AU668160B2 - Preparation useful for prevention and treatment of intestinal infections in animals and human beings - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): PHARMALETT DANMARK A/S Invention Title: PREPARATION USEFUL FOR PREVENTION AND TREATMENT OF INTESTINAL INFECTIONS IN ANIMALS AND HUMAN BEINGS

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The following statement is a full description of this invention, including the best method of performing i.t known to me/us: PREPARATION USEFUL FOR PREVENTION AND TREATMENT OF INTESTINAL INFECTIONS IN ANIMALS AMD HUMAN BEINGS Background of the Invention It is known that Psyllium Husk (also known as Isphagula Husk), in common with many other compounds, such as charcoal, kaolin, and pectins, is capable of binding bacteria to some extent.

To the best of our knowledge, this is a physical binding, similar to the adsorption of chemicals to the surface of activated charcoal. It is also known that divalent ions may increase the adhesion of bacteria to pectins. In this context, it will be clearly understood that adsorption 15 means adhesion to a surface, as distinct from absorption in which something is absorbed into the interior of the absorbing material. To the best of our knowledge, the prior art contains no description of the above substances being capable of adsorbing very effectively some bacteria 20 in preference to others, with the help of a combination of monovalent ions.

ooo Intestinal Flora Normally, many different types of bacteria are present in 25 the intestinal canal. Some are harmless and are important *o in homeostasis, while others are pathogenic or potentially pathogenic under certain conditions. The pathogenic organisms may be present in the intestinal canal of the host in a "dormant state", and do not become pathogenic until they are exposed to some influence, such as for example stress, change of foodstuffs or virus infection.

Two types of pathogenic bacteria present in the intestinal canal are of particular interest: 1) the invasive bacteria that infiltrate into the blood of the host organisms causing septicaemia, and 2) the toxin producing bacteria, that cause toxaemia.

The coliforms and in particular Entero Toxic E. coli (ETEC) that produce colitoxin are of special interest in the present invention.

An activated ETEC is mobile,'and seeks the internal surface of the intestine and adheres to a receptor on the surface of the intestinal wall where it starts to produce toxin.

It is the toxin that is dangerous as in its presence it is 15 more difficult and sometimes no longer possible for the host animal to retain water in the intestinal canal, nor to absorb water or nourishment therefrom. As a result the animal host will dehydrate and in many cases die. Known agents for treatment of this condition include Diaproff 20 (see U.S. patent 5,038,396).

Standard treatments of ETEC and other undesirable bacteria include prophylaxis by administering small amounts of antibiotics in the feedstuff to control the infection, or 25 to reduce the pH of the intestinal canal by means of various acids administered orally. Improving hygiene is also of assistance.

Alternatively, or in addition to the foregoing, therapeutic doses of antibiotics may be given by injection or orally to kill the undesirable bacteria. However, such administration of antibiotics is not specific to the undesirable bacteria, and will kill all bacteria, including 4 the bacteria normally residing in the intestine which are protective of the host, resulting in further complications.

The Invent ion We have found that administration of Psyllium Husk combined with sodium and potassium ions, is unexpectedly effective in binding ETEC.

Although we do not wish to be limited by any hypothetical or postulated mechanism for the observed beneficial effects, we believe that Psyllium Husk in the presence of sodium and potassium ions selectively adsorbs undesirable bacteria and in particular coliforms such as ETEC from the intestinal contents to such an extent that the solution 15 will be almost totally purified therefrom, while at the *same time the normal intestinal flora are substantially unaffected. Although the addition of either sodium or potassium ions has some effect, we have found that by adding both sodium and potassium ions simultaneously, we 20 achieve a synergistic effect exceeding the additive effective of either sodium or potassium ions independen~tly.

The compositIon of Psyllium Husk containing the said sodium and potassium ions may be administered either dry or suspended in water, and may further be used in combination 25 with other biological, organic or inorganic active substances.

The breaking out of diarrhoea is thus prevented by selective removal of the undesirable bacteria, such as ETEC from the site, leaving the normal flora unaffected, and thus optimwum conditions of growth and continuing heal~ily existence of the animal are secured. The present invention greatly reduces the need for consumntion of antibiotics and by doing so also minimises the attendant problem of the generation of antibiotic resistant bacteria that may be potentially pathogenic under certain conditions.

Detailed Description of the Invention Psyllium Husk, also known as Isphagula Husk, hereinafter referred to as PH, contains mucopolysaccarides which in combination with water form a gel (see British Pharmacopaeia 1980, vl. 2:246-247). The MP consist of a polyxylose backbone with side chains containing galactose, mannose, glucose, fructose, rhamnose and arabinose (see Sandh, J.S. 1981. The gel structure of carbohydrate of Isphagula husk ex Plantago ovata Forsk. Carbohydr. Res.

93:247-259, and Dzuyuba, N.P. et al. 1977, Estab3ishment of S" 15 the qualitative and quantitative composition of the polysaccharide of vegetal material and preparations by physico chemical methods Farm Zh. 32: 56-59). The polyxylose backbones are resistant to breakdown by intestinal bacteria or enzymes, with the exception of 20 Bacteroides ovatus (see Salyers. D. at al. 1978, Breakdown of Psyllium hydrocelloid by Strains of Bacteroides ovatus from the human intestinal tract, Can. J. Mic'obiol. 24: 336-338).

25 Isphagula has been used in the past as a local traditional medicine against diarrhoea (see Singh, B.H. 1940 Indian Med. Gaz.: 733).

More recently it has been shown that PH added to electrolyte glucose mixtures stimulates the absorption of glucose and consequently stimulates rehydration in dehydrated animals and humans patent 5,038,396).

We have found that PH together with a combination of sn'41c 6 and potassium ions is far more effective that PH alone. It is believed that this is due to an improved affinity for pathogens which are selectively adsorbed, avoiding the disadvantages of the prior art.

Examples The bacterial strains used in this study was E. coli K88ab (ETEC strains).

Four solutions were made: Solution PH was made in Nutrient Broth (Oxoid CM) and tap water (1 gram/15 ml).

15 Solution was made as solution however 80 mmol/L sodium was added as sodium chloride.

Solution was made as solution however 15 mmol/L potassium was added as potassium chloride.

Solution was made as solution however 80 mmol/L sodium and 15 mmol/L potassium was added as sodium chloride and potassium chloride.

25 In general bacterial suspensions containing 50 million colony forming units per ml were made.

Qualitative adherence Equal amounts of the solutions and bacterial suspensions were homogenized and incubated aerobically at 37 0

C

0.1 0 C) in a water bath. The suspensions were gently shaken for 15 min. Just before and during incubation samples were taken at regular intervals from the bacteria/solution mixtures.

7 Adherence to PH particles was determined in both native preparations under phase contrast microscope and methyleneblue stained preparations under normal light microscope with 400 and 1000 times magnification (Zeiss, 40 and 100 phase-contrast and standard oil jimersion lenses).

Semi-q-uantitative adherence Centrifugation was used to separate the bacteria-PH mixtures into a precipitation fraction containing bacteria adhered to PH and a supernatant fraction with only free bacteria. The PH-bacteria mixtures were centrifugated (Labofuge GL, radius=150 mm) in 10 ml tubes at 600 rpm for 15 min. Before and after centrifugation the optical density of the mixtures and the supernatants were 15 measured (wavelength 541 nm). The difference in optical :::o:density before and after centrifugation is a semi quiantitative measure for the numbers of bacteria adhered to PH particles.

20 The precipitates were resuspended in the original volumes to measure the optical density. Besides, native and methylene-blue stained preparations were made for (phasecontrast) miscroscopy to verify the findings.

25 Results Qualitative adherence The results showed that the adherence of E. coli K88ab to PH particles started after 2 hours of incubation. The adherence was most pronounced for the mixture containing sodium and potassium ions in comparison with the other mixtures.

After three hours the difference between mixture was even more pronounced. Mixture showed the least I I 8 adherence, mixture and did increase the adherence to some extent. The methylene-blue stained slides confirmed the findings of the native preparations.

Semi-quantitative adherence Table 1. Optical density before and after 3 hours incubation of the ;inxtures the supernatants after centrifugation and the difference between and Before incubation After incubation M S M-S M S M-S *o e SMixture 0.98 0.83 0.15 0.96 0.69 0.27 15 Mixture 1.13 0.97 0.16 1.02 0.51 0.51 Mixture 1.15 0.98 0.17 1.16 0.58 0.58 Mixture 1.05 0.93 0,12 1.10 0.14 0.96 Before and after incubation the optical densities of the 20 mixtures and the supernatants were measured in duplicate.

The results indicate that mixture shows strong adherence capability; the supernatant is almost clear of the ETEC E. coli.

Obviously the combination of sodium ions and po'assium ions strongly enhance the capability of the PH particles to adhere to the bacteria. It is envisaged that this combination can replace antibiotics in a lot of cases, such as in medicated feeds.

It is possible that this combination, by strongly adhering to the bacteria, prevents such bacteria from docking onto I 9 the receptor on the surface of the intestinal wall. This .may be as a result of acting as a receptor analogue.

We do not however wish to be limited by any hypothetical or postulated mechanism for the observed beneficial effects of this combination.

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Claims (3)

1. 2. A preparation according to claim 1 wherein the sodium containing substances and potassium containing substances comprise sodium chloride and potassium chloride.
2. 3. A method of preventing and treating animals or human beings suffering from intestinal infections which 15 comprises administering thereto a preparation according to S* any one of claims 1 to 2. DATED THIS 22ND DAY OF FEBRUARY 1996 PHARMLETT DANMARK A/S O0 By Its Patent Attorneys: S 20 GRIFFITH HACK CO., 1 Fellows Institute of Patent 0 Attorneys of Australia a statvhrordkeepspeciPHARM.OANMARK 22.2 ABSTRACT A preparation useful for prevention and treatment of intestin...l infections in animals and human beings, comprises Psyllium Husk together with sodium and potassium ions, for example in the form of sodium chloride and potassium chloride. 4* 4**4 4 44*4 4*404s 4 4 *4 4 4 4* *444 6*
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