This invention relates to chelating compositions for the treatment of Crohn's and Johne's diseases and other similar medical conditions; all of which may be associated with the bacterium Mycobacterium Avium (subspecies) Paratuberculosis (MAP) which is considered to be pleomorphic and in the nano-bacteria classification for size, and other nano bacteria species.

MAP, on a genetic basis, is virtually identical to

Mycobacterium Avium (a bird bacteria variety) ; however it grows very slowly, and using an iron transport chemical called mycobactin it infects mammals instead of birds .

While sharing many genetic similarities with Mycobacterium Avium, MAP is closely linked genetically to pathogenic mycobacteria for Tuberculosis. Mycobacterium Tuberculosis is the cause of tuberculosis in humans, and Mycobacterium Bovis, the cause of tuberculosis in cattle and other animals. Paratuberculosis appears to have a relationship, although not clearly defined/understood, with the cause of leprosy in humans, Mycobacterium leprae.

It has been shown that MAP can be present in a broad host range inducing a condition called "Johne's Disease" - a chronic tissue wasting disease, which can result in the animal's death. Ruminants (animals with chambered stomach that chew their cud) are the type of animal most affected. These are cattle, sheep, goats, and deer. MAP has also been found in non-ruminant species such as pigs, rabbits and humans where it shows up as Crohn's disease.

In humans, Crohn's disease is an inflammatory digestive tract and bowel disease. It can be difficult to diagnose,

as its symptoms are similar to Irritable Bowel Syndrome,

Ulcerative Colitis, Colitis and Slim disease, a chronic wasting condition often associated with HIV/Aids patients. It is thought that MAP may be responsible for these conditions as well.

With Crohn' s disease MAP can cause inflammation of a part of, or the whole of, the digestive tract from the mouth to the anus resulting in extreme pain and frequent diarrhoea. Humans with Crohn' s disease often end up not been able to consume or digest solid food and are then placed on a liquid food diet, a situation that can seriously affect the activities of the person concerned.

The cause of the actual medical condition associated with Crohn' s is that the body' s immune system reacts to the MAP bacterium by causing ongoing inflammation of the stomach and intestine walls. Persons with this condition can experience abdominal pain, rectal bleeding, diarrhoea, severe weight loss and fevers.

A lot of people with chronic Crohn' s and other digestive tract disorders are at times confined for long periods of time to bed due to the severe pain and the worst cases are hospitalised. In these situations the quality of the person's lifestyle is completely eroded.

It has been shown that the MAP bacteria once present in a person in the intestinal system is also present in their blood. The transmission from the intestinal system into the blood appears to be through lesions or bulges which develop in the intestine where the blood vessels are damaged due to this condition allowing the MAP to enter the blood system.

Animals that contract Johne' s disease experience constant diarrhoea and deterioration in their physical shape, so that it is often referred to as chronic wasting disease. Animals that experience this do not survive very long as the weight loss is extremely rapid once the disease becomes established in the animal.

There is no known treatment to cure Crohn's or Johne's Diseases. The current alleviative treatments for Crohn's can include drugs, nutritional supplements, surgery or a combination of these options, but all they do is attempt to control the disease rather than cure it. With Johne's there is no cure and the beast is left to die whilst experiencing severe debilitating weight loss.

Patients with Crohn's are frequently prescribed corticosteroids to control the inflammation but they leave the person with greater susceptibility to infections in the damaged tissue within the intestinal system. This could mean massive doses of antibiotics as well to reduce or control any infection resulting from the steroid treatment.

MAP has been found to be present in the majority of Dairy cattle and this bacterium is then subsequently present in the milk produced. Control of MAP in pasteurised milk has been attempted in some areas by increasing the pasteurisation temperature and dwell time, however this is not universal and certainly does not apply to un- pasteurised milk which is available for consumption and also is used in dairy products like cheese and yogurt.

The life span of the MAP bacterium has been shown to be over 18 months in some circumstances. It is thought that the primary form of transmission of MAP into humans is

through the MAP being present in dairy products and additionally, in some cases, via meat. A study carried out recently showed that lactating human females that were MAP positive passed this via their milk. MAP was also present in the lactating female's blood. Their babies also became MAP positive at an early stage of their lives, leaving them with the potential legacy of a future likelihood of Crohn' s disease. MAP was also present in the babies' blood.

MAP positive animals produce MAP positive dung. MAP bacterium sourced from dung can survive in the soil for periods up to 18 months allowing the possibility of infection of other animals simply by them eating contaminated grass. This means that an entire herd of animals can be affected by just one initial MAP positive animal .

Dairy industry cattle dung is collected into slurry tanks from the milking sheds and later distributed over the grass land. Due to the high incidence of MAP among dairy cattle, MAP will be present in the slurry which, once it is distributed over the grassland, means that the MAP bacterium is again available for fresh re-acquisition.

Among humans the Crohn' s incidence worldwide is considered to be approximately 0.9 cases per 100,000 persons, but in some areas the incidence is up to 11.6 cases per 100,000. With Ulcerative Colitis and Irritable Bowel Syndrome no exact figures are available on a population basis but the incidence figure is believed to be between 0.6 to 1.4 cases per 100,000. In respect of Johne's, it has been reported that over 70% of Dairy cows are MAP positive and that this figure is increasing. With Beef cattle the incidence varies, but in some countries up to 25% of the breeding

stock die each year from Johne's disease. Johne's disease is now found in a majority of farming countries and the incidence is increasing significantly.

MAP bacterium is an unusual bacterium as it is slow growing. A normal bacterium can reproduce with seconds to hours; Mycobacterium can take from 3 hours to 3 days with some isolates taking even up to 12 months to reproduce. Nano-bacterium can take 3 to 6 days to reproduce. This places MAP either in-between the other two types of bacteria or categorically in the nano-bacterium area.

MAP bacterium also appears to be pleomorphic, which indicates that it changes shape; typically from a cocolithic structure to a rod shaped bacterium, and the reverse, while between these states appearing to have no cell wall structure. At the same time there have been indications that the MAP bacterium has a biofilm coating of calcium around its polysaccharide membrane. Nano-bacteria also exhibit this unusual property of having a calcium biofilm around each cell and this is one reason why these generally prove difficult to kill. These similarities could mean that MAP is really a nano-bacteria variety that has unigue survival properties making it especially difficult to treat.

The calcium biofilm around each cell may be of calcium carbonate or calcium phosphate and this biofilm makes the MAP and nano-bacteria impervious to a wide range of antibiotic and other treatments.

It is an object of the present invention to avoid or minimize one or more of the above problems.

There has previously been proposed in our earlier Patent publication WO 03/0329.44, various topical chelating compositions suitable for use in combating antibiotic- resistant infections and contamination of the skin and open wounds. There has been no previous suggestion, however, of the possible utility of such compositions for other conditions .

We have now found that compositions based on the use of chelating compounds as disclosed in WO 03/032944 (the contents of which are hereby incorporated herein by reference thereto) , have the properties of alleviating the conditions of Crohn's and Johne's diseases and at the same time killing the MAP bacterium while reducing the inflammation condition present in the digestive system over a period of time.

Thus in a first aspect the present invention provides the use of at least one metal ion chelating agent for the manufacture of a medicament for the treatment or prophylaxis of a MAP-mediated intestinal condition.

The said at least one metal ion chelating agent preferably provides a metal ion chelating capacity for calcium ions and/or for at least one metal ion on which said MAP species is dependent for viability.

The present invention also provides the use of at least one metal ion chelating agent for the manufacture of a medicament for the treatment or prophylaxis of Crohn' s disease.

The present invention also provides the use of at least one metal ion chelating agent for the manufacture of a medicament for the treatment or prophylaxis of Johne's disease.

The processes within MAP species that are dependent on metal ions and that are required for microbial species viability are generally numerous and include processes of nutrition and reproduction such as DNA replication, cell division, protein synthesis, RNA synthesis. Preferred chelating agents can chelate various different metal ions and thereby attack microbes dependent on such different metal ions, by multiple routes. In the case of MAP it is also essential that there is used a chelating agent that will absorb calcium present in a calcium based coating on the MAP bacterium membranes in order to make the MAP cell available to be attacked by a chelating agent that has a chelating capacity for one or more metal ions necessary for the viability of the MAP cell - such as Mg ²⁺ , Fe ²⁺ , Fe ³⁺ , Cu ²⁺ , Zn ²⁺ , Mn ²⁺ , Ni ²⁺ , and Se ²⁺ . Desirably also there is used a chelating agent that has a chelating capacity for ferric ions in order to attack the mycobactin mediated ferric ion dependence of the MAP cell.

It is in principle possible to use a single chelating agent which provides two or more of the above essential or desirable requirements . In practice though we have found that different chelating agents have different chelating capability or strength for different metal ions, and it is generally preferable to use two or more different chelating agents wherein the chelating capability for the various different metal ion targets is maximized to a greater or lesser extent.

Having regard to the above, 8-hydroxyquinoline has been found to have a particularly broad spectrum of activity, chelating, with a high degree of effectiveness, most metal ions (including ferric and ferrous) , apart from sodium, potassium and calcium. It is accordingly necessary to use this chelating agent in combination with a secondary, calcium ion-chelating, agent. Suitable, calcium ion- chelating, agents are well known in the art, and include inter alia EDTA.

By providing a metal ion chelating agent that removes a variety of metal ions, which may conveniently be referred to as the target metal ions, the effectiveness of the compositions of the invention may be increased. Thus, in relation to attacking the basic "nutrient" metal ion requirement of the bacterial cell, it is preferable to use a metal ion chelating agent which can form a chelate with a plurality of metal ions selected from Mg ²⁺ , Fe ²⁺ , Fe ³⁺ , Cu ²⁺ , Zn ²⁺ , Mn ²⁺ , Ni ²⁺ , and Se ²⁺ .

It appears that at least part of the effect of the primary - nutrient metal ion, chelating agent may be through a coating of the MAP bacteria, by the primary chelating agent, thereby effectively smothering the bacteria.

Typically the metal ion and the metal ion chelating agent form together a stable complex such that the metal ion is effectively removed for a sufficient period of time to prejudice microbe viability and overcome the infection before the metal ion chelate dissociates. In general the metal ion and metal ion chelating agent should form a

stable chelate under physiological conditions, and especially intestinal conditions.

Preferably the primary "nutrient" metal ion chelating agent is a heteropolar compound comprising at least one unsaturated heterocyclic six-membered ring in which at least one heteroatom moiety acts as a hydrogen acceptor and in which said compound also comprises at least one hydrogen donor moiety, conveniently a hydroxy1 group, said heteropolar compound having no substituent which by itself or together with another substituent or substituents creates such steric hindrance and/or renders the molecule so basic or acidic or so alters the steric geometry of the molecule as to prevent interaction of the hydrogen donor and acceptor moieties of one molecule of heteropolar compound with the hydrogen donor and acceptor moieties of another molecule of said heteropolar compound. In general the preferred "nutrient" metal ion chelating agent is a hetero aryl compound having at least one nitrogen in the ring structure and at least one hydroxyl substituent disposed on the ring structure so as to provide together, a chelating function. Preferred metal ion chelating agents are selected from optionally substituted 2 , 3 -dihydroxypyridine ; 4 , 6-dihydroxypryrimidine ; 2- pteridinol; 2, 4-quinolindiol; 2, 3-dihydroxyquinoxalin; 2,4- pteridinediol; 6-purinol; 3-phenanthridinol; 2- phenanthrolinol; 2-phenazinilol, and most preferred is 8- hydroxyquinoline. 8-hydroxyquinoline has the advantage of forming metal ion chelates with a particularly broad range of different metal ions.

A further unexpected benefit of the present invention is that we have found that the at least one chelating agent used in the medicaments provided by the present invention, also appears to coat the nerve receptor ends that induce the pain from the inflammation associated with MAP-mediated intestinal conditions, and once this coating has taken place, the pain appears to disappear, thereby reducing stressing of the body and thereby further aiding recovery from the condition. It is also observed that the inflammation associated with these conditions is gradually reduced until it is cleared.

Another further unexpected benefit of the present invention is the ability of a liquid medicament of the metal ion chelating agent to enter the blood stream and prejudice the nano bacteria and MAP bacterium viability. This means that upon treatment all locations of the MAP and nano bacteria can be eliminated through this treatment.

In a further aspect the present invention provides an orally ingestible pharmaceutical composition suitable for use in the treatment or prophylaxis of a MAP-mediated condition selected from Crohn's disease and Johne's disease, said composition comprising at least one physiologically acceptable metal ion chelating agent providing a "nutrient" metal ion chelating capability and a calcium ion chelating capability, and a pharmaceutically acceptable carrier therefor, in which composition said metal ion chelating agent has a metal ion chelating capacity for calcium ions and for at least one metal ion on which said MAP species is dependent for viability.

The compositions of the present invention for internal application will generally comprise from 0.0031% to 0.05% w/w, preferably from 0.005 to 0.02 % w/w, of said at least one chelating agent.

Various forms of orally ingestible composition may be used in accordance with the present invention. Preferably there is used an aqueous based liquid composition, as this can provide a relatively rapid coating of the inflamed parts of the intestines, and thereby provide a relatively rapid onset of pain relief and at the same time enter the blood stream to eliminate any nano-bacteria and MAP bacteria present. Alternatively there may be used a paste formulation, which may be more convenient for use in drenching cattle or other animals. Such formulations may conveniently be prepared by adding a suitable physiologically acceptable thickener. Various suitable thickeners are known in the art, including inter alia hydroxypropylcellulose thickeners such as that commercially available under the Trade Name KLUCEL H CS from Aqualon of Wilmington, DE, USA. Conveniently there is used a Dehydroxanthan Gum anionic polymer thickener such as that commercially available under the Trade Name AMAZE ^™ XT from National Starch & Chemical Company of Manchester, England. As a further alternative, the compositions of the invention may be in encapsulated form e.g. using capsules of suitable material which is more or less readily soluble in the stomach and intestines, and which are readily available, e.g. based on starch, gum and alginate. It is also possible to use slow or delayed release capsules, which is generally preferred in veterinary applications, and/or which has the advantage of delivering more of the chelating agent further down through the intestinal system.

It will be appreciated that the choice of components of the composition may be limited by the nature of the metal ion chelating agent. For example the preferred metal ion chelating agent 8-hydroxyquinoline is generally insoluble or only poorly soluble in aqueous solution. Suitable aqueous based compositions can be prepared by using an intermediate solvent such as a polyol, including glycols, preferably propylene glycol, glycerine, or sorbitol, and a wetting agent. Those skilled in the art will appreciate that a wide range of wetting agents are available that may be used which would give solubility of the metal ion chelating agent in glycol, including inter alia Polyoxyethylene Sorbitan Fatty Acid Ester T20, T40, TβO and T80 (Polysorbate) , and C9-C11 Alcohol ethoxylate (Synperonic 91/8 and Synperonic 91/6) .

It will be appreciated that a range of different proportions of the various components of the aqueous based compositions may be used depending on the solubilities of the metal ion chelating agents used, the final concentration required etc. In general we have found that the amount of wetting agent used is relatively sensitive. In the case of the intermediate solvent or diluent (glycol etc) , once a required minimum amount sufficient for solubilisation of the metal ion chelating agent in the water is present, then the amount of this intermediate solvent or diluent can be readily increased further, though there is normally no particular advantage in doing so.

Advantageously there is also included a pH controller, in order to ensure an alkaline pH in the composition, most preferably a pH in the region of 9.2 to 9.4, as we have

found that this provides the fastest onset of pain relief and the inflammation itself is reduced most quickly. The pH controller may simply be KOH or NaOH. Preferably, though, there is used EDTA, conveniently in the form of the DiSodium or, most preferably, TetraSodium salt.

In the case of 8-hydroxyquinoline we have found that suitable proportions which may be used in a liquid, aqueous-based, composition of the invention suitable for use in the treatment of MAP bacterium to reduce Crohn's and

Johne's disease, would in general have the following composition :

Component Weight

Metal ion chelating agent 1 part Secondary chelating agent 0.1 part

(EDTA)

Wetting agent 4 +/- 5% parts

Intermediate Diluent at least 20, preferably

40 parts De-ionised Water as required to obtain the final concentration required.

In the case where an alkaline pH formulation is required, then the composition would include a suitable additional component, which preferably would be as follows: pH Controller (DSEDTA or TSEDTA) as required to achieve a pH of 9.2 to 9.4

In the case where a Gel and Paste formulation is required, then the composition would include a suitable additional component as follows : Thickener as required for the desired viscosity.

For human application the composition is conveniently taken twice daily at the rate of 5 to 20 mis per dosage. This dosage rate has to be maintained for a period of time, initially probably 3-6 days before any relief is noticed, (normally the pain is reduced initially) and then for a period that can be determined only by the patient's progress until the inflammatory condition has been finally brought under control .

This remedial action in humans can take from several weeks to several months to achieve, depending on the severity of the inflammation, rate of progress, and the length of time the person has had the disease, and the appropriate duration of the treatment in any given case would normally be as directed by the patient's physician. Once the remedial action has concluded, consumption of solid food can recommence, thus allowing the person to revert once again to a normal lifestyle.

Thus in yet another aspect the present invention provides a method of treatment or prophylaxis of a MAP-mediated intestinal disease comprising the administration, to a human or animal in need of such treatment, of an effective dose of at least one metal ion chelating agent.

The said at least one metal ion chelating agent preferably provides a metal ion chelating capacity for calcium ions and/or for at least one metal ion on which said MAP species is dependent for viability.

The present invention, in another aspect, provides a method of treatment or prophylaxis of inflammatory conditions selected from Crohn's, IBS (Irritable Bowel Syndrome),

Colitis or Ulcerative Colitis, e.g. in humans, or Johne's disease in animals, comprising the administration, to a human or animal in need of such treatment, of an effective dose of at least one metal ion chelating agent.

Application of the composition to animals to treat Johne's can be either on a group or individual basis. Incorporation of the composition in the drinking water has the advantage that all animals sharing the drinking source are all treated at the same time to reduce the possibility of reinfection. Alternatively, or additionally, the composition can be administered by individually dosing the animals with an effective dosage of the composition into the digestive system. Various forms of composition may be used for this purpose including liquid drenches, and more or less viscous paste compositions. In general it will be necessary for the animals to be dosed repeatedly, preferably at least daily, for a suitable period of time, which typically should be at least 2 weeks, conveniently from 3 to 4 weeks.

Another method of reducing the MAP infection in the farm fields is by spraying the pastures with the composition to reduce the incidence of MAP present. This may require repeated spraying over a period of time to overcome the problem.

Another contributory factor to the spread of Johne's in Dairy farms is that the animal waste slurry that is collected, is normally spread over pastures that the dairy cows will graze over on a rotational basis. As the MAP bacteria has a long life in the soil a Dairy herd will become infected with MAP over time, to the point where no

animal will be MAP free due to acquisition thereof through grazing. Treatment of the slurry with a composition of the invention will eliminate MAP from the slurry before it is distributed over the fields and provide a degree of infection control.

Further preferred features and advantages of the present invention will appear from the following detailed examples given by way of illustration of some preferred embodiments.

Example 1 - Method of Preparation of concentrate 10 gm of 8-hydroxyquinoline was dissolved at 70 degrees centigrade in 50 grammes of a wetting agent selected from: Polyoxyethylene Sorbitan Fatty Acid Ester T20, T40, TβO and T80 (Polysorbate) , and C9-C11 Alcohol ethoxylate

(Symperonic 91/8 or 91/6) , with 200 grammes of a water soluble non-aqueous diluent selected from Propylene Glycol, Glycerine and Sorbitol. Once solution has been achieved, a further quantity of the glycol or glycerine or Sorbitol diluent was added to make up to a solution of 500 grammes and then cooled giving 500 g of 2% w/w 8-hydroxyquinoline concentrate .

Example 2 - Preparation of Human Treatment Composition Take one part of the 2% w/w 8-hydroxyquinoline concentrate from Example 1 and dilute in 159 parts of deionised water, then the pH of this composition is adjusted to 9.2 to 9.4 by the addition of Tetra Sodium Ethylene Diamine Tetra Acetic Acid. The strength of this preparation is 125 ppm of 8-Hydroxyquinoline, and it provides 5 ml dosage units each containing 625 microgrammes of the 8-hydroxyquinoline.

Using a typical dose rate of this composition of 10 mis per day providing a dosage of 1250 microgrammes and, based on an average body weight of a human adult of 70 kilogrammes, this corresponds to an approximate dosage rate of 17 to 18 microgram per kilogram body weight/day. Naturally this can be varied according to the individual patient's requirements .

Example 3 - Preparation of Animal Treatment Composition Take one part of the 2% 8-hydroxyquinoline concentrate of Example 1 and dilute with 79 parts of deionised water, then the pH of this composition is adjusted to 9.2/9.4 by the addition of Tetra Sodium Ethylene Diamine Tetra Acetic Acid.

The strength of this preparation is 250 ppm of 8- hydroxyquinoline providing a dosage unit of 5OmIs containing 12.5 milligrammes. An average beast of greater than 2 years of age will be approximately 350-400 kilogrammes, whereby ingesting 50 mis per day of the composition will give between 31 to 36 microgrammes of 8- hydroxyquinoline per kilogram body weight each time.

If the water troughs are dosed then they are dosed on a prescribed strength material per the quantity of water in the trough at a given time.

Example 4 - Preparation of Paste Composition for Animals One part of a Dehydroxanthan Gum anionic polymer thickener (AMAZE ^™ XT available from National Starch & Chemical Company of Manchester, England) is added to 99 parts of an aqueous liquid composition prepared according to Example 3. The resulting paste composition is introduced via the animal's

mouth, using a conventional applicator, at a dosage of 50 mis .

Example 5 - Preparation of Pasture Spray Take one part of the 2% 8-hydroxyquinoline concentrate of Example 1 and dilute with 159 parts of deionised water, then the pH of this composition is adjusted to 9.2/9.4 by the addition of Tetra Sodium Ethylene Diamine Tetra Acetic Acid. The strength of this preparation is 125 ppm of 8- hydroxyquinoline . The liquid is sprayed over the pasture at a rate of 0.10 litres/m ² . Spraying is desirably repeated at least 3 times at regular intervals of A weeks while treatment of the animals is ongoing.

Example 6 - Preparation of a Soluble Pessary or Tablet.

Take one part of the 2% 8-hydroxyquinoline concentrate of Example 1 and dilute with 74 parts of deionised water, then the pH of this mixture is adjusted to 9.2/9.4 by the addition of Tetra Sodium Ethylene Diamine Tetra Acetic

Acid. Then 2.5 parts of Alginate powder is added along with 2.5 parts of Starch. This produces a thickened mass which can be pressed into tablets or pessaries ready to be given to humans or animals.