WO1996028176A1

WO1996028176A1 - Composition for oral tolerance induction and use thereof

Info

Publication number: WO1996028176A1
Application number: PCT/EP1996/001004
Authority: WO
Inventors: Dominique Rigal; Claude Pinon; Jean-Claude Truffier
Original assignee: TRUFFIER, Francis
Priority date: 1995-03-09
Filing date: 1996-03-08
Publication date: 1996-09-19
Also published as: FR2731353A1; AU5004296A

Abstract

The present invention relates to a composition for enteral administration comprising a quail's egg lyophilisate, characterized in that the quail's egg lyophilisate is produced in the presence of 5 to 40 % by weight of carbohydrate, based on the weight of the quail's egg content. The invention also relates to the use of quail's egg or of such a composition in the preparation of a medicament or nutritional formulation for the induction of oral tolerance in mammals, whereby the quail's egg or said composition improve the oral tolerance.

Description

Composition for oral tolerance induction and use thereof

The present invention relates to the use of quail's egg or quail's egg lyophilisate as an adjuvant for induction of oral tolerance (OT).

Oral tolerance, or systemic unresponsiveness to ingested antigen, was first described at the turn of the century. It is usually effected by oral administration of minimal and very progressively increasing doses of antigen under medical surveillance favouring the systemic unresponsiveness to the ingested antigen. OT may thus be employed to treat or cure allergies, auto-immune diseases, pathological situations where auto-immunity is an aggravating factor, induction of tolerance prior to organ transplants, etc.

However, the rules which govern the induction of OT are still not well known. OT could be induced in several experimental animal models but its induction varied e.g. with: the structure of the antigen and its sensitivity to proteolysis the dose of antigen ingested the chronology of administration the method and duration of administration the species and strains of the animals employed the age and sex of the animals the mode of feeding.

It is very difficult to control all these parameters and consequently to control induction of OT. What is needed is a stabilizing factor which provides the required reliability to the treatment of induction of OT so that it can be used on a large scale basis as a conventional treatment for allergies, auto-immune diseases, etc. The present invention provides a solution to this problem by providing an adjuvant for the induction of OT which improves the outcome of the treatment, i.e. which enhances the effect on OT, and gives it the required reliability.

The use of the quail's egg iyophilisates for the treatment of allergy-related illnesses was the object of EP 0 032 344. However, while EP 0 032 344 describes a therapeutic effect with quail's egg lyophilisate in the treatment of immuno-allergic diseases it is silent with respect to the induction of oral tolerance.

Furthermore, according to the present experimental systems when quail's eggs are lyophilised according to standard methods from the agro-alimentary industry, e.g. according to the method described in EP 0 032 344, they lose their capability to enhancing the OT-inducing effect of an antigen administered to obtain an immune response of the tolerance type.

We have now surprisingly found that the property of quail's egg to enhance the OT-inducing effect of an antigen is well maintained when carbohydrates are added to quail's eggs in a proportion of 5 to 40% by weight, based on the weight of the quail's eggs, before subjecting the eggs to the lyophilisation process.

The present invention therefore provides for a composition for enteral administration comprising a quail's egg lyophilisate, characterized in that the quail's egg lyophilisate is produced in the presence of 5 to 40% by weight of carbohydrate, based on the weight of the quail's egg content, (hereinafter referred to as composition of the invention)

Quail's eggs or the composition of the invention are used in combination with an antigen for which an immune response of the tolerance type, i.e. systemic unresponsiveness, is to be achieved, in order to enhance the OT-inducing effect. The antigen can be administered prior to the administration of the adjuvant, subsequently or simultaneous with the adjuvant. Preferably the antigen is mixed together with the composition of the invention and administered simultaneously.

The invention further provides for the use of quail's eggs or of a composition of the invention in the preparation of a medicament or nutritional formulation for the enhancement of induction of oral tolerance in mammals.

Preferably, the mammals are humans.

The invention also provides a method for enhancing the OT-inducing effect of an antigen in a mammal, which comprises administering enterally, prior to, subsequent to or simultaneous with the antigen to which an immune response of the tolerance type is to be achieved, an amount of quail's egg or of a composition of the invention which is effective for enhancing the OT-inducing effect of the antigen.

Quail's egg as used herein refers to egg white and/or yolk, OT-inducing components of egg white and/or yolk or mixtures thereof. Preferably quail's egg refers to egg white and yolk, i.e. whole egg content.

Any quail's eggs may be used according to the invention. Typical examples of eggs suitable as an adjuvant for the induction of OT include those from the following quail types: Coturnix-coturnix, Coturnix-delagorguei, Coturnix- novaeZealandiae, Coturnix-chinensis, Coturnix-coturnix japonica, Excalfactoria and strain MINA B. The eggs may be fertilized or unfertilized, fertilized eggs being preferred. Preferably fertilized eggs of the quail strain MINA B are used in accordance with the invention, whereby MINA B is a cross of the wild quail Coturnix-coturnix-coturnix and the quail Coturnix-coturnix-japonica. Whereas eggs of other quail species have revealed the same properties as the fertilized eggs of MINA B, this effect was never obtained with eggs of chickens, ducks, guinea fowl or pheasants.

The dosage of the composition of the invention for humans per day is preferably from 2 to 12 g. It is normally administered as cure of nine consecutive days followed by nine consecutive days without treatment and an optional further cure of nine consecutive days. The corresponding dosage of fresh quail's egg is from 1 to 6 eggs per day.

Antigen as used herein refers to soluble or particulate antigen such as allergens, auto-antigens, allo-antigens, xeno-antigens, proteins, glycoproteins, cells such as red blood cells, bacteria, viruses, etc. The dosage of antigen to be employed to obtain the desired effect will depend on the particular treatment and the desired oral tolerance. Such dosages are known or can be established by the skilled person in a manner known per se for conventional treatment with antigen to induce OT. In general, the amount of antigen to be administered lies in the range of from 1 ng to 1 g, preferably from 1 mg to 500 mg, particularly preferred about 100 mg. The antigen may be administered enterally such as by oral feeding or intragastric feeding or by inhalation. The term OT is also used herein when reference is made to administration of antigen by inhalation or intragastric feeding because OT is a term known in the art and describes the same effect that is observed with the present invention.

For a successful induction of OT administration of antigen and quail's egg or the composition of the invention should be started at least five days before the challenge with the immune response inducing agent, whereby the duration of administration may vary. Usually induction of OT can be effected after treatment according to the invention for three weeks, however, improvements of the clinical condition may already be observed after one week.

Induction of OT can further be enhanced through the "idiotypic network" if auto¬ antibodies are administered in combination with the quail's egg or the composition of the invention in combination with the antigen as set out above. Depending on the quantity and frequency of administration of auto-antibodies a higher rate of induction of OT is achieveable. Generally, for humans the dosage of auto¬ antibodies having an enhancing effect on induction of OT is within the range of 1 μg to 10 mg per day during 1 to 10 days. The autoantibodies can be administered prior to, subsequent to or simultaneous with the quail's egg or the composition of the invention in combination with the antigen, whereby mixing the quail's egg or composition of the invention with antigen and auto-antibody and administering all these ingredients simultaneously is preferred.

A recent study carried out in vitro by the present inventors showed that it is possible to induce the production of a lymphokine, the Transforming Growth Factor (TGF beta 1), by means of the lymphomonocytes in the presence of the quail's egg. This cytokine is known to have very immunosuppressive properties.

Accordingly, the invention further provides the use of quail's eggs or of a composition of the invention in the preparation of a medicament or nutritional formulation for the induction of production of lymphokines with immunosuppressive properties.

Apart from the addition of carbohydrates prior to lyophilisation the composition of the invention can be prepared by lyophilisation of egg contents according to known methods. Some preferred methods of lyophilisation are as follows:

The batches of eggs are steeped in an antiseptic solution, broken en masse, filtered, centrifuged, and mixed with carbohydrates in a proportion of 5 to 40% (w/w), preferably 5 to 20% (w/w). Suitable examples of carbohydrates that may be used in this application are disaccharides such as maltose, trehalose or lactose, monosaccharides such as glucose, fructose, galactose or carbohydrate mixtures such as honey. The carbohydrate is preferably saccharose or honey. The mixture may then optionally be pasteurized at a temperature not exceeding 37°C.

Lyophilization is carried out with a freezing stage which should not drop below -42°C, at a rate of cooling of 5°C/min, at a pressure of 0.01 torr. An alternative consists in freezing the egg contents abruptly with liquid nitrogen. Drying should be effected at less than 25°C at a pressure of 0.003 to 0.006 torr. Afterwards, the powder is divided into sachets of 6 g which corresponds to 3 eggs.

Alternatively, the quail's eggs are separated into white and yolk according to a manual or industrial procedure especially adapted to quail's eggs and egg white and yolk are mixed separately with the carbohydrates. Then the whites are lyophilised according to a standard procedure and the yolks are lyophilized by freezing in liquid nitrogen. Drying should be effected at less than 25°C at a pressure of 0 003 to 0.006 torr. Afterwards, the powder is divided into sachets of 6 g which corresponds to 3 eggs.

A standard procedure for lyophilising the egg whites is e.g. as follows: the egg whites are put into a petri dish whereby the thickness of the egg layer should not exceed 2 cm, then the petri dish is put in a -80°C-freezer overnight, whereafter it is transferred to a dryer chamber where for the primary drying of 24 hours the condenser temperature is -45°C and the chamber pressure 0.01 torr and for the secondary drying of 6 to 12 hours the shelf temperature is +5°C and the pressure 0.01 torr.

Generally, methods including a freezing step in liquid nitrogen are preferred.

These preparations preserved all the properties of the raw egg regarding the adjuvant effect on the induction of OT in methods used with mice.

Accordingly, the invention further provides for a process for manufacturing quail's egg Iyophilisates comprising the steps of: a) separating egg contents from eggshells, b) adding 5 to 40% (w/w) of carbohydrate and mixing it with the egg contents, c) freezing and drying the mixture.

The composition of the invention may be formulated into conventional enteral administration forms, such as granules, tablets, capsules, liquids, powders, etc. When formulated in a physiologically acceptable formulation form such as a capsule or tablet form, such formulations will conveniently contain 0.2 to 90 % by weight, preferably from 30 to 50 % by weight of the composition of the invention. Typical pharmacologically acceptable formulation forms for oral administration will further comprise pharmacologically acceptable diluents, carriers, vitamins, spices, pigments and/or other adjuvants well known to the skilled person to be suitable for incorporation into such formulation and preferably also antigen. Preferably, the composition of the invention is administered in powder form as this form is most convenient to mix the lyophilisate with the appropriate antigen.

In order to illustrate the invention, five examples on the mouse are described, in the course of which OT was induced with the assistance of the quail's egg lyophilisate produced according to the process described under A) below. In all these experiments, fertilized eggs of the strain MINA B - a cross of the wild quail Coturnix-coturnix-coturnix and the quail Coturnix-coturnix-japonica were used.

A) Preparation of quail's egg lyophilisate

Batches of eggs are steeped in an antiseptic solution, broken en masse, filtered, centrifuged, and mixed with saccharose in a proportion of 5 to 20% (w/w). Then the mixture is pasteurized at a temperature not exceeding 37°C.

Subsequently the egg contents are frozen abruptly with liquid nitrogen and dried at less than 25°C at a pressure of 0.003 to 0.006 torr. Afterwards, the powder is divided into sachets of 6 g which corresponds to 3 eggs.

JU Adjuvant action of the quail's egg on the induction of OT

Example 1:

Production of anti-transferrin antibody in C3H. Balb/c. OF1 mice

Balb/c mice do not produce OT to soluble antigens, whereas it is very easy to induce OT with C3H mice. Human transferrin was used as antigen. Thus, 5 Balb/c mice (group A) were given orally 20 mg of transferrin dissolved in 1 ml of isotonic salt serum at 0.9% on day 0. Then, on day 15 they were given 1 mg of transferrin mixed in 100 μl of complete Freund's adjuvant (CFA) by injection, finally on day 30 the anti-transferrin antibody titre is evaulated by a passive haemagglutination technique according to the method of Johnson et al (Int Arch Allergy 1968, 33, 511-520).

A group B of 5 Balb/c mice underwent the same treatment, but were given the transferrin by digestive means, i.e. enterally, with 100 mg of a mixture of quail's egg lyophilisate before challenge with transferrin on day 15. In addition, a control series of 5 mice were given orally 20 mg of bovine albumin with quail's egg lyophilisate; then on day 15 they were given 1 mg of transferrin mixed in 100 μl of complete Freund's adjuvant (CFA) by injection, finally on day 30 the anti-transferrin antibody titre is evaulated as set out above (group C).

Another control group D of 5 Balb/c mice underwent the same treatment as group A but were given fresh chicken's egg instead of quail's egg lyophilisate.

The same experimental pattern was carried out with C3H mice (groups E, F, G, H) and OF1 mice (groups I, J, K, L).

Balb/c C3H OF1

transf p.o./ day 0; Tf+CFA day 15 A E I

idem + quail's egg (100mg) B F J

Bov. Alb. p.o./ day 0; Tf+CFA day 15 C G K + quail's egg (100mg)

transf p.o./ day 0; Tf+CFA day 15 D H L + chicken's egg (100mg)

The results of these experiments are given in the following table. The values indicated represent the geometrical average of the titre of antitransferrin antibodies of each animal in each group. Balb/c C3H OF1

transf p.oJ day 0; Tf+CFA day 15 13 5 16

idem + quail's egg ( 00mg) 4 1 3

Bov. Alb. p.oJ day 0; Tf+CFA day 15 16 18 17 + quail's egg (100mg)

transf p.oJ day 0; Tf+CFA day 15 15 7 14 + chicken's egg (100mg)

These results show that the addition of the quail's egg to the antigen at the moment of oral administration increases the induction effect of OT in Balb/c mice and in OF1 which have little sensitivity to this phenomonen in the basal state.

Example 2

Delayed hypersensitivity of contact (OHO of OF1 mice

In this series of experiments, outbred mice were used in order to show that the induction of a OT with the quail's egg was independent of genetic background.

The hapten used is DNCB (2,4-dinitrochlorobenzene) obtained from Sigma. This hapten is diluted in a mixture of acetone/olive oil (4 vol / 1 vol). To induce a state of hypersensitivity of contact, after shaving the backs of the mice, a 5% solution of DNCB was applied thereto on day 0. Then, the reaction was revealed 5 days later by application to the ear of a 1% suspension. Finally, the reaction is read 48 hours later by measuring the thickness of the ear with a micrometer. The controlateral ear serving as a control. Under these conditions, a specific DHC is observed, which may be easily evaluated.

OT was induced by the ingestion of 500 μl of a 1.25% suspension of DNCB with or without 100 mg of quail's egg 7 days before immunisation. The following table summarises the results of the groups of 5 mice. The values indicate the difference in thickness in mm between the test ear and the reference ear.

DNCB per os day -7 DNCB per os + quail's egg test

group 1 0 0 50±6

group 2 + 0 36±8

group 3 0 + 16±9

It is evident that the simultaneous administration of the antigen with the quail's egg induces much greater OT with all the animals.

Example 3

Prevention of experimental autoimmune haemolvtic anaemia (AIHAi of the OF1 mouse

The intraperitoneal injection of 10⁸ rat erythrocytes once per week for 4 to 5 weeks causes AIHA of the mouse with the presence of auto-antibodies and anti rat- erythrocyte xeno-antibodies. This AIHA occurs with 100% of the animals (group A). If 10⁹ rat erythrocytes are ingested 3 days before each of the injections, a reduction in the number of animals affected by AIHA is obtained (60%) with titres a little lowered (group B). A mixture of 10⁹ rat red blood cells with 100 mg quail's egg increases this effect dramatically and brings about an important reduction in the number of animals having AIHA, since only 10% of the animals are affected with very weak titres of anti mouse erythrocyte auto-antibodies (group C). These results are shown in the following table. % AIHA titre of auto-antibodies

group A 100 15±2

group B (rat erythrocytes per os) 60 12±3

group C (rat erythrocytes per os + quail's 10 2±1 egg)

This work demonstrates that it is possible to induce OT with the quail's egg in current auto-immunisation situations.

Example 4

Prevention and treatment of experimental AIHA of the mouse by ingestion of auto¬ antibodies with the quail's egg

The procedure described in example 3 was used. However, it was established that the ingestion of mouse anti-erythrocyte auto-antibodies [200 μl of sera with high titer (1/1000) of autoantibodies] together with 100 mg quail's egg significantly reduced the percentage of animals having AIHA (group C). In fact, depending on the quantity of auto-antibodies ingested and the frequency of ingestions, only 25 to 45% of OF1 mice have AIHA, against 100% in the reference group A.

Furthermore, when AIHA has been established with OF1 mice, the ingestion of auto-antibodies with the quail's egg very significantly reduces the duration of auto- immunisation after stopping the injection of rat erythrocytes. In fact, it changes from 150±35 days in the case of reference animals (group A) to 54±23 days in the case of animals treated with auto-antibodies and quail's egg (group C).

Example 5

Prevention of the production of reaαin antibody in the case of Balb/c mice Balb/c mice are resistant to the induction of OT according to standard procedures. They even have an anaphylactic shock at the moment of ingestion of the antigen if, first of all, they have been immunised with it. In a series of experiments, it was demonstrated that the antigen (human transferrin) mixed with the quail's egg induces OT in these mice. On the other hand, the same ingestion with immunised animals no longer induces anaphylactic shock.

This work demonstrates that the antigen in the presence of the quail's egg no longer induces a manifestation linked to phenomena of direct hypersensitivity.

Various observations made in the induction of OT in the presence of the quail's egg enabled us to verify that the active ingredient(s) was or were present at the same time in the white and the yolk of the egg, since experiments made with each of these constituents gave results that were less conclusive, but however still statistically significant.

Claims

1. Composition for enteral administration comprising a quail's egg lyophilisate, characterized in that the quail's egg lyophilisate is produced in the presence of 5 to 40% by weight of carbohydrate, based on the weight of the quail's egg content.

2. Composition according to claim 1 wherein the amount of carbohydrate added is 5 to 20% by weight.

3. Composition according to claim 1 or claim 2 wherein the carbohydrate is saccharose.

4. Composition according to claim 1 or claim 2 wherein the carbohydrate is honey.

5. Composition according to any one of the preceding claims, characterized in that it further contains antigen.

6. Composition according to any one of the preceding claims, characterized in that it further contains auto-antibodies.

7. Composition according to any one of the preceding claims, characterized in that the quail's eggs are fertilized eggs of the quail strain MINA B.

8. Use of quail's egg or of a composition as defined in claim 1 in the preparation of a medicament or nutritional formulation for the induction of oral tolerance in mammals.

9. Use of quail's eggs or of a composition as defined in claim 1 in the preparation of a medicament or nutritional formulation for the induction of production of lymphokines with immunosuppressive properties.

10. Use according to claim 9, wherein the lymphokine is Transforming Growth Factor (TGF beta 1 ).

11. Process for manufacturing quail's egg Iyophilisates comprising the steps of: a) separating egg contents from eggshells, b) adding 5 to 40% (w/w) of carbohydrate and mixing it with the egg contents, c) freezing and drying the mixture.