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Field of the invention

The present invention relates to a method and composition for the prevention, cure and/or soothing of gastro-intestinal ulcers and non-ulcer dyspepsia in mammals in need of such treatment. More particularly the invention relates to a method and composition that can utilise only naturally occurring food grade ingredients. PecKqround Art

One of the amazing features of the mammalian body is how it can digest meat without digesting the wall of the gastro-intestinal (GI) tract in which that process occurs. The protective mechanism is a subject of very active research and much debate but break-down of the mechanism results in ulcers. Although ulcers can form in several parts of the GI tract, e.g. duodenal ulcers, those causing the greatest problem clinically are those forming in the highly acidic conditions of the stomach where the secretion of hydrochloric acid from glands in the stomach wall can lower the pH to about 2. There is general agreement that stomach ulcers are caused by the back- diffusion of hydrogen ions, i.e. acid, into the stomach lining. The major question to GI physiologists and gastroenterologists has been what is the natural protective mechanism. Many physiological parameters of the lining are found to change with ulcer formation and each has been adopted by one group or another as the basis of a theory for "gastric mucosal protection". At least 16 different theories can be found in the GI literature. The real problem is one of sorting out which changes are cause and which are effect.

In the absence of a well accepted theory of gastric mucosal protection, the clinical approach has been based upon the axiom that stomach ulcers will not form if the

acidity of the contents is reduced. Hence the clinical approach has been essentially one of neutralising the acid with "ant-acids" such as bicarbonate or acting upon the cells which produce the acid and glands which deliver it to suppress acid secretion. Hence a class of drug used for this purpose are known as H2 blockers since they act upon histamine receptors in the stomach lining to reduce acid secretion by about 85%. Drugs such as cimetidine and ranitidine have proven very effective for this purpose. Recently a variation of this theme, termed the hydrogen-ion-pump inhibitors such as omeprazole, has been released onto the market and these drugs can virtually suppress any acid secretion by the stomach.

The hydrogen-ion-pump inhibitors and H2 blockers are particularly effective in treating stomach ulcers acutely, but the relapse rate can be as high as 100% over two years (Brit. Med. J., 284: 621, 1982). The patient can be maintained ulcer-free by continually dispensing the acid- suppressing drugs but that raises a new set of problems. Firstly there is the very high cost of continued medication. Secondly there are the side-effects which include excessive gain of weight and gynaecomastia (very unacceptable to males) while there are reports of carcenoid formation in animals subjected to prolonged elevation of pH. Intuitively, it is undesirable to suppress the normal secretion of acid needed to kill bacteria and other unwanted pathogens present in the flora and fauna normally ingested.

The major clinical need is for natural foods or a mixture thereof which can be used to maintain the serious ulcer patient who has been successfully treated in the acute phases with H2 antagonists or hydrogen-ion-pump inhibitors. In the general population there is also a real need for a simple remedy to avoid the mild ulceration which can form during stressful periods when our stomachs secrete more acid than usual.

The present invention has previously found that gastro-intestinal ulcers may be prevented, cured and/or soothed by the internal administration to a patient of an effective amount of a composition containing a dispersion of ripe banana in milk (see Australian patent specification B29902/89). This previous invention was predicated upon the discovery of lamellar bodies of phospholipid in bananas. It is believed that these surface active phospholipids induce or stabilise a replacement layer of phospholipid on the mucosa of the gastro-intestinal tract and thereby relieves the symptoms of gastro-intestinal ulcers which are caused by the breakdown of the naturally occurring phospholipid layer. The present invention relates to a new composition and method that builds on and extends the previously described invention. Disclosure of the Invention

The present invention consists, in a first aspect, in a method for the prevention, curing and/or soothing of gastro-intestinal ulcers or other gastro-intestinal erosion in mammals in need of such treatment, comprising administering orally to such a mammal an effective amount of a composition containing an effective proportion of a naturally occurring phospholipid present in the form of the lamellar bodies and an effective proportion of a naturally occurring hydrophobic protein, the composition being dispersed in a naturally occurring and nutritionally acceptable carrier. In another aspect the present invention consists in a composition for the prevention, curing and/or soothing of gastro-intestinal ulcers in mammals comprising a composition containing an effective proportion of a naturally occurring phospholipid present in the form of lamellar bodies and an effective proportion of a naturally occurring hydrophobic protein. The present methods are of particular utility in the treatment of gastro-intestinal ulcers and other gastro-

intestinal erosion such as gastro-oesophageal reflux (heartburn) and non-ulcer dyspepsia in man, however, it could be used to treat other mammalian animals. The method and composition may be used to ensure the maintenance of a satisfactory protective phospholipid layer in the gastro-intestinal tract and thereby prevent gastro-intestinal ulcers forming. It may also be used to re-establish such a protective layer and thus cure gastro¬ intestinal ulcers. In a third alternative there may be a partial re-establishment of the protective phospholipid layers which ameliorates the pain and discomfort associated with the gastro-intestinal ulcer and thus can be said to be soothing.

The composition according to this invention and utilised in the method includes a naturally occurring phospholipid present in the form of lamellar bodies. Lamellar bodies are biological stores of highly surface active phospholipid found in animal and plant cells and are described in more detail in B.A. Hills "The Biology of Surfactant" Cambridge University Press 1988 pp 48-51. The most suitable source of lamellar bodies is fruit or vegetables containing such bodies. Most preferably the lamellar bodies are obtained from ripe bananas and are included in the composition by the inclusion of the flesh of the ripe banana fruit in the composition.

Desirably the banana fruit will have a ripeness of at least stage 7, more preferably stage 8, of the banana growers ripeness scale which ranges from 1 for green to 8 for brown fully mature bananas (see Von Loesecke H.W., "Economic Crops" in Kertesz Z.I, Ed. "Bananas" 2nd Edition, New York, Interscience 1950:108-109). An alternative index of ripeness is the sugar content and on this basis a sugar content of at least 17% by weight, and preferably 20% by weight, is preferred. In the case that banana or other fruit or vegetables are used as the source of the phospholipid it is desirable

that the fruit or vegetables are dried or frozen rapidly after being peeled or otherwise prepared. The fruit are preferably steamed, rapidly heated or dipped in dilute hypochlorite solution to prevent enzymes turning it brown. This drying or freezing may take place either before or after mixing of the phospholipid components with the other components of the composition.

The phospholipid will be naturally occurring and will therefore vary to some extent in composition, however, the phospholipids will typically be straight chained and largely saturated. The predominant and most surface active component will normally be L- α - dipalmitoyl phosphatidyl choline (DPPC) . Typically phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol will also be present.

The composition also contains a naturally occurring source of a hydrophobic protein. The hydrophobic proteins are also called proteolipids in the literature. They are proteins typically extractable from their source with a strongly non-polar solvent such as chloroform or ether. The hydrophobic proteins are associated inter alia with the myelination of axons (nerve fibres) as well as the expansion of the lung at birth. In the latter situations the hydrophobic proteins play a major role in the location of phospholipids at the air-aqueous interface of the neonatal lung upon switching to ventilation by air.

The present inventor postulated that these hydrophobic proteins may be useful in assisting in the adsorption of phospholipids at a solid surface. He further postulated that such hydrophobic proteins may be present in birds eggs as the developing embryo in the egg must have a rapidly developing nervous system with myelination proceeding very rapidly. He found by experiment that such eggs did indeed contain such hydrophobic protein and that hydrophobic protein does facilitate the adsorption of phospholipids onto solid surfaces. It is therefore a

particularly preferred aspect of the invention that the hydrophobic protein component of the composition comprises or is derived from the eggs of birds. The eggs may be whole eggs or egg yolks and may be added to the composition in a wet or dried form. If desired the eggs may be fractionated to increase the proportion of hydrophobic protein in the egg derived material added to the composition. The most readily obtained and economically most advantageous eggs are those derived from chickens, however, eggs from any other avian species could be used with substantially equal effect. Other sources of hydrophobic protein may also be used such as the eggs of non-avian species or the lungs and other suitable tissue of young mammalian animals or the embryos thereof. The components of the composition are preferably dispersed in a suitable carrier for oral administration to the consumer. This carrier may be an aqueous solution of any suitable type but preferably comprises milk, cream or another dairy based liquid. The milk or other dairy based liquid is preferably mixed with the other ingredients in a fresh state and is then rapidly frozen or dried to yield a saleable product. Alternatively powdered milk produce may be mixed with the other components in a dried state. It is preferred that the banana and milk are mixed prior to drying with the egg being added either fresh before drying or as a dried powder after drying.

The preferred milk based liquid is a fat and cholesterol reduced and calcium enriched milk product sold in New South Wales, Australia under the registered trade mark "Shape". The milk and banana are most preferably mixed in a weight/volume ratio of 1:1 i.e. 1 gm of banana is mixed with 1 ml of milk and then dried however this ratio may be varied widely, preferably within the range 8:1 to 1:8. Obviously if other sources of phospholipid are used and other carriers used the ratio could change dramatically, however, the ratios described above may be

used as a guide for further routine experimentation of suitable ratios.

The egg content may vary widely within the preferred range of 20:1 to 1:20 but, more preferably one part by weight of dried egg yolk powder is added to nine parts by weight of dried banana milk powder. If other sources of hydrophobic proteins and phospholipid are used similar effective ratios would form a starting point for the routine determination of the most effective dose and ratio.

If desired polyvalent metal cations such as zinc or calcium may be added to the composition as such polyvalent cations are believed to stabilise the protective phospholipid layer in the gastro-intestinal tract. While freeze drying is an appropriate method for treating the banana/milk or egg/banana/milk mixture it is preferably spray dried. The mixture is preferably rapidly heated at the inlet valve of the spray drier. This temperature may not be optimal but it does afford a degree of pasteurisation which is desirable. The heated pureδ is preferably passed through the inlet nozzle directly onto a rapidly spinning disc. This disc disperses the jets of puree into a fine mist in which droplets lose water as they are held in the upward current of air flowing through the dryer. The dried droplets collect at the base of the dryer as a powder. Some very fine particles become entrained in the upward air current and are recovered as "fines" in a cyclone separator. These "fines" have a similar efficacy to the coarser material and are mixed with that material. The dried material preferably has a moisture content of 5% or less as this reduces any tendency for the powder to "cake". When packed in sachets material of this dryness has remained viable for a prolonged period. Best Mode of Carrγinσ out the Invention

The present invention is further illustrated by reference to the following examples. Methods

The animal model used was the standard non-pylorus- ligated rat. Wistar rats were starved for 18h. before experimentation during which coprophagy was prevented by keeping each animal in a tube just too small for it to turn around. There was no ligation of the pylorus in these trials so as to avoid variability in acid secretion induced by surgical intervention. The rats were lightly anaesthetised in an atmosphere of 50:50 02:C02 when the test product was administered as a suspension in water via a stomach tube. Control rats were administered 1ml. of water. The rats were given a dose of 5% thiopentone sodium as a maintenance anaesthetic. After lh. the acid insult was administered by stomach tube, consisting of lml. of 0.8N.HC1. The dose of the test product and of the acid insult was calculated in each case based upon the weight of the rat using the formula dose = 0.015 wO-75 when W is the weight of the rat. As an example the dose was 250mg in lml water for a 270gm rat. The rats were killed 2h. later by a lethal dose of sodium pentobarbitone after which the stomachs were excised and laid flat on a light box with the mucosal side uppermost. The damage was then assessed by strongly illuminating the mucosa and projecting an image into a video-camera from which the ulcerated area could be clearly defined by colour enhancement. The ulcerated area was then determined as a percentage of total mucosal area by computerised topography. This process, together with identical colour enhancement of controls, was felt to minimise, if not eliminate, any operator bias.

The protection rate was calculated using the formula : - 100(ulcerated area of control - ulcerated area of test)% prolection rale =

(ulcerated area of control)

Materials

Ripe bananas were rapidly dispersed in a dairy product being a calcium enriched fat reduced milk sold under the registered trade mark "SHAPE" when the temperature is immediately raised and the puree injected into a spray-drier to produce a banana/dairy (BD) product. In a variation of this process whole eggs (minus shell) or egg yolks are also incorporated into the ρure6 to be sprayed to produce an egg- banana-dairy (EBD) product. The spray-dried powder is continuously removed from the drier. If egg was not added to the initial puree, then egg-yolk powder is mixed with the BD product by tumbling the two powders together to produce a mixed powdered egg/banana/dairy product (PEBD) . Results

The following trials have been completed on rats. I. The addition of one raw egg yolk to fresh banana (50gm.) dispersed in dairy product (50ml.) enhances efficacy, further reducing the mean ulcerated area by 41%

TABLE 1

PROTECTIVE AGENT N MEAN ULCERATED PROTECTION

AREA* RATE

Fresh banana + dairy 15 21.36 ± 4.37 38% products (X)

X + raw egg yolk 16 12.64 + 3.27 63%

None (water control) 17 34.63 ± 3.46 -

* Mean + sem

II. Commercially available spray-dried egg-yolk powder

(PE) is more effective than raw egg yolk (E) in enhancing the efficacy of fresh banana dispersed in dairy product, further reducing mean ulcerated area by

XA£ £_2

PROTECTIVE AGENT N MEAN ULCERATED PROTECTION AREA* RATE

Raw yolk(E) + BD ⁺ 12 10.79 ± 2.37 36%

Egg yolk powder 12 5.51 ± 1.93 67% (PE)+BD#

None (water control) 14 16.79 + 2.41

* Mean + sem

+ 1 yolk to lOOgm. BD

# lOgm. powdered egg + lOgm. water added to lOOg . BD

III.Commercially available spray-dried egg-yolk powder (PE) is more effective in enhancing spray-dried banana/dairy product (BD) when it is added as a simple mixture (PEBD) than when it is reconstituted and added to the puree for co-spray-drying (EBD), further reducing mean ulcerated area by 54%.

TABLE 3

PROTECTIVE AGENT N MEAN ULCERATED PROTECTION AREA* RATE

PE+BD puree co-spray- 16 14.55 + 3.36 56% dried (2:5) ⁺ (EBD) #

PE mixed with BD as 19 6.65 + 1.48 80% powders (PEBD) #

None (water control) 18 32.81 + 3.97

*Mean +. sem

+Powdered egg-yolk was reconstituted in water (lgm/ml) before addition to the puree of fresh banana/dairy product. The new puree is now spray-dried to give the product tested. (EBD). Ratio equivalent to 2:5 PE:BD #Applied as 250mg/ml in both cases.

IV. There is a synergism between egg and banana/dairy product because egg-yolk powder alone (lgm/10ml.H2θ) is not as effective as either the mixture of powders (PEBD) or the co-spray-dried product (EBD) .

ABLE 4

PROTECTIVE AGENT N MEAN ULCERATED PROTECTION AREA* RATE

⁺ PE mixed with BD 8 ^~ 11.15 + 2.72 68% as powders (PEBD) ⁺ PE+BD puree 7 17.15 ± 7.26 50% co-spray-dried (EBD) ^" • ^" Powdered egg(PE) 17 31.32 + 4.03 9% alone (lml. ) None (water control) 17 34.32 + 4.46

*Mean ± sem

+Equal quantities of egg-yolk powder (PE:BD 2:5) administered at 250 mg/ml.