、微生物カウントをAS/NZ（オーストラリア/ニュージーランド）スタンダード 3666.3(Int):1998のガイドライン内および生物分散剤（通常、非常に暑い夏期の厳しい試験条件には不適切であると考えられる）を含む隣接するタワーにおけるカウント以下に維持したことを示す。 (b) 1200 g of poly (2-propenal, 2-propenoic acid) is dissolved in 7680 g of PEG200 at 60 ° C. and then 96 g of Na ₂ CO ₃ in 3024 g of water is added. The solution is heated at 100 ° C. for 6 hours.
Place the product in the container of the intake ventilation cooling tower to a concentration of 300ppm (30ppm polymer). Dosing three times a week in the evening and resume operation after contact time of 8-12 hours; the residual concentration is expected to halve every 3-6 hours of operation. Recycled water on average has a temperature of 27 ° C., a pH of 8.5, and a conductivity of 3000 microS. Microbial counts are determined and compared to adjacent identical towers administered daily with biodispersant. The results are shown in Table 13.

Data show that the treatment program is inadequate for microbial counts within the AS / NZ (Australia / New Zealand) Standard 3666.3 (Int): 1998 guidelines and biodispersants (usually in very hot summer conditions) It is kept below the count in the adjacent tower including (possible).

Example 10
Comparative Example 10 (a)
This example illustrates a method for producing an acrolein polymer without using the method of the present invention.
1.0 0.8w / w% sodium hydroxide
9.90kg deionized water is placed in a 10L stainless steel bowl and 0.08kg sodium hydroxide is added to the water and stirred until dissolved.
2.0 Polymerization
Place 100.1 kg of deionized water in a 200 L stainless steel bowl and add 0.8 w / w% sodium hydroxide solution to the 200 L tank. The solution is equilibrated at 15-20 ° C. At the same time, 20 kg of acrolein monomer and the remaining 0.8 w / w% sodium hydroxide solution are added to the 200 liter over 1 hour such that the pH is maintained at 10.5-11.0 and the temperature does not exceed 30 ° C. The polymerization is continued for another 90 minutes.
3.0 Wash The polymerization mixture is filtered / centrifugated and the polymer is washed with deionized water until the wash pH is below 7.0. The approximate yield is 8 kg.
4.0 Drying The polymer is air dried and then heated in an oven using the following program.
Step Time Temperature
1 2 hours 25 ℃
2 1 hour 40 ℃
3 1 hour 70 ℃
4 1 hour 75 ℃
5 2 hours 85 ℃
5.0 Dissolution
Add 400L of water to a 500L jar, add 4kg of sodium carbonate and stir until dissolved. Slowly add 8 kg of dry, heated polymer and stir for 30 minutes.
The resulting polymer is found to have an approximate solubility in 1 w / w% sodium carbonate of 90-95 w / w%.

Example 10 (b)
This example illustrates a method for producing an acrolein polymer in which the polymer of the comparative example is superactivated by the method of the present invention.
1.0 Base manufacture In a suitable container, 0.4 kg of sodium carbonate is dissolved in 30.6 kg of water and 64 kg of polyethylene glycol 200 is added to the mixing container. Start stirring with a mechanical stirrer and heat PEG200 to 65 ± 3 ° C. To the PEG 200 is added 5 kg of the dry acrolein polymer of Example 10a and stirred until a homogeneous mixture is obtained.
Note: At this stage, the solid does not dissolve completely.
Sodium carbonate solution is slowly added to the glycol mixture at such a rate that the pH of the solution is maintained in the range of 3.5-9.0.
The solution is stirred at 65 ± 3 ° C. for 45 minutes.
Note: The pH should be in the range 7-9. The temperature should be in the range of 65 ± 3 ℃.
2.0 Superactivation Cap the mixing vessel and heat at 100 ° C for 4 hours. The resulting polymer is found to have an approximate solubility in water of 99.5-100 w / w%.

Example 11
This example examines the antibacterial activity of the dry, normally activated poly (2-propenal, 2-propenoic acid) polymer of Example 10a and the anti-activated acetal derivative of Example 10b.
The chickens treated with each antimicrobial agent are compared to the control group according to the following procedure.
In each test, 20 Cob chickens (line 53) are purchased from commercial hatcheries. They are weighed, gendered, and randomly assigned to adjacent cages in an isolated zoo room. The distribution of male and female birds is the same. Water and feed are freely available. The feed is a commercial crumble (Chick Starter, Milne Feeds: 18% crude protein) supplemented with an anti-coccidial agent (125 ppm dinitramide).
Chickens are administered 0.1 w / w% of the normally activated aqueous antimicrobial solution formulation of Example 10a via a static water dispenser for 14 days; the dose is 30 mg / kg / day. Another 10 chickens are a control group.
Both groups of chickens are weighed on days 0, 4, 7, 11 and 14. At the completion of the study, all chickens are euthanized and the treated chickens are necropsied. Perform a gross examination of the chest and abdominal cavity.

コントロールグループと比較して、処置グループは、１４日間の期間の終わりに測定可能的に体重が増加する。
試験完了時の検死時、処置グループのニワトリにおいて、肉眼的検査では、毒性の臨床的または病理的徴候は明らかでない。

残りのニワトリにおける試験完了時の検死時、両グループのニワトリにおいて、肉眼的検査では、毒性の臨床的または病理的徴候は明らかでない。 result:

Compared to the control group, the treatment group gains measurable weight at the end of the 14 day period.
At necropsy at the completion of the study, clinical or pathological signs of toxicity are not evident in the treatment group of chickens by gross examination.

At necropsy at the completion of the study in the remaining chickens, no clinical or pathological signs of toxicity are evident in macroscopic examinations in both groups of chickens.

Conclusion:
There was a significant difference in weight gain of the treated group compared to the control group p (χ ² ; P <0.015). At study completion, the treatment group is 23% heavier than the control group.
In the following examples, a significant improvement in weight gain of the superactivated acetal derivative when compared to the control compared to the normally activated poly (2-propenal, 2-propenoic acid) Demonstrate that it provides a significant improvement in activity.

Example 12
This example evaluates the polymer antibacterial agent of Example 10b in field conditions against control of porcine post-weaning colitis (PWC).
Method:
146 piglets that were administered orally with various formulations of the superactivated antimicrobial agent, APRALAN® (Elanco) in feed or water, or self-vaccinated or “no-weaned” The “piggy” is challenged with weaning-related stress in a large commercial pig house with a long history of PWC problems.
Their responses in diarrhea incidence, weight gain and mortality are evaluated and are shown in Table 15.

注：
１．処置の符号化：
ｉ．グループ１＝飼料中の0.1w/w%の超活性化ポリマー抗菌剤
ii．グループ１＝水中の0.02w/w%の超活性化ポリマー抗菌剤
２．試験中にPWCで死亡したグループのパーセンテージ
３．便スコア１または２が記録される場合のグループ内の各ブタの平均日数；便スコアは下痢の強度の尺度である。
４．ブタ毎に観察されたサンプルの数で割った便スコアの総和
５．Ｆ＝フィッシャーの直接確率検定

note:
1. Treatment encoding:
i. Group 1 = 0.1 w / w% super-activated polymer antibacterial agent in feed
ii. Group 1 = 0.02 w / w% superactivated polymer antibacterial agent in water 2. Percentage of groups that died of PWC during the study. Average days for each pig in the group when stool score 1 or 2 is recorded; stool score is a measure of the intensity of diarrhea.
4). 4. Sum of stool scores divided by the number of samples observed per pig F = Fisher's exact test

Conclusion:
This field trial shows an acetal derivative (superactivated antibacterial) of poly (2-propenal, 2-propenoic acid) for use with piglets in commercial pig farms when challenged with β-hemolytic E. coli after weaning. The following points were positively considered for the efficacy of the drug.
1. Mortality Mortality is lower in any group treated with a superactivated polymer antibacterial than any of the untreated, vaccinated or Apralan® groups.
2. Diarrhea days:
There is significantly fewer days of diarrhea in any group treated with the superactivated antimicrobial than any of the untreated, vaccinated or Apralan® groups [F; P <0.0001].
3. Stool score The mean stool score is significantly lower in any group treated with superactivated antimicrobial than any of the untreated, vaccinated or Apralan® groups [F; P <0.0001].

Example 13
This example examines the effect of using the antimicrobial agent of the present invention and certain additives.
Method:
Prepare an overnight broth of the challenge culture and evaluate the total survival count (TVC) of the overnight culture.
Dilute the sample serially 1: 1 with sterile standard saline (5 mL). When testing samples containing EDTA, use sterile hard water (SHW) as the diluent.
Dilute 1 part of each overnight culture with 9 parts of diluent. The diluted suspension obtained is used as the inoculum solution.
Inoculate each sample dilution containing 100 microL of diluted overnight culture. (1 culture / 1 test tube). Mix well.
Incubate the tubes for ~ 24 hours at 37 ° C (A. nigera is incubated for ~ 24 hours at 28 ° C).
Subculture 1 mL from each tube into 9 mL recovery broth and mix well (recovery broth: nutrient broth for polymer antibacterial and EDTA + 3% Tween 80 (NBT); nutrient broth for glutaraldehyde + 3% Tween 80 + 0.1% ammonia (NBTA); and, for methylparaben, resene broth (LB)).
Samples are incubated for an additional ˜48 hours at 37 ° C. (A. nigera ˜5 days at 28 ° C.).
Examine each tube for growth. All tubes are then subcultured on selective agar and incubated for ˜24 hours at 37 ° C. (A. nigera for ˜5 days at 28 ° C.). Grow on selective agar to confirm growth of test microorganisms.
Perform positive control using 5 mL of the diluted inoculum of the culture, incubate, collect and confirm.
Perform a negative control with a 5 mL dilution of non-inoculum, incubate, collect and confirm.

凡例：
1) ポリマー抗菌剤 0.025w/w%
2) ポリマー抗菌剤 0.2w/w%
3) グルタルアルデヒド 0.025w/w%
4) ポリマー抗菌剤 0.025w/w%＋グルタルアルデヒド 0.025w/w%
5) ポリマー抗菌剤 0.2w/w%＋グルタルアルデヒド 0.025w/w%
6) ポリマー抗菌剤 0.2w/w%
7) EDTA 0.1w/w%
8) ポリマー抗菌剤 0.2w/w%＋EDTA 0.1w/w%
9) ポリマー抗菌剤 0.2w/w%(80w/w% グリセロール中)
10) メチルパラベン 1w/w%(80w/w% グリセロール中)
11) ポリマー抗菌剤 0.2w/w%(80w/w% グリセロール中)＋メチルパラベン 1w/w%(80w/w% グリセロール中)。 Results: The results are shown in the table below.

Legend:
1) Polymer antibacterial agent 0.025w / w%
2) Polymer antibacterial agent 0.2w / w%
3) Glutaraldehyde 0.025w / w%
4) Polymer antibacterial agent 0.025w / w% + glutaraldehyde 0.025w / w%
5) Polymer antibacterial agent 0.2w / w% + glutaraldehyde 0.025w / w%
6) Polymer antibacterial agent 0.2w / w%
7) EDTA 0.1w / w%
8) Polymer antibacterial agent 0.2w / w% + EDTA 0.1w / w%
9) Polymer antibacterial agent 0.2w / w% (in 80w / w% glycerol)
10) Methylparaben 1w / w% (in 80w / w% glycerol)
11) Polymer antibacterial agent 0.2w / w% (in 80w / w% glycerol) + methylparaben 1w / w% (in 80w / w% glycerol).

注：相乗効果＜1.0、相加的＝1.0、拮抗的＞1.0
SI＝CD/A＋CE/Bにより算出
A＝MKC ポリマー抗菌剤
B＝MKC 保存剤
C＝MKC 混合物
D＝Bに対するAの比率
E＝Aに対するBの比率

Note: Synergistic effect <1.0, additive = 1.0, antagonistic> 1.0
Calculated by SI = CD / A + CE / B
A = MKC polymer antibacterial agent
B = MKC preservative
C = MKC mixture
D = Ratio of A to B
E = Ratio of B to A

Conclusion:
Acetal derivatives of poly (2-propenal, 2-propenoic acid) have a synergistic effect with A. Nigella, C. albicans, E. coli, P. aeruginosa and S. aureus with glutaraldehyde, EDTA and methylparaben, respectively. It is clear to have.

Example 14
This example demonstrates the activity of the antimicrobial agent of the present invention in combination with the “Dettol” brand antimicrobial agent.
Dilute the sample serially 1 to 1 with sterile saline (5 mL).
Each sample dilution is inoculated with 100 microL diluted overnight culture (one culture per tube). Mix the sample well. Incubate tubes at 37 ± 2 ° C. for ˜24 hours (test tubes inoculated with Aspergillus nigera are incubated at 28 ± 2 ° C. for ˜24 hours).
From each tube, subculture 1 mL into 9 mL recovery broth and mix thoroughly by vortexing (for A. nigera, NBT or Sabouraud + 3% Tween 80 (SABT).
Tubes are incubated at 37 ± 2 ° C. for ˜48 hours (A. nigera at 28 ± 2 ° C. for an additional 5 days).
Examine the recovery broth for turbidity (growth) and streak into selective agar to confirm growth.

凡例：
1) 0.1w/w% ポリマー抗菌剤
2) 0.2w/w% ポリマー抗菌剤
3) 「デットール」4.8% w/v(1:20希釈)
4) 0.1w/w% ポリマー抗菌剤＋「デットール」(1:20希釈)
5) 0.2w/w% ポリマー抗菌剤＋「デットール」(1:20希釈)

Legend:
1) 0.1w / w% polymer antibacterial agent
2) 0.2w / w% polymer antibacterial agent
3) `` Detall '' 4.8% w / v (1:20 dilution)
4) 0.1w / w% polymer antibacterial agent + "Detol" (1:20 dilution)
5) 0.2w / w% polymer antibacterial agent + "Detole" (1:20 dilution)

注：SI＞１ならば拮抗的、SI＝１ならば相加的、SI＜１ならば相乗的
SI＝CD/A＋CE/B
A＝MKC ポリマー抗菌剤(ppm)
B＝MKC デットール(ppm)
C＝MKC of ポリマー抗菌剤／「デットール」混合物(ppm)
D＝デットールに対するポリマー抗菌剤の比率
E＝ポリマー抗菌剤に対する「デットール」の比率
注：「デットール」中の活性抗菌剤は、クロロキシレノールである。

Note: antagonistic if SI> 1, additive if SI = 1, synergistic if SI <1
SI = CD / A + CE / B
A = MKC polymer antibacterial agent (ppm)
B = MKC debt (ppm)
C = MKC of polymer antibacterial agent / "Detole" mixture (ppm)
D = ratio of polymer antibacterial to dettle
E = Ratio of “Detall” to polymer antibacterial agent Note: The active antibacterial agent in “Detol” is chloroxylenol.

Conclusion:
It is clear that the polymer antibacterial agent of the present invention has a synergistic effect with “Dettor” against E. coli, S. aureus, P. aeruginosa, C. albicans and A. nigera. This indicates that "Detall" and the polymeric antimicrobial agent perform substantially better when used together as a mixed solution than when used alone.

Example 15
Poly (2-propenal, 2-propenoic acid) [superactivated] as a disinfectant of poly (2-propenal, 2-propenoic acid) [superactivated] Poly (2-propenal, 2-propenoic acid) [superactivated] is followed for disinfectant quality. Many bacteria present on the subject's hands are measured before and after applying the antimicrobial agent and wearing surgical gloves. Compare the disinfection effect of poly (2-propenal, 2-propenoic acid) [superactivated] with the commonly used surgical disinfectant 4% Chlorhexidine Surgical Scrub (Western Australia, Perth, Orion Laboratories) .
A 3 w / w% aqueous solution of poly (2-propenal, 2-propenoic acid) [superactivated] reduces the baseline count of the bacterial population on the gloves after 3 hours.
Poly (2-propenal, 2-propenoic acid) [superactivated] 2 w / w% aqueous solution + 70% ethanol is similar to 4% chlorohexidine in the persistence of baseline counts of bacterial populations on gloves after 3 hours Indicates a decrease.
3.2 w / w% aqueous solution of poly (2-propenal, 2-propenoic acid) [superactivated] + 3.1% sodium lauryl sulfate applied to the hand before wearing surgical gloves, then 70% A 4 w / w% aqueous solution of poly (2-propenal, 2-propenoic acid) [superactivated] in ethanol significantly reduces the baseline bacterial count after 3 hours.
The results show that poly (2-propenal, 2-propenoic acid) [superactivated] has good residual antibacterial activity necessary for sustained control of bacterial count in surgical aseptic conditions. Inclusion of 70% ethanol in the formulation helps with an early rapid decrease in bacterial count.

Example 16
Biocidal activity of 0.125 w / w% and 0.05 w / w% superactivated polymer against the reference strain Helicobacter pylori NCTC 11637 at pH 7 and pH 4 Established against the strain Helicobacter pylori NCTC 11637. Two variables are chosen as variables; one is a 5% solution of the superactivated polymer prepared in Example 2 that gives a concentration of 0.125 w / w% of the superactivated polymer that mimics dilution in the stomach. The other is a 100-fold dilution that gives a concentration of 0.05 w / w% of the superactivated polymer. Two pHs are chosen as the baseline: pH 7 and pH 4, which is a mimic state in the stomach.

  Cultures of H. pylori NCTC 11637 are grown microaerobically on selective agar plates at 37 ± 2 ° C. until full growth is observed. The growth is aseptically removed from the plate, diluted with sterile saline and prepared as a 10% T standardized turbid suspension as indicated on the Vitek colorimeter. Weigh 19.9 g sample and inoculate 100 microL culture suspension. Immediately transfer 1 mL of sample to inactivated / recovered broth (Nutrient Broth + 3% Tween 80) and then serially dilute. A 100 micro aliquot is placed on a selective agar plate and spread using a sterile disposable spreader. Repeat the moving step at time intervals of 5, 10, 15 and 20 minutes. All plates are microaerobically incubated at 37 ± 2 ° C. until sufficient growth is achieved (approximately 5-7 days). Count all colonies and determine the descending slope of the population over time. The test is repeated using sterile saline as a sample to determine the natural solids depletion rate at atmospheric conditions.

注：コロニー形成ユニット(cfu)におけるカウント／失活ブロスmL
参考菌株に対する結果（第２０表）は、超活性化ポリマーが、pH 7において0.125w/w%および0.05w/w%の両方の濃度で有効であり、またpH4において0.125w/w%の濃度が有効であることを示す。 Legend:
Culture 1. Superactivated polymer, pH 7, 0.125w / 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 saline, pH 7.
Culture 6. Sterile saline, pH 4.

Note: Count / inactivated broth in colony forming unit (cfu)
The results for the reference strain (Table 20) show that the superactivated polymer is effective at both concentrations of 0.125 w / w% and 0.05 w / w% at pH 7, and a concentration of 0.125 w / w% at pH 4 Indicates that is valid.

0.125w/w%の超活性化ポリマーでpH7にて処置すると、すべての菌株が速やかに殺され、抗生物質耐性菌株は特に脆弱であり10分以内に死亡する（第２１表）。コントロール菌株は、無処置である。 Example 17
Following the analysis of Example 16, three additional H. pylori strains are examined at pH 7 and pH 4: H. pylori 01/303 resistant to clarithromycin and metronidazole; possible animal models isolated in Sydney H. pylori SS1 is a clinical strain with high colony-forming ability that is of interest to H. pylori; and H. pylori ATCC 700392, a genome from which the genome has been sequenced.

When treated with 0.125 w / w% superactivated polymer at pH 7, all strains are killed rapidly, antibiotic resistant strains are particularly vulnerable and die within 10 minutes (Table 21). The control strain is untreated.

胃を模倣しているpH4であって0.125w/w%の超活性化ポリマーの試験の結果、すべての菌株が20分以内に殺される（第２２表）。H.ピロリを殺すための時間枠が胃を通過する時間（40分〜1時間）以下なので、この結果は重要である。これは、胃内でのH.ピロリ感染の処置に一致するpH、濃度および時間枠における活性化ポリマーの有効性を実証する。コントロール菌株は、無処置である。 Example 18
The method of Example 3 is repeated in testing the biocidal activity of 0.125 w / w% superactivated polymer (pH 4) against all H. pylori strains.

Testing of 0.125 w / w% superactivated polymer at pH 4 mimicking the stomach kills all strains within 20 minutes (Table 22). This result is important because the time frame for killing H. pylori is less than the time through the stomach (40 minutes to 1 hour). This demonstrates the effectiveness of the activated polymer in pH, concentration and time frame consistent with treatment of H. pylori infection in the stomach. The control strain is untreated.

Example 19
This example demonstrates the intestinal antimicrobial activity of an acetal derivative of poly (2-propenal, 2-propenoic acid) prepared according to the procedure of Example 10b.
Materials and methods:
Sixteen freshly weaned piglets (age: 18 days ± 2 days and weight: 5.5 kg ± 1.0 kg) are purchased from a commercial pig farm. They are randomly assigned to two groups of 8 (same gender distribution) and housed in an environmentally controlled and isolated animal house.
Water and feed are freely available in the animal house, and the feed is commercially available anti-bacterial-free baby food pellets [19% crude protein].
All new weaned piglets are euthanized by intravenous injection of sodium barbiturate and then necropsied. At autopsy, DNA from the stomach and esophagus area of the abdomen of 24 piglets is extracted using the Qiagen Dneasy tissue kit according to the instructions for use. 3 microL of extracted DNA is used to test the presence of Helicobacter in biopsy tissue samples. Perform polymerase chain reaction (PCR) twice for each sample and the seven control DNA samples included in each PCR run. There is no discrepancy between the PCRs performed.

グループ1（無処置）では、ヘリコバクター属に対する5つのポジティブなPCR結果(1つ−胃、4つ−食道)があるが、グループ2(0.1w/v% of ポリマー抗菌剤)では、ポジティブなPCRの結果はない。
結論：
0.1w/v%のポリ(２−プロペナール，２−プロペン酸)のアセタール誘導体は、離乳したての子ブタの胃および食道粘膜におけるブタヘリコバクター属の出現を有意に減少させる(χ²: P<0.025)。 result:
Treatment encoding:
Group 1: No treatment (negative control)
Group 2: Superactivated polymer antimicrobial agent prepared according to Example 10b 0.1 w / v%; 30 mg / kg / day.

In group 1 (no treatment), there are 5 positive PCR results (1-stomach, 4-esophagus) for Helicobacter, but in group 2 (0.1 w / v% of polymeric antibacterial agent) There is no result.
Conclusion:
An acetal derivative of 0.1 w / v% poly (2-propenal, 2-propenoic acid) significantly reduces the appearance of Porcine Helicobacter in the stomach and esophageal mucosa of freshly weaned piglets (χ ² : P < 0.025).

Example 20
Comparative Example 20 (a)
This example demonstrates a method for producing non-superactivated poly (2-propenal, 2-propenoic acid).
0.8w / w% sodium hydroxide
9.90kg deionized water is placed in a 10L stainless steel bowl and 0.08kg sodium hydroxide is added to the water and stirred until dissolved.
polymerization
Place 100.1 kg of deionized water in a 200 L stainless steel bowl and add 0.8 w / w% sodium hydroxide solution to the 200 L tank. The solution is equilibrated at 15-20 ° C. At the same time, 20 kg of acrolein monomer and the remaining 0.8 w / w% sodium hydroxide solution are added to the 200 liter over 1 hour such that the pH is maintained at 10.5-11.0 and the temperature does not exceed 30 ° C. The polymerization is continued for another 90 minutes.
Wash Filter / centrifuge the polymerization mixture and wash the polymer with deionized water until the pH of the wash is below 7.0. The approximate yield is 8 kg.
Drying The polymer is air dried and then heated in an oven using the following program.
Step Time Temperature
1 2 hours 25 ℃
2 1 hour 40 ℃
3 1 hour 70 ℃
4 1 hour 75 ℃
5 2 hours 85 ℃
Dissolution
Add 400L of water to a 500L jar, add 4kg of sodium carbonate and stir until dissolved. Slowly add 8 kg of dry, heated polymer and stir for 30 minutes.
The resulting polymer is found to have an approximate solubility in 1 w / w% sodium carbonate of 90-95 w / w%.

Example 20 (b)
This example illustrates a method for producing an acrolein polymer in which the polymer of Comparative Example 20 (a) is superactivated.
Base production In a suitable container, 0.4 kg of sodium carbonate is dissolved in 30.6 kg of water and 64 kg of polyethylene glycol 200 is added to the mixing container. Start stirring with a mechanical stirrer and heat PEG200 to 65 ± 3 ° C. To the PEG 200 is added 5 kg of the dry acrolein polymer of Example 10a and stirred until a homogeneous mixture is obtained.
Note: At this stage, the solid does not dissolve completely.
Sodium carbonate solution is slowly added to the glycol mixture at such a rate that the pH of the solution is maintained in the range of 3.5-9.0.
The solution is stirred at 65 ± 3 ° C. for 45 minutes.
Note: The pH should be in the range 7-9. The temperature should be in the range of 65 ± 3 ℃.
Cap the superactivated mixing vessel and heat at 100 ° C for 4 hours. The resulting superactivated polymer is found to be miscible with water in all proportions.

Example 21
In Example 14 of PCT / AU9600328, poly (2-propenal, 2-propenoic acid) polymer in 0.5 w / w% sodium carbonate solution has anti-cancer activity against Ehrlich ascites cell line in a mouse model Has been demonstrated.
Anti-cancer activity of poly (2-propenal, 2-propenoic acid) polymer [Example 20 (a)] against the anti-cancer activity of superactivated polymer [Example 20 (b)]. An in vitro model of gastrointestinal cancer is performed in the human colon cancer cell line HT-29. Poly (2-propenal, 2-propenoic acid) is used at a concentration of 5 w / w%. In tests with, in order to obtain a plot can establish IC _50, the cancer cells are incubated with polymers of various concentrations.

methodology
HT-29 cells (human colon cancer cells) are seeded (100 microliters each) in a 96-well culture plate and incubated overnight at 37 ° C. in a humidified CO ₂ 5%, air 95% atmosphere. Polymers [poly (2-propenal, 2-propenoic acid) polymer of Comparative Example 20 (a) and superactivated polymer of Example 20 (b)] were dissolved in water and then in the 4-log range in the medium. Dilute to 10 concentrations. 100 microliters of each solution is then placed in 5 wells each. Plates were further incubated for 72 hours before 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) is used to measure viable cells. Cells were then fixed with 10% cold trichloroacetic acid for 1 hour at 4 ° C., plates were rinsed with distilled water, air dried and then 0.4% sulforhodamine B (Aldrich) (v / v) in 1% acetic acid. Stain for 30 minutes. Unbound dye is removed by washing twice with distilled water and finally with 1% acetic acid. The protein-bound dye is then solubilized in 10 mM unbuffered Tris base and the absorbance at 550 nm is measured using an automated plate reader. The average absorbance of each drug is expressed as a percentage of the absorbance of untreated control wells.

IC_５０は、細胞成長を50%阻害するのに必要な濃度である。
最後に、当然のことながら、本明細書に概略した本発明の範囲を逸脱することなく、種々の他の修正および／または変更をなすことができる。
The test results are shown in Table 24.
The average IC ₅₀ in two tests of the poly (2-propenal, 2-propenoic acid) polymer of Comparative Example 20 (a) is 0.030%; the assigned poly (2-propenal, 2-propenoic acid) polymer is The value is 100%. This corresponds to 0.0015 w / w% active polymer.
The average IC ₅₀ in four tests of the superactivated polymer of Example 20 (b) is 0.025%. This corresponds to 0.00125 w / w% superactivated active polymer, indicating that the superactivated polymer has potent anti-cancer activity.

IC ₅₀ is the concentration required to inhibit cell growth by 50%.
Finally, it should be understood that various other modifications and / or changes can be made without departing from the scope of the invention as outlined herein.

Claims (17)

  Poly (1 to 1400 hours in the temperature range of 40 ° C. to 150 ° C. to form a protective carbonyl group for treating or preventing gastrointestinal diseases selected from gastroenteritis, gastric ulcer, diarrhea, gastrointestinal cancer and dysentery Preparation of a pharmaceutical for oral administration to animals, comprising as an active ingredient a polymer derivative of poly (2-propenal, 2-propenoic acid) formed by a reaction between 2-propenal, 2-propenoic acid) and polyethylene glycol Formed by reaction between poly (2-propenal, 2-propenoic acid) and polyethylene glycol for 1 to 1400 hours in the temperature range of 40 ° C. to 150 ° C. to form a protected carbonyl group Use of polymer derivatives of (2-propenal, 2-propenoic acid).   The use according to claim 1, wherein the gastrointestinal disease is at least one of diarrhea, gastroenteritis and dysentery.   Use according to claim 1, wherein the animal is selected from dogs, pigs, sheep, horses, goats, cattle, cats, poultry, ducks, turkeys and quails.   Use according to claim 1, wherein the animal is selected from poultry and pigs.   The use according to claim 1, wherein the animal is a poorly growing pig.   Use according to claim 1, wherein the medicament is an oral medicament and the gastrointestinal disorder is post-weaning porcine post-weaning colibacillosis. Use according to claim 1 in the manufacture of a medicament comprising a polymer derivative in a dose for administration at 0.05 to 5000 mg / kg / day . Use according to claim 1 in the manufacture of a medicament comprising a polymer derivative in a dose for administration at 0.05 to 500 mg / kg / day . Use according to claim 1 in the manufacture of a medicament comprising a polymer derivative in a dose for administration at 0.05 to 50 mg / kg / day .   Gastrointestinal diseases are coliforms, Salmonella, P. aeruginosa, Helicobacter, Enterobacteria, Yeasts, Protozoa, Clostridium ( The use according to claim 1, resulting from one or more microorganisms selected from Clostridia, Shigella and Coccidia.   The use according to claim 1, wherein the gastrointestinal disease is a disease of the gastrointestinal tract caused by Helicobacter infection.   The use according to claim 1, wherein the gastrointestinal disease is caused by at least one of E. coli and β-hemolytic E. coli.   The use according to claim 1, wherein the gastrointestinal disease is necrotizing enterocolitis in poultry.   The use according to claim 1, wherein the gastrointestinal disease is poultry coccidiosis.   The use according to claim 1, wherein the derivative comprises a number of protected carbonyl groups selected from at least one hemiacetal and acetal groups.   The use according to claim 15, wherein the protected carbonyl group comprises an acetal.   The use according to claim 1, wherein the polyethylene glycol is a polyethylene glycol having a number average molecular weight of 200 to 2,000.