COMPOSITIONS FOR COMBATTING DIARRHOEA

Abstract: A therapeutical composition for combatting and preventing diarrhoea. Said composition comprises one or more substances which block adhesine receptors present on the intestinal epithelium. Preferably said substances are peptides, in particular Ser-Leu-Phe, Ile-Ala-Phe and/or Ala-Ile-Phe.

Description: Compositions for combatting diarrhoea The most important procreators of neonataldiarrhoea with human beings and animals are the enterotoxical E. coli stems. With babies or very young children and with new-bo cattle this infection almost always has a lethal course. It appears from recent figures from the WHO that each year 500 million babies and young children (in particular in the developing countries) suffer from diarrhoea which causes over 5 million victims. Besides V. cholearae Ξ.coli appears to be the main procreator. No reliable figures are known with respect to slaughter cattle but the loss will certainly run into milliards a year. Enterotoxic E.coii stems distinguish themselves from commensal non-pathoσeneous stems in that they comprise two important virulence factors, to wit: the production of enterotoxins and the presence of adhesion antigens. The adhesion antigens of Ξ.coli consist of electron microscopically visible thread-like appendixes on the bacterium surface indicated as fi briae or pili. Said fimbriae appear to be consisting of hundreds of identical protein sub-units having a molecular weight in the order of magnitude of 25,000. Through the fimbriae the bacterium appears to be capable of colonizing .the intestinalepitheliumand of proliferating the intestine in this way. As soon as the colonisation is effected the produced and excreted enterotoxin causes the development of diarrhoea. Experiments with mutated stems which cannot produce either adhesion antigens or enterotoxin anymore proved that said stems cause no or a very slight, passing form of diarrhoea. For some time various research teams have been occupied wi the question whether prevention of this type of infections • through induction of protecting antibodies is possible. It appeared that administration of toxoid does raise antitoxin immuno-globulins , but that these antibodies do not or only partly give protection. A second approach is based on inducing antibodies directed against the adhesion antigens. This approach appeared to be successful. Vaccination with purified adhesine preparations in most cases appears to give a sufficiently high respons of protectinσ antibodies to protect new-born descendants against the infe ion. . All these experiments relate to infections with piglets, calves or lambs. Various companies bring this type of vaccir.s on rhe market. It is a problem, wit rhe vaccination of slauσhter cattle with purified antigen, preparations that Ξ.coli can produce various different adhesion antigens, so that now it is clear that more and more different adhesion antigens have to be added to the commercial vacci s. Some stems only produce oneadhesion antigen, others can synthetize 2 or even 3 adhesion antigens simultaneously. It is a second problem relating to this type of combatting diarrhoea that vaccination of the parent animals (cows, sows) indeed has a preventive effect and protects the descendants, but, in view of the costs that go with it, this usually can only be used in companies in trouble. From a therapeutical point of view vaccination at the moment at which the infection manifests itself is useless, of course. In regard of the fact that vaccination is usually only used if problems are to be expected diarrhoea is still combatted on a large scalewith antibiotics, which has L many disadvantages (costs, side-effects, etc.). Therefore, other forms of therapeutics are searched for. The approach chosen is directed to the prevention of colonization, i.e. attaching of the bacteria to the intestinal~epithelium.lt appeared to be possible to combatdiarrhoea by oral administration of (monoclonal) antibodies directed against the adhesion antigens on the bacteria surface. A first composition for combatting neonatal diarrhoea with calves is now commercially available (Molecular Genetics) but relatively little is known about its success. Disadvantages are for instance the costs and the tenabilitv of the preparation. The above mentioned therapeutical approach is directed against the infecting microorganism. Now it has appeared that a novel therapy can be directed to blocking the achnsir.e receptors resen on the intestinal epithelium. Until recently of none of the adhesines of enterotoxic E.coli-stems it was known which part (or parts) of the structure is (are) of importance for the receptor recognition. Surprisingly it has appeared now that here it concerns spatially very restricted structures. One of the adhesion antigens in enterotoxic E.coli- stems from piglets is indicated as K88. Three variants of the K88 adhesine are known which can properly be immunologically distinguished. The primary structure of the subunit,of which the adhesine is composed, of each of said variants has been completely elucidated by us. Besides conserved areas (identical amino acid sequences in the subunit variants) variable amino acid sequences appear to be present on certain sites in which the three variants distinguish themselves from - . - each other. With digestions of denatured subunits with proteol tic enzymes it has appeared now that of each variant fragments can be isolated which can block the binding of the adhesine carrying bacteria to erythrocytes (haemagglutination reaction) or to intestinal epithelial cells in low concentrations. Said fragments originate from the conserved parts of the subunit variants. Analysis of three isolated fragments showed that there is question here of the following tripeptides: serine-leucine-phenylalanine, isoleucine-alanine-phenylalanine and alanine-isoleucine- phenylalanine. Said peptides are resistant to decomposition by proteolytic enzymes from the gastro-intestinai tract. All of the three isolated and identified tripeptides have been synthetized. The synthetic peptides have the same effect and activity as the peptides isolated from the adhesine. Therefore, in the K88 adhesine known amino acid sequences are present on certain sites which show a strong affinity for the adhesine receptors. Said amino acid sequences, therefore, possibly form an essential part of the natural receptor binding site. Provisional research with respect to the adhesine antigen K99 of enterotoxic E.coli-stems from calves and lambs points out that here spatially very restricted structures are responsible for attaching to the receptor as well. As the above-mentioned peptides as it were form an imitation of the natural receptor binding site as it occurs on the adhesine at the bacterium surface, said peptides will compete in the intestine with the bacterium for attaching to the intestinal epithelium. Once bound bacteria can also be removed from the intestinal epithelium by the peptides. With respect to the short duration of the infection (1-2 days) blockade of the intestinal epithelium receptors will only have to last for a short period of time. This limitation of the duration of the blockade is effected by the host himself as intestinal epithelial cells (enterocytes) are replaced and released fast (usually within 24 hours) by new making. On account of the above invention substances' with the spatially very restricted structures meant will be inserted into food or into therapeutical compositions. The possibly occurring absorption of said substances by the intestinal epithelium, can be prevented by coupling said substances to inert macromolecular carriers. The above mentioned spatially very restricted structures such as tripeptides can be isolated or synthetized cheaply and can be preserved very well. They do not have any disadvantageous effect upon the host. Besides use with new-born descendants the therapeutical compositions according to the invention can also be used with adults to combat diarrhoea caused by enterotoxic E.coli-stems.

Claims: V_ A I :. S 1. Therapeutical composition for combatting and preventin diarrhoea, characterized by the presence of one or more substances which block the adhesine receptors present on the intestinal epithelium. 2. Composition according to claim 1, characterized by the presence of substances which block specifically adhesine receptors. 3. Composition according to claim 1 or 2, characterized by the presence of one or more substances, the molecules of which comprise one or more amino acid units or derivatives. 4. Composition according to claim 3, characterized by the presence of one cr more peptides. 5. Composition according to claim 3 or 4, characterized by the presence of one or more substances having in their molecules at least one amino acid unit belonging to the group consisting of serine, leucine, isoleucine, alanine and phenyl-aianine. 6 . Composition according to claim 5, characterized by the presence of serine-leucine-phenylalanine, isoleucine-alanine-phenvlalanine and/or alanine- isoleucine-phenylalanine. 7. Composition according to one of the preceding claims, characterized in that the adhesine receptors blocking substances are used in combination with an inert macromolecular carrier.

Inventors: DE GRAAF FRITS KAREL, JACOBS ANTONIUS ARNOLDUS CHRIS, DE GRAAF, FRITS, KAREL, JACOBS, ANTONIUS, ARNOLDUS, CHRISTIAAN
Applicants: VERENIGING VOOR CHRISTELIJK WE, VERENIGING VOOR CHRISTELIJK WETENSCHAPPELIJK ONDER
Application year: 1986
Main classification: A61K37
IPC codes: A61K38, A61K39, C07K5, C07K14