AU9066891A

AU9066891A - Lectins in probiosis

Info

Publication number: AU9066891A
Application number: AU90668/91A
Authority: AU
Inventors: Arpad Janos Pusztai
Original assignee: Rowett Research Services Ltd
Current assignee: Rowett Research Services Ltd
Priority date: 1990-12-14
Filing date: 1991-12-16
Publication date: 1992-07-08
Also published as: CA2098305A1; JPH06504533A; EP0561912A1; WO1992010204A1; GB9027185D0

Description

Lectins In probiosis

This invention relates to probiosis, and especially to the use of lectins in blocking attachment sites on tissue to prevent the attachment of undesirable entities on these sites.

Bacteria, other micro-organisms, parasites and other undesirable entities attach or bind to tissue through sites on the tissue which express sugar residues. The binding of the entities is sugar-specific as a result of the activity of lectins (also known as adhesins and lectin-adhesins, particularly in the context of bacteria, and agglutinins) which are carried by the entities. Lectins are proteins or glycoproteins of a non- immunoglobulin nature which recognise specific sugar residues and bind to them, generally in a reversible manner without causing any covalent modification to the structure of the carbohydrate ligand.

Once attached or bound, for example in the small intestine of an animal, the lectin-carrying microorganism or parasite can multiply and thereby poison or cause damage to the animal. The binding of lectins to sugar residues is known, but in general the studies that have been undertaken have considered only the nature of the binding and not any practical benefits and uses that may derive therefrom.

It has now been found that considerable benefit can be obtained by blocking attachment sites of the lectin- carrying undesirable entities by means of innocuous or beneficial lectin-carrying entities, or lectins themselves, in order to deny the sites to the undesirable entities. According to a first aspect of the present invention there is provided the use of a substantially non-toxic lectin or lectin-carrying entity in the manufacture of a medicament for the treatment or prevention of a disease or condition caused by undesirable entities capable of lectin-mediated binding to tissue.

Substantially non-toxic lectins are innocuous or beneficial to animal tissue, when compared with the toxic, harmful or other undesirable lectin or lectin- carrying entity whose attachment to tissue is sought to be prevented. This should not be taken to suggest that the only lectins which can be used in the invention are those which are free of side effects (although they are of course preferred) ; rather, any lectin or lectin- carrying entity can be used provided that, overall, the consequences of attachment to tissue are preferable to those resulting from the attachment of the microorganism or parasite.

Lectins suitable for use in this invention are generally of plant origin. They include those obtainable from

(particularly the bulbs of) plants of the families

Amaryllidacea , Lilaceae , Iridaceae and Alliaceae. Preferred members of the families Amaryllidaceae include

Galanthus and Narcissus spp. such as the snowdrop

(Galanthus nivalis) and narcissi (daffodils) in general.

Preferred members of the family Iridaceae include irises while those of the family Liliaceae (or Alliaceae) include Allium spp. such as garlic.

The precise lectin preferred for any particular application of the invention will depend on the microorganism or parasite whose attachment to tissue is unwanted. The therapeutic (or prophylactic) lectin may compete with bacterial or other microorganism or parasite lectin for attachment to surface receptors on tissue, or it may cause the expression of surface receptors to change. Whatever the mechanism, the therapeutic lectin should be matched to the undesirable lectin, for example by reference to the sugars to which both of them can bind. Thus for example if an undesirable entity targets mannose or lactose, a mannose-specific or lactose- specific lectin can be used. Specificity of lectins can readily be matched by agglutination studies, which therefore provide a means for determining suitable lectins for use in the invention.

The mannose-specific Galanthus nivalis agglutinin (GNA) has been shown to bind to sites in the small intestine and is therefore a useful lectin for inclusion in the invention with the specific aim to be used for the prevention of mannose-specific lectin-carrying microorganisms, parasites or other harmful entities. In this way, mannose-specific E. coli poisoning can be prevented or controlled. GNA can be isolated from snowdrop bulbs by the method of Van Damme et al . (FEBS Letters 215 140-144 (1987)).

Other sugars (generally in the D-configuration) for which plant lectins have specificity include glucose, galactose, lactose, N-acetylamino-sugars such as N- acetylglucosamine, N-acetylgalactosamine and others, such as α-2,6-neuraminylgalactose. More than one sugar may be implicated in the binding of a lectin. The invention is not, at its broadest, limited to any particular sugar or combination of them. Even though lectins are proteins or glycoproteins, some of them (including GNA) seem to survive passage through the gut almost quantitatively (Van Damme et al . , loc. cit. ) . Although others are less resistant to breakdown, with most plant lectins the survival rate might be at least 20% or more. This fact has implications for the formulation or presentation of lectins in the use of the invention. One implication is that the lectin may of course be administered orally, as will be discussed below. Secondly, there is usually no need to protect or carry the lectin being administered in any particular way to ensure that it survives passage through the gut; however, the provision of a suitable carrier or means of protection is not ruled out and may be desirable under certain circumstances, such as when the less gut-stable lectins are to be used. The therapeutic lectin may therefore be administered as a "lectin-carrying entity", which overall should have appropriate toxicity characteristics for the particular circumstances of use.

As indicated above, lectins of various specificities can be isolated from plants. However, the invention is not restricted to those lectins which have naturally been synthesised in a growing plant. For example, tissue culture may be used to produce suitable lectins. Further, recombinant DNA technology means that lectins can be produced in heterologous host cells and also enables the structure of lectins to be varied to suit particular circumstances. The use of synthetic lectins, whether or not corresponding to natural lectin, is not ruled out. Many natural lectins are available commercially, for example from Sigma Chemical Co. Ltd. , Poole, Dorset, United Kingdom. The invention is useful in combatting the effects of lectin-carrying microorganisms, parasites and, generally, other undesirable organisms and entities. Frequently, the invention will be used to counter or prevent the effects of lectin-carrying bacteria.

Bacteria against which the invention may be used with considerable effect are varied. They include Escherichia spp. (such as Escherichia coli) , Salmonella spp. (such as Salmonella enteriditis , S. typhi and S. typhimiurium) , Lactobacillus spp. and non-lactose-fermenting coliforms including Proteus spp. (such as Proteus mirabilis) , to give only a few examples.

It is important to note that the invention may give protection not only against microorganisms usually regarded as pathogens but also against harmful effects of those not normally so regarded. For example, high bacterial numbers of commensal and/or opportunistic bacteria, such as non-toxic E. coli , can cause severe diarrhoea (a symptom of the so-called "non-specific colitis") because they produce many extracellular metabolites which in high concentration are harmful. Some substances are toxic because they cause bacterial overgrowth, and the harmful consequences just mentioned; included within this group of substances are, paradoxically, certain plant lectins such as Phaseolus vulgaris agglutinin (PHA) , which will be discussed below.

Bacterial "lectins" (more often called adhesins in this context) are usually carried by fi bria. Such fimbrial adhesins can have specificity for a variety of sugars (including but by no means limited to glucose, galactose, lactose, N-acetylamino-sugars such as N- acetylglucosamine, N-acetylgalactosamine and others, such as α-2,6-neuraminylgalactose) , but in the case of at least some strains of E. coli and certain Salmonella spp. they are mannose-specific.

Bacteria such as E. coli bind to the cells of the small intestine wall through mannose or other sugar residues on the wall which are targeted by lectin carried by the bacterium. These mannose sugar residues occur mainly in the crypt cells which are not accessible to bacteria. However, with the addition of other sugar residues during the normal course of differentiation and maturation of crypt cells, as the cells move up the intestinal villi, the original mannose groups become gradually submerged in more complex structures. These then no longer serve as attachment points for the mannose-specific lectin-adhesin of E. coli . Thus under normal conditions relatively few E. coli cells will be bound to the villus tip cells.

The toxicity of some compounds can be explained by an understanding of these events. For example if the pathogenic lectin Phaseoluε vulgaris agglutinin (PHA) is present in the systemic circulation of the body, the supply of cells from the crypts to the villi will speed up considerably and, therefore, the cells will have less time to differentiate and mature in the crypts. As a result, the villi will be populated with an increasing proportion of immature cells, still expressing mannose residues, similar to those in the crypt. Thus, a greater number of E. coli can be bound to the absorptive villi and thereby multiply. Evidence in support of this theory comes from the observation that PHA is non-toxic for germ-free (gnotobiotic) rats. By means of the present invention this effect, and the toxic effects of similarly acting agents, can be prevented or treated as the substantially non-toxic (that is to say innocuous or beneficial) lectins occupy sites on the villi thereby denying these sites to the bacteria which then travel on through and out of the intestine.

It will be appreciated from what has been said above that tissues to which lectin-carrying entities bind, by means of interaction with sugar residues, include the gastrointestinal tract in general and the gut in particular. From the point of view of nutrition and health, the small intestine is the most important part of the gut, but this consideration does not mean that other tissues which may be either part of or separate from the gastrointestinal tract cannot be treated by the invention. Tissues forming part of the gut to which the invention may be applied include the caecum and the large intestine; other tissues include the urethra, the trachea and other (wounded or healthy) tissue open to invasion and infection from the outside world.

The invention finds application in both human and veterinary medicine, as well as in animal husbandry.

It has been common practice in recent years routinely to include antibiotics in the diet of some animals which are reared and slaughtered for food. However, this practice is becoming unacceptable for a variety of reasons; for example, the presence of antibiotics can encourage the production of resistant strains of bacteria and can disturb the intestinal balance. The present invention provides an alternative to the use of antibiotics without the associated social and biological disadvantages: animals can be raised on a diet containing effective amounts of the non-toxic lectins useful in the invention.

The present invention may be employed in the treatment or prophylaxis of disease or other medical or veterinary conditions. When used in treatment the lectin of the blocking agent is preferably administered in quantities sufficient to compete effectively with, and preferably to exceed, the quantities of the undesirable entities already bound or attached to the tissue. In prophylaxis, the lectin of the blocking agent binds to sugar residues on tissue to prevent or reduce attachment of harmful lectin-carrying entities. Doses for treatment of an existing condition will therefore be likely to be lower than prophylactic doses (which would include doses routinely or occasionally added to feeds) . While the most appropriate dose and treatment regime will be determined by the clinician, physician, veterinary surgeon or nutritionist depending on the nature and severity of infection, the type of lectin used and other factors, likely doses include those in the range of from 0.001 to 0.5 g/kg body weight (per dose or per day), possibly about 0.05 g/kg body weight.

Preferably the lectin in this invention is administered over a period of days instead of in a single dose. In experiments it has been found that when a single dose of lOmg of GNA was administered to rats and the small intestine examined after one hour, no binding of GNA to the intestine wall was observed. This has been confirmed with other lectins of the same family, for example from daffodil bulbs, iris bulbs and garlic cloves. However, a dose of 42mg administered each day for 10 days resulted in strong binding being observed. This finding may be explained by material already bound to mannose sites in the rats' intestines preventing binding by the Amaryllidaceae lectin until these materials have become detached and washed through the intestine in the course of time as the cells on the villi are replaced by fresh cells from the crypt. The new sites are then occupied by the continuously-administered lectin instead of the bacteria or other organisms.

Lectins are preferred for oral administration. Their ability to withstand the conditions of the stomach and other regions of the gastrointestinal tract make them particularly appropriate for administration by this route. However, other routes of administration are also contemplated. Rectal administration and, at least in some indications, parenteral administration may sometimes be appropriate. Formulations for parenteral administration will generally be sterile.

Treatment (or prophylaxis) in accordance with the invention may be accompanied by fasting or a lesser dietary restriction. Under such circumstances the substantially non-toxic lectins are observed to be more effective. The reasons for this are varied, but are consistent with the common human experience of loss of appetite coupled to gastrointestinal infection.

According to a second aspect of the invention, there is provided a substantially non-toxic lectin or lectin- carrying entity for use in medicine, particularly in the prevention or treatment of infection or infestation by infectious or infesting agents whose attachment to tissue is lectin-mediated. According to a third aspect of the present invention there is provided a pharmaceutical or veterinary composition comprising a lectin or lectin-carrying entity and a pharmaceutically or veterinarily acceptable carrier.

Such compositions may be in solid or liquid form and will contain suitable excipients, according to the intended mode of administration

According to a fourth aspect of the invention, there is provided a method of rearing an animal, the method comprising administering to the animal a suitable diet supplemented with one or more substantially non-toxic lectins capable of preventing or reducing the attachment of lectins carried by an undesirable microorganism or parasite.

According to a fifth aspect of the invention, there is provided an animal foodstuff comprising one or more substantially non-toxic lectins capable of preventing or reducing the attachment . of lectins carried by an undesirable microorganism or parasite.

It can be seen that the invention involves, in general, a method of blocking attachment sites on tissue, the attachment sites on tissue comprising one or more sugar residues, the method comprising contacting the tissue with a lectin or lectin-carrying entity which is innocuous or beneficial to the body containing said tissue.

Preferred features of the various aspects of the invention are as for the first aspect mutatis mutandis . Modifications and improvements may be made without departing from the scope of the invention, which will now be illustrated by the following example.

EXAMPLE

In this Example, the overgrowth of non-pathogenic, mannose-fimbriated E . coli is induced by increasing the number of mannose-receptors in the small intestinal brush border by dietary exposure to the lectin PHA, from the seeds of kidney bean (Phaseolus vulgaris) . This lectin induces extensive hyperplastic growth of the small intestine and, by speeding up the migration of cells from the crypts into the absorptive villi, reduces the time available for cells to differentiate and mature. With the consequent increase in the number of mannose- receptors of epithelial cells, more points of attachment for E. coli are generated leading to their overgrowth in the small intestine. With the input of 30-40mg of PHA/rat/day levels of E. coli of 10⁸ to 10⁹ per g of wet tissue are usually achieved. By pre-treatment with the mannose-specific lectin, GNA, the brush border mannose- receptor sites are blocked by this lectin and made unavailable for the binding of E. coli . If administered together with PHA, GNA blocks out competitively the new mannose-sites generated and, thus, arrests the proliferation of the bacteria.

Animals: Male Hooded-Lister rats of the Rowett colony were used. Their average weight was 80-85g and their pre-experimental diet was the control diet (6 g/rat/day) .

Control Diet: The lactalbumin protein-based control diet (lOOg protein/kg) was made up according to the following formulation.

(50g of vitamin mix and 50g of mineral mix added per kg of diet.)

Protocol: 4 groups of rats (5 animal/group) . Rats in the control group were fed lactalbumin diet (group 1) during the whole experiment and intragastrically intubated with saline for the last four consecutive days. The second control (GNA) group were fed control diet to which GNA is added (0.7g/100g diet) for 6 days (pretreatment) and then intragastrically intubated with GNA (50-60mg GNA/rat/day) for the last four days. Group 3 were treated exactly as Group 2 but on the last four days the rats were also intragastrically intubated with about 60mg of PHA/rat/day. Group 4 were fed lactalbumin control diet throughout the whole experiment (6 days pretreatment + 4 days experimental period) and intubated for the last four days with about 60mg PHA/rat/day. Rats were killed, dissected and 10cm of their small intestine taken for bacterial counting. The numbers of lactose-fermenting (-57. coli) and non-lactose-fermenting coliforms and Lactobacilluε spp. were determined and expressed as numbers per gram of wet small intestinal tissue.

Bacterial counting methodology: At the end of the feeding period, rats were killed by decapitation, their small intestine removed aseptically from the abdominal cavity. A piece of 10cm (between 5-15cm from the pylorus) was cut, weighed and homogenized by a short burst (5-10 sec) at 20,000rpm with an Ultra-Turrax homogenizer in a Maximum. Recovery Diluent (Oxoid; Code CM733) at 1:10 working dilution. This was further serially diluted to final dilution of 1 x 10⁷. From each dilutions drops were taken in duplicate by a measured volume pipette (50 drops = 1ml) onto MacConkey Agar No. 3 plates and incubated at 37°C for 24h. Colonies were counted from the dilution which gave between 10-50 colonies. For confirmation of E. coli , Eosin Methylene Blue Agar (Oxoid; Code CM69) was used for the bacteria subcultured from the MacConkey plates. Further confirmation was done with api 20E. Results: Compared with control values of about 10⁸ E. coli/q wet proximal small intestinal tissue in the PHA- fed positive control rats, the number of E. coli was reduced to about 10⁴-10⁵/g wet tissue in the rats fed GNA before and after PHA treatment. GNA was particularly effective if, during the period of treatment with the two lectins, the rats' dietary input was restricted (1-2 g diet/rat/day, compared to 6 g/rat/day before the experiment) .

Claims

1. The use of a substantially non-toxic lectin or lectin-carrying entity in the manufacture of a medicament for the treatment or prevention of a disease or condition caused by undesirable entities capable of lectin-mediated binding to tissue.

2. The use as claimed in claim 1, wherein the substantially non-toxic lectin is obtainable from the plant family Amaryllidaceae , Lilaceae , Iridaceae or AZliaceae.

3. The use as claimed in claim 1, wherein the substantially non-toxic lectin is obtainable from a plant of the genus Galanthus or Narcissus .

4. The use as claimed in claim 1, wherein the substantially non-toxic lectin is obtainable from the snowdrop (Galanthus nivalis) .

5. The use as claimed in claim 1, wherein the lectin has specificity for mannose, glucose, galactose, lactose, N-acetylglucosamine, N-acetylgalactosamine and/or α-2,6- neuraminylgalactose.

6. The use as claimed in claim 1, wherein the undesirable entity is a microorganism.

7. The use as claimed in claim 6, wherein the microorganism is a bacterium.

8. The use as claimed in claim 7, wherein the bacterium belongs to the genus Escherichia , Salmonella or Proteus .

9. The use as claimed in claim 7, wherein the bacterial infection is toxin-mediated.

10. The use as claimed in claim 1, wherein the tissue is part of the gastrointestinal tract.

11. The use as claimed in claim 10, wherein the tissue is the small intestine.

12. The use as claimed in claim 1, wherein the medicament is intended for administration over a period of days.

13. The use as claimed in claim 1, wherein the medicament is intended for oral administration.

14. The use as claimed in claim 1, wherein the medicament is intended for administration with dietary restriction or fasting.

15. A substantially non-toxic lectin or lectin-carrying entity for use in medicine.

16. A lectin or lectin-carrying entity for use in medicine as claimed in claim 15, for use in the prevention or treatment of infection or infestation by infectious or infesting agents whose attachment to tissue is lectin-mediated.

17. A pharmaceutical or veterinary composition comprising a lectin or lectin-carrying entity and a pharmaceutically or veterinarily acceptable carrier.

18. A method of rearing an animal, the method comprising administering to the animal a suitable diet supplemented with one or more substantially non-toxic lectins capable of preventing or reducing the attachment of lectins carried by an undesirable microorganism or parasite.

19. An animal foodstuff comprising one or more substantially non-toxic lectins capable of preventing or reducing the attachment of lectins carried by an undesirable microorganism or parasite.

20. A method of blocking attachment sites on tissue, the attachment sites on tissue comprising one or more sugar residues, the method comprising contacting the tissue with a lectin or lectin-carrying entity which is innocuous or beneficial to the body containing said tissue.