JPH06504533A - Lectins in symbiosis - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Lectins in symbiosis The present invention aims to improve symbiosis, particularly on these sites by blocking tissue attachment sites. The invention relates to the use of lectins to prevent the adhesion of undesirable organisms.

Bacteria, other microorganisms, parasites and other undesirable organisms are Attach or bond to tissue through woven areas. The combination of these organisms is this lectins (and, especially in the case of bacteria, adhesins and As a result of the activity of lectins (known as adhesins and agglutinins), sugars - Specific. Lectins are covalently bound structures of their ligands, usually carbohydrates. Recognizes specific sugar residues and binds to them in a reversible manner without causing modification. A protein or glycoprotein that has the properties of a non-immunoglobulin.

For example, when attached or bound to the small intestine of an animal, it carries lectins (Iecji n-carrying) microorganisms or parasites can multiply, thereby poisoning the animal. Kill or injure animals. Although the binding of lectins to sugar residues is known, , research that has been conducted to date has generally only considered the nature of the bond, and No consideration was given to the actual benefits and uses to be derived from it.

Delivering harmless or beneficial lectins to avoid giving sites to undesirable organisms. (Ieetin-carr7ing) organisms 1 or the lectin itself. Leclin-carrying attachment sites for undesirable organisms It has been thought that significant benefits can be gained by blocking.

According to the first concept of the invention, preferred lectin-mediated binding to tissues is provided. For the manufacture of drugs for the treatment or prevention of diseases or conditions caused by harmful organisms. carry substantially non-toxic lectins or lectins (lecjin-ca) rr7ing) biological uses.

Substantially non-toxic lectins can be used to prevent toxic adhesion to tissues. , carrying harmful or other undesirable lectins or lectins (Iecti n-carrying) that are harmless or beneficial to animal tissue compared to living organisms. be.

This means that the lectin used in the present invention is the only one without side effects. (Of course, something like this is preferable.) Rather, overall, the result of adhesion to tissue is the attachment of microorganisms or parasites. carry any lectin or lectins if it is favorable compared to the results resulting from ( It can also be used in lectin-carBing) organisms.

Lectins suitable for use in the present invention are typically of plant origin. the above As a lectin, specifically, Amaryllidacea (Amaryllidacea) (AmarYl) Lidaceae) family, Lilaceael family, Iridacea (Iri) plants of the daceaeJ family and the aliacea family (especially this Examples include those obtained from the bulbs of the same species. Amaryllidacea Preferred members of the family idaceae are generally snowdrops (s nowdrop) (Galanjhus niv direct) 1is)) and narcissi (dxf l.

Galanthus such as dils) and na +cissus) subspecies. of the Iridaceae family Preferred examples include Irises, and Liracea ( Lilacexelidae (or Alliaceaelidae) preference Examples of potatoes include Allium subspecies such as garlic.

Lectins particularly preferably used in the present invention are those that do not adhere to tissues. dependent on microorganisms or parasites that are Rectification for treatment (or prevention) The antimicrobial agent is directed against bacterial or other microorganisms for attachment to surface receptors on tissues. compete with parasite lectins, or this alters surface receptor expression. It is like becoming

Regardless of the mechanism, therapeutic lectins, e.g. It must be able to combat undesirable lectins, such as sugars that can be combined. this Thus, for example, undesirable organisms can target mannose or lactose. If so, use a mannose-specific or lactose-specific rectifier. Chin is used. The specificity of lectins can be easily challenged by agglutination studies. , this provides a method for determining suitable lectins for use in the present invention. It will be done.

Gaianthus n1va specific for mannose lis) agglutinin (GNA) is known to bind to sites in the small intestine. Therefore, this agglutinin carries a mannose-specific lectin (lectin). n-carrying) to prevent microorganisms, parasites or any other harmful organisms This is an example of a lectin useful in the present invention that has a special purpose of being used. Ru. This allows Escherichia coli (E. coli), which is specific for toxic mannose, to inhibited or controlled. GNA is based on Van Damme et al. FEBS Letters (FEBS Levers), Volume 215, Page 14 Snowdrop (tn.0-144 C1987) method.

It can be isolated from the bulbs of P. wdrop.

Other sugars (usually monolithic) for which plant lectins have specificity include glucose, Galactose, lactose, N-acetylglucosamine, N-acetylgalactose N-acetylamino-sugars such as samin, α-2,6-neuraminylgalactose, etc. can be mentioned. One or more sugars are involved in lectin binding. The present invention maximizes Broadly speaking, it is not limited to specific sugars or combinations thereof.

Although lectins are proteins or glycoproteins, lectins (such as GNA) In some cases, it is thought that most of the amount remains even after passing through the intestines. Other lectins are degraded However, with most plant lectins, the survival rate is low. Both are 20% or more. This means that in the use of the present invention, lectin related to the formulation or publication of - Examples include Recreation, as described below. Chin is naturally administered orally. Second, in a special way that allows it to remain even after passing through the intestines. There is generally no need to protect or carry the administered lectin; however, if appropriate A suitable carrier or protection method may be used to protect the lectin, which is less stable in the intestine. This may be desirable depending on the environment, such as when using a machine. therefore, Therapeutic lectins are [lecjin-carr7ing] Overall, it has appropriate toxicity characteristics for the particular environment of use. must have.

As mentioned above, lectins with various specificities can be isolated from plants. but However, the present invention is not limited to lectins naturally synthesized in growing plants. . For example, tissue culture may be used to produce suitable lectins. Furthermore, recombinant Using DNA technology, lectins can be produced in heterologous host cells and adapted to specific environments. The structure of the lectin may be changed to Is it the same as natural lectin? Regardless, synthetic lectins can also be used. For example, Sigma Chemical C.O. Eltidi (Sigma Chemical C.

, LNf, ), Poole, Dorset, UK Many natural lectins are commercially available.

The present invention provides lectin-carrying microorganisms and parasites. and are typically useful in counteracting the effects of other undesirable organisms and organisms. Ru. Often, the present invention also carries lectins (leclin-cgrryi ng) Used to overcome or prevent the effects of bacteria.

There are a variety of bacteria against which the present invention can be used with considerable effectiveness. These details As a bacterium, Escherichia (EscheTichia) subspecies (Escherichia coli (Esch e+1chia coli, etc.), Salmonella subspecies ( Salmonella enteritidis (5a1m.

netla enterilidis), Salmonella typhoid ((Salmone lla 1ypb i) and Salmonella typhimurium (Salmonella typhimurium). a typhimiu "ium", etc.), Lactobacillus (Lacjobacillus lusl subspecies and e.g. Proteus subspecies (Proteus subsp. Non-lactose such as Proleus m1rabilis etc.) - Fermentative E. coli type is specifically mentioned, but these are just a few examples.

The present invention not only targets microorganisms that are generally regarded as pathogens, but also It can also protect against the harmful effects of microorganisms that are not recognized as such. It is important to note that For example, non-toxic Escherichia coli (E.

High numbers of commensal and/or opportunistic bacteria, such as E. coli, At high concentrations, many extracellular metabolisms occur that are harmful, resulting in severe diarrhea (so-called symptoms of non-specific colitis). Also, depending on the substance, bacteria may Toxic as it causes excessive growth and even the harmful consequences mentioned above; Paradoxically, substances included in the group of listed substances include kidney beans, which are listed below. (Phaseolus vulgaris) Certain plant lectins include agglutinin (PHA).

Bacterial “lectins” (often referred to herein as adhesins) are commonly It is carried by the fireworks. Such fruit-like adhesins contain various sugars (e.g. but not limited to, glucose, galactose, lactose, N-acetylgluco N-acetylamino-sugars such as samine, N-acetylgalactosamine, α-2,6 - specificity for neuraminylgalactose, etc.), but with little Some strains of E. coli and some species of Salmonella monella) subspecies are in some cases mannose-specific.

Bacteria such as Escherichia coli (E. coli) are targeted by lectins carried by the bacteria. It binds to the cells of the small intestine wall through mannose or other sugar residues in the wall of the small intestine.

These mannose sugar residues are primarily present in vesicular gland cells, which are inaccessible to bacteria. . However, other sugar residues are added during normal differentiation and maturation of vesicle gland cells. As the cells migrate to the intestinal villi, the original mannose groups gradually become more complex. buried in the combined structure. In addition, these are E. coli manno can no longer function as an attachment site for lectin-adhesins specific to the base.

In this way, under normal conditions, a fairly small number of Escherichia coli (E. coli) cells binds to the villus tip cell.

The toxicity of some compounds can be explained by understanding the above points. example For example, common bean (Phaseolut yu1ga+) is a pathogenic lectin. When isl agglutinin (PHA) is present in the intercorporeal ring of the body, it is transmitted from the vesicular gland to the villus. The rate of supply of cells to the vesicular glands increases considerably, and this allows the cells to differentiate and differentiate within the vesicular glands. and the time to maturity is shorter. As a result, as in the vesicular gland, the manno As the proportion of immature cells increases despite expressing S residues, So, the villi start to occupy the area. In this way, a larger number of Escherichia coli (E, coli) can bind to the resorptive villi, thereby increasing the number of absorptive villi. Supports the above theory The reason for this is that PHA does not contain microorganisms (germ-tree) (germ-tree). This comes from the fact that it is not toxic to rats.

By using the present invention, substantially non-toxic (i.e., harmless or beneficial) lectins occupy sites on the villi, which allow bacteria to bind to these sites. The above-mentioned effects, and the toxic effects of similarly acting substances can be prevented or treated.

From the above, lectin-carrying organisms bind Tissues include the gastrointestinal tract in general, and the intestine in particular, due to interactions with sugar residues. It is thought that From a nutrition and health perspective, the small intestine is the most important part of the intestine. However, before the above, part of the gastrointestinal tract or other tissues in the non-gastrointestinal tract are This does not mean that the invention cannot handle it. This invention is applicable Tissues that form part of the intestine include the cecum and large intestine; other tissues These include the urethra, trachea, and other organs that are open for invasion and infection from the outside world ( damaged or healthy) tissue.

The invention can be used in both human and animal medicine, as well as in dairy farming.

Depending on the animal that is raised and then slaughtered for food, antibiotics may not be added to the feed. This is a common practice in recent years. However, this For example, the presence of antibiotics It promotes the development of resistant strains of bacteria, which can upset the balance of the intestines. The present invention , an alternative to the use of antibiotics that does not have the associated social and biological disadvantages In other words, a non-toxic lectin that can be used in the present invention Animals can be raised on feed containing effective amounts of

The present invention is useful for the treatment or prevention of disease or other medical or veterinary conditions. It is used in When used in therapy, the or the amount of undesirable organisms attached to it. It is preferable to administer the blocking agent, lectin, preferably in an amount greater than that. prevention In this case, the blocker lectin binds to sugar residues in tissue-L and removes harmful lectins. Preventing or reducing the adhesion of 1ectin-carrying organisms Ru.

Therefore, dosages to treat symptoms should be combined with preventive dosages (in feed). (including doses added regularly or as needed) considered to be less than . The most appropriate dosage and therapeutic regimen will depend on the nature and severity of the infection, the use Depending on the type of lectin used and other factors, the clinician, internist, prison surgeon or Typical dosage is 0.001 to 0.5 g /kg body weight (-dose or per day) range, approximately 0.05 g/k The weight is g.

The lectin in the present invention is preferably administered not in one time (in several consecutive days). given. In an experiment, 10 mg of GNA was administered once to rats, and 1 hour later It is known that when the small intestine was examined, no binding of GNA to the intestinal wall was observed. There is. This applies, for example, to the bulbs of daffodils, It has also been confirmed in other lectins of the same family, such as the bulbs of succulents and the bulbs of garlic. ing. However, by administering for 10 days at a dose of 42 mg daily , strong binding was observed. ” Bipedal findings reveal the location of mannonose in the rat intestine The material already bound to the Amaryllidacea (of the family Amaryllidaceie J) After binding is inhibited by lectins, cells on the villi become new cells from the vesicular glands. During displacement, these materials are dislodged and flushed through the intestines. It is explained. Additionally, this new site may be replaced by bacteria or other microorganisms. occupied by sequentially administered lectins.

Preferably, the lectin is administered orally. Maintains the condition of the stomach and other areas of the gastrointestinal tract It is particularly preferred for administration by the above routes. however, Other routes of administration may also be used. Rectal administration and, at least in some indications, parenteral administration. Oral administration is also preferred in some cases. Formulations for parenteral administration are usually sterile .

Treatment (prevention) according to the present invention is carried out by fasting or restricting food intake. will be accomplished. Under these circumstances, virtually non-toxic lectins may be more effective. It is known that There are various reasons for the above, but appetite loss due to gastrointestinal infection is This is consistent with the common human experience of decline.

According to the second concept of the present invention, in medicine, adhesion to tissues is particularly mediated by lectins. Infection or infestation by infectious or ectoparasitic substances that are present Substantially non-toxic lectins or lectins used in prophylaxis or therapy This provides an organism that carries (Iecjin-carrying).

According to a third concept of the invention, carrying lectins or lectins (Iectin- carBing) from biological and pharmaceutical carriers or veterinary use carriers. The present invention provides a pharmaceutical composition or a pharmaceutical composition for animals.

Such compositions may be in solid or liquid form, depending on the mode of administration used. contains suitable excipients.

According to the fourth concept of the invention, the one or more that can prevent or reduce the adhesion of lectins From feeding the animal an appropriate diet supplemented with the above substantially non-toxic lectins, The present invention provides a method for raising animals, which is a method for raising animals.

According to the fifth concept of the invention, the one or more that can prevent or reduce the adhesion of lectins -1- Provides animal feed consisting of substantially non-toxic lectins. .

The present invention typically provides that the tissue attachment site consists of one or more sugar residues; Lectins or lectins that are harmless or beneficial to the body containing tissues Bringing the tissue into contact with a lectin-causing organism The method shall include a method of blocking the attachment site on the tissue, which is a method of is understandable.

Preferred of the various concepts of the invention are described mutatis mutandis. is mutandig) as described in the first concept.

Modifications and improvements are possible without impairing the concept of the invention and are described below. This will be described by way of examples.

Example In this example, kidney bean (Phaseolus vulgaris) Adding food to PHA, a lectin obtained from the seeds of P. lus vulgaris) Increases the number of mannose receptors in the brush border of the small intestine by exposing This reduces the overabundance of non-pathogenic mannose-fruited Escherichia coli (E. coli). induces the growth of This lectin induces the growth of extensive hyperplasia in the small intestine and Furthermore, by increasing the rate of migration of cells from the vesicular glands into the absorptive villi, Decrease the time it takes cells to differentiate and mature. Mannose in epithelial cells As a result of the increased number of receptors, more Escherichia coli (E. coli) Adhesion sites occur, leading to hyperplasia in the small intestine. 30-40mg Administration of PHA/rat/day reduced E. coli (E. , coli) are typically obtained. Mannose-specific lectin, GNA By pre-treating the brush border with mannose-receptor sites, this It is blocked by Chin and is no longer available for E. coli binding. P When administered together with HA, GNA competes and blocks the new mannose sites created. in this way, stopping the growth of bacteria.

Animals: Male two-headed dog from Rowett colony Hooded-Lister rats were used. child Their average weight was 80-85g, and their diet before the experiment was control diet. (6g/rat/day).

Control feed: lactalbumin protein-based control feed A sample (100 g protein/kg) was prepared according to the following formulation.

― Corn starch 374 Potato starch 100 Glucose 150 Silicic acid 0.4 Pyridoxine (Bi9min B@l 1,000mg Iron sulfate 25,000mg Riboflavin 1,000mg Manganese Sulfate 20. OQOmg N Amino Rest Fragrance acid f,000tog Zinc sulfate 18,000ml/g Nicotine II 3,0 00mg Potassium iodine 200mg Calcium pantothenate 2,000 mg Potassium iodide 200mg Confectic acid 500mg Sodium fluoride 60 0mg vitamin A I, 200mg stannous chloride 600mg vitamin D 25 0mg Sodium selenate 30mg Vitamin E 6,000mg Chromium Alum 4,800mg Choline chloride 80.000mg Chloride IJ UA 110g (50g vitamin mixture and 50g mineral mixture per kg) Protocol: 4 groups of rats (5 animals/group). control group of rats were fed lactalbumin chow for the entire experimental period (group 1) and for the last 4 days. Subsequently, the stomach was intubated and saline was supplied. The second control (GNA) group included Feed control feed supplemented with GNA (0.7g/100g feed) for 6 days. Furthermore, for the last 4 days, GNA (50-60mg) was intubated into the stomach. /rat/day).

Group 3 received approximately 60 mg PHA/rat/mouth by intragastrically intubating the rats during the last 40 hours. The treatment was carried out in exactly the same manner as the second group except that . Group 4 was used throughout the entire experiment. (60-hour pretreatment + 40-hour experimental period) lactalbumin control diet During the last 4 days, approximately 60 mg of PHA/rat/day was supplied through intragastric intubation.

Rats were sacrificed and dissected, and 10 cm of small intestine was removed for bacterial counts.

Lactose-fermenting (E. coli) and non-lactose-fermenting The number of sexual E. coli types and Lactobacillus subspecies were measured and expressed as numbers per gram of wet small intestine tissue.

Bacterial measurement methodology: At the end of the feeding period, rats were killed by decapitation and their small The intestine was aseptically removed from the abdominal cavity.

Cut a 10cm section (between 5 and 15cm from the pylorus), weigh it, and make a 1:1 Maximum recovery with working dilution of 0 Diluent (Muximum Recover7 Diluent) Ultra-tura in Oxoid (code CM733) 20.00 Or pm for a short period of time (5 to 10 seconds) It was homogenized by Furthermore, this was continued until the final dilution of IX107. Diluted. Each diluted solution was poured into pine conkey agar (McConkey A). gar) No. 3, place a measuring pipette (mensured volume) on the plate. (5Q drops = 1 ml) and incubated at 37°C for 24 hours. It was cultured for a period of time. Colonies of dilutions between 10 and 50 colonies were counted. E. coli ( E. coli), use eosin methylene blue a Gar (Eosin Mrlhylene Blue Agar) (Oxoy Using McConk (manufactured by Oxoid; code CM69), Bacteria were subcultured from the plates of ey). Nibeye 20E (api) 20E) was used for further confirmation.

Results: 1 g of hydration in positive control rats fed PHA. Approximately 108 Escherichia coli (E, col) per proximal small intestine tissue. Compared to the comparison value in i), the number of E. coli was lower than that before treatment with PHA. In rats that were later given GNA, it decreased to approximately 10-105/g wet tissue. . GNA restricts the diet of rats during treatment with two lectins. (1-2g feed/rat/day, but 6g/rat/mouth before the experiment) and effective.

Copy and translation of written amendment) Submission (Article 184-8 of the Patent Law) May 31, 1993

Claims (20)

[Claims] 1. caused by undesirable organisms whose binding to tissues can be mediated by lectins. Substantially toxic substances in the manufacture of drugs for the treatment or prevention of diseases or conditions asexual lectins or lectin-carrying organisms use of things. 2. The substantially non-toxic lectin is Amaryllidacea (Amaryllidacea). eae) family, Lilaceae family, lridacea e) obtained from plants of the family or Alliaceae family. The use according to claim 1. 3. The substantially non-toxic lectin is derived from Galanthus sp. is obtained from a plant of the genus Narcissus. Uses as described in Scope 1. 4. The substantially non-toxic lectin is found in snowdrops. Obtained from Galant'hus nivalis) The use according to claim 1, which is 5. The lectin contains mannose, glucose, galactose, lactose, Cetylglucosamine, N-acetylgalactosamine and/or α-2,6- Claim 1 has specificity for soyraminylgalactose. Use as described. 6. The use according to claim 1, wherein the undesirable organism is a microorganism. 7. The use according to claim 6, wherein the microorganism is a bacterium. 8. The bacteria are of the genus Escherichia, Salmonella belonging to the genus Monella or Proteus , the use according to claim 7. 9. 8. The use according to claim 7, wherein the bacterial infection is toxin-mediated. 10. Use according to claim 1, wherein the tissue is part of the gastrointestinal tract. 11. 11. The use according to claim 10, wherein the tissue is the small intestine. 12. Use according to claim 1, wherein the medicament is administered for several days in succession. 13. The use according to claim 1, wherein the medicament is administered orally. 14. Claim 1, wherein the drug is administered while restricting or fasting. Use of. 15. Carrying substantially non-toxic lectins or lectins used in pharmaceuticals (l ectin-carrying) organisms. 16. Infectious or exoparasitic, where attachment to tissue is lectin-mediated Claimed for use in the prevention or treatment of infections or infestations caused by The lectin or lectin-carrying agent used in the drug according to scope 15 n-carrying) organisms. 17. Lectins or lectin-carrying organisms and a pharmaceutical or veterinary carrier. Pharmaceutical composition for use in medicine. 18. Prevents attachment of lectins carried by undesirable microorganisms or parasites one or more substantially non-toxic A method that consists of administering to the animal a suitable diet supplemented with lectins. Breeding method. 19. Prevents attachment of lectins carried by undesirable microorganisms or parasites one or more substantially non-toxic Animal feed consisting of lectins. 20. Attachment site on tissue consists of one or more sugar residues and has tissue Carrying lectins or lectins that are harmless or beneficial to the body ectin-carrying) organism and said tissue. A method of blocking attachment sites on tissue.