WO1999053947A1

WO1999053947A1 - Th2-MIGRATION PROMOTERS CONTAINING W/O EMULSIONS

Info

Publication number: WO1999053947A1
Application number: PCT/JP1999/002008
Authority: WO
Inventors: Kazuyoshi Masuda; Kazutoshi Horie; Ryuji Suzuki
Original assignee: Shionogi & Co., Ltd.
Priority date: 1998-04-20
Filing date: 1999-04-15
Publication date: 1999-10-28
Also published as: JP4375773B2; AU3344299A

Abstract

Th2-migration promoters for helper T cells characterized by containing W/O (water-in-oil type) emulsions containing various antigens which are developed based on a finding that the migration of helper T cells into Th2 can be promoted by the oral (or transmucosal) administration of various antigens in the form of W/O emulsions. These preparations are useful in treating various diseases wherein the Th1/Th2 balance of helper T cells participates, for example, autoimmune diseases and allergic diseases.

Description

Specification

Th2 migration accelerator containing WZO emulsion

The present invention provides a balance between Th1 type (hereinafter, referred to as Th1) and Th2 type (hereinafter, referred to as Th2), which are subsets of helper T cells (Th) involved in an immune reaction in a living body. A formulation that promotes the transition from Th1 to Th2. This preparation is useful as a prophylactic or therapeutic agent for, for example, autoimmune diseases, various allergic diseases, or rejection at the time of organ transplantation. Background art

Autoimmune diseases (eg, organ-specific autoimmune diseases, etc.), allergic diseases, organ transplant rejection, etc. are hyperimmune reactions to endogenous or foreign antigens, and these diseases are immune to self or non-self. This can be explained as a phenomenon in which reactivity breaks down. Medications (immunosuppressants, corticosteroids, antiallergic agents, antihistamines, etc.) are mainly used as treatments for these diseases. However, immunosuppressants cause non-specific immunosuppression, and are accompanied by serious side effects such as concurrent infection and the development of malignant tumors. At present, antiallergic agents and the like are simply used as symptomatic treatments. On the other hand, specific immunotherapy such as desensitization therapy has been attempted, but the mechanism of action is unknown in many parts, and it is hard to say that it is an established treatment method.

Under such circumstances, attention has recently been paid to attempts to apply oral tolerance (oral tolerance) to the treatment of autoimmune diseases and the like. For example, a method for treating the above-mentioned diseases using oral tolerance induced by orally administering a substance that induces the proliferation of regulatory cells that secrete inhibitory cytokines such as TGF-β, which is called bystander antigen, (W093 / 16724, WO96 / 40232, Tokuhyo Hei 8-504745, etc.). However, in these methods, little consideration has been given to DDS preparations for antigens to be orally administered, and basically, only an aqueous solution or dispersion of an antigen protein is simply administered. You. Therefore, it is expected that the protein preparation to be prepared is extremely unstable, so that the administration efficiency is poor and that the effect of stable immunological tolerance is hardly reproduced. Attempts have also been made to use cytodynamics, cholera toxin B chain or lipopolysaccharide in combination to increase the efficiency of inducing oral immunity tolerance, but none of them is practical.

On the other hand, the relationship between the balance of Thl and Th2, a subset of helper T cells (Th) (TMZTh2 balance), and various diseases has also been studied. As one of the treatment methods, conversion from a Thl-dominant immune response to a Th2-dominant immune response is considered (Nippon Rinsho 55 5 6 (6, 1997); History of Medicine Vol.182 No.9) (1997), P523). However, to date, there have been few attempts to promote the transition from Thl to Th2 and efficiently reach the state of Th2 deflection. For example, WO93 / 00160 and ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Vol. 778 (1996), etc., state that the antigenic protein is a W / 0 type emulsion or a W / O / W type polyphasic emulsion to improve oral immune tolerance. A method for induction is described, but only the change in total antibody titer is examined experimentally. INFECT. AGENTS DIS. (USA), 2/2 (55-73) (1993) states that Th2 predominates when orally immunized with microspheres. The ThlZTh2 balance during oral tolerance has not been studied. Proc. So Exp. Biol. Med. 133, 423-427 (1970); Enteral. Nutr. 14, 459-462 (1990); Clin. Immunol. Immunopat. 25, 196-202 (1982); Ausl. NZJ Surg. 57, 323-329 (1987) and others state that parenterally administered W / 0 emulsion functions to suppress the immune system. However, these emulsions do not contain antigens, and no changes in ThlZTh2 balance when administered orally are discussed. In this way, by controlling the Thl-Th2 balance in the immune response, and particularly by promoting the transition to Th2 and preferably reaching a Th2-biased state, various diseases related to the Thl / Th2 balance are considered. Methods for efficiently preventing or treating pneumonia have not been sufficiently studied. Disclosure of the invention

The present inventors have conducted intensive studies and found that administration of various antigens in a water-in-oil emulsion (W / 0 emulsion) facilitates the transfer of ThlZTh2 balance of helper T cells to Th2. The present invention described below has been completed.

(1) An agent for promoting the transfer of helper T cells to Th2, comprising a W / 0 emulsion containing an antigen.

(2) The promoter according to the above (1), wherein the antigen is a protein or a peptide.

(3) The promoter according to the above (1) or (2), which induces immune tolerance by transmucosal administration.

(4) The promoter according to any one of the above (1) to (3), which is an agent for preventing or treating a disease associated with TMZTh2 balance of helper T cells.

(5) The promoter according to the above (4), wherein the disease is a disease caused by a Thl-based immune reaction, an allergic disease or a rejection reaction at the time of organ transplantation.

(6) The promoter according to the above (5), wherein the disease caused by a Thl-based immune reaction is an autoimmune disease.

(7) The value of the antibody titer ratio represented by "IgGl / IgG2a" at the time of oral administration to mice is about twice or more as compared with the case where the control antigen-containing physiological saline is administered. (6) The accelerator according to any one of (1) to (4).

(8) A method for promoting the transfer of helper T cells to Th2, comprising orally administering an antigen contained in W / 0 emulsion.

(9) A method for promoting the transfer of helper T cells to Th2, which comprises administering the promoter according to any one of claims 1 to 7.

(10) A method of using W / 0 emulsion as a dosage form of an antigen to promote the transfer of helper T cells to Th2.

(11) A method of using an antigen-containing W / 0 emulsion for producing a prophylactic or therapeutic agent for a disease associated with the balance of ThlZTh2 in helper T cells. BRIEF DESCRIPTION OF THE FIGURES (Figure 1 )

Fig. 4 is a graph showing the total Ig anti-titer titer against OVA of the serum collected on day 21 after oral administration of ovalbumin (OVA) -containing W / 0 emulsion to mice daily for 5 days and subcutaneous immunization with OVA. See Example 1).

(Figure 2 )

FIG. 9 is a graph showing the antibody titer to OVA (IgGl / IgG2a) of serum collected on day 21 after subcutaneous immunization with OVA after oral administration of ovalbumin (OVA) -containing W / 0 emulsion to mice daily for 5 days. (See Example 1).

(Fig. 3)

A graph showing the antibody titer to CII ratio (IgGl / IgG2a) of serum collected on day 21 after subcutaneous immunization with CII after oral administration of a type II collagen (CII) -containing W / 0 emulsion to mice for 5 consecutive days. Yes (see Example 2).

(Fig. 4)

The antibody titer to OVA (IgGl / IgG2a) of the serum collected on day 21 after oral administration of ovalbumin (OVA) -containing W / 0 microemulsion to mice daily for 5 days and subcutaneous immunization with OVA is shown. It is a graph (refer to Example 3).

(Fig. 5)

Sai Tokerachin (CK) containing W / 0 Emarujiyon _{(0.01 mg / m 0 use /} day) and dermatitis score in the case of performing three weeks intervals up once continuous oral administration of 5 days for mice (5-6 7 is a graph showing the transition of the average value of the example (see Example 4).

(Fig. 6)

The pruritus when mice were given a 5-week continuous oral administration of W / 0 emulsion (0.01 ing / raouse / day) containing cytokeratin (CK) at 3-week intervals. 7 is a graph showing a change in the cumulative number of seconds of behavior: average value of 5 to 6 cases (see Example 4). BEST MODE FOR CARRYING OUT THE INVENTION

Various W / 0 emulsions can be used in the present invention. Margillon may be used. Its size is usually about 0.001 to 100 μπι. Although there is no particular limitation on the composition, as the aqueous phase, water or any buffer of weakly acidic, neutral, or weakly basic can be used, and gelatin or the like is used for stabilization. The aqueous phase may be solidified.

Normally, physiologically acceptable oils or waxes may be used alone or in combination as the oil layer. As the oils, vegetable oils, animal oils, fatty acid esters and the like exemplified below can be used.

(Vegetable oil) Hardened castor oil, pine nut oil, castor seed oil, almond oil, jojoba oil, peanut oil, coconut oil, palm oil, safflower oil, kukui oil, malt oil, grape seed oil, rapeseed oil, corn oil Olive oil, cottonseed oil, apogado oil, camellia oil, corn oil, wheat germ oil, rice oil, cocoa butter, sesame oil, evening primrose oil, southern oil, and the like, but preferably sesame oil.

(Animal oil) Squalene, lanolin, mink oil, egg yolk oil, turtle oil, pristane and the like.

(Fatty acid ester) butylyl stearate, hexyl laurate, octyldodecyl oleate, oleyl oleate, isostearyl octanoate, cetyl octanoate, isopropyl myristate, octyl dodecyl myristate, neopentyl glycol dioctanoate, neo dicaprate Pentyl glycol, isopropyl palmitate, diisopropyl adipate, diisopropyl sebacate and the like.

Examples of the waxes include vegetable wax and animal wax, preferably carnauba wax, beeswax and whale wax.

The content ratio of the water phase and the oil phase is usually water phase: oil phase = 1: 1 to 1:50 (volume ratio), preferably 1: 5 to 1:20 (volume ratio). is there.

The emulsion of the present invention may optionally contain pharmaceutically acceptable additives such as surfactants, alcohols, fatty acids, polysaccharides, preservatives, ρ regulators, viscosity modifiers, and the like.

As the surfactant, ionic or nonionic surfactants can be used alone or in combination. Preferably, the HLB value is 2 to 20. Vitan fatty acid ester, polyoxyethylene sorbin fatty acid ester, glycerin fatty acid ester, propylene glycol fatty acid ester, sucrose fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene hydrogenated castor oil, lecithin, monoglyceride of caprylic acid, etc. More preferably, a combination of sorbitan sesquioleate and polyoxyethylene hydrogenated castor oil 60 or a combination of carboxylate monoglyceride, lecithin and polyoxyethylene hydrogenated castor oil 50 is used.

The surfactant is not a necessary component for promoting the transfer to Th2, but is preferably added in view of the stability of the emulsion and the like. The amount of the surfactant is usually about 1 to 75% by weight, preferably about 1 to 30% by weight, based on the whole oil phase. The antigen contained in the aqueous phase of the W / 0 emulsion according to the present invention may be any substance that has high water solubility and can cause an immune reaction in a living body. Examples of the antigen include proteins, peptides, and glycoproteins. Although it can be used, it is preferably a protein or a peptide. Specifically, ovalbumin (OVA), cytokeratin, type I collagen, type II collagen (CII), type IV collagen, transgluminase, myelin basic protein, insulin, glucagon, S-antigen, MHC An antigen can be used. The antigen content varies depending on the type of antigen, target disease, route and timing of administration, and cannot be unconditionally specified, but is usually about 0.001 to 20% by weight based on the total amount of the aqueous phase, preferably Is about 0.001 to 10% by weight.

The method for producing the emulsion is not particularly limited, but is carried out, for example, as follows. That is, a mixture comprising an antigen, an aqueous phase component, and an oil phase component is mixed, if necessary, while heating, subjected to ultrasonic treatment for several minutes to several tens of minutes, and then cooled on ice. Alternatively, a method such as shaking the mixture at room temperature may be used.

In the present invention, the ThlZTl ^ balance is determined by determining the ratio of each antibody titer of IgG2a, which is an antibody produced by TM activation, and IgGl, produced by Th2 activation, as shown in the following test examples. Was. In other words, it was judged that when the value of IgGlZlGg2a was increased, the ratio of Th2 in helper T cells was increased, and the transfer to Th2 was promoted. As a result, when the emulsion preparation of the present invention is administered to mice, It was found that the translocation to Th2 was promoted as compared with the case where the control saline preparation was administered. At that time, the total IgG antibody titer was lower than that of the control preparation, indicating that humoral immune reaction was also suppressed and oral immune tolerance was induced.

This effect of promoting the transfer to Th2 was confirmed in a plurality of W / 0 emulsions having different compositions, suggesting that this is a pharmaceutical effect common to the W / 0 emulsion. The promotion of the transfer to Th2 by this preparation means a change in the Thl / Th2 balance of the immune response system in which the proportion of Thl decreases and the proportion of Th2 increases, and preferably the proportion of Th2 Means the change to the Th2 deflection state.

This product is used for ThlZTh2 balance-related diseases, especially for autoimmune diseases thought to be due to a Thl-dominant immune response (eg, multiple sclerosis, rheumatoid arthritis, reactive arthritis, psoriasis, juvenile diabetes, chronic thyroid gland) Inflammation, uveitis, etc.), various allergic diseases mediated by or dependent on T cells (eg, contact dermatitis, etc.), or as a preventive or therapeutic agent for rejection during organ transplantation. is there. Also, atopic dermatitis, etc., may involve a Th2-dominant immune response depending on the disease state, and may activate Th2 cells to inhibit cytokines (IL-4, (IL-10, TGF-3, etc.) is secreted, etc., so that the prophylactic or therapeutic effect of this formulation can be expected. The route of administration of this formulation is not particularly limited.For example, one of the concomitant effects of using this formulation is expected to be the induction of immune tolerance induced through mucosa-associated lymphoid tissue (MALT). In this case, transmucosal administration such as oral administration, nasal administration, or pulmonary administration is performed, and oral administration is preferred. Example 1 (oral administration of ovalbumin-containing W / 0 emulsion)

Using ovalbumin (OVA) as an antigen protein, 7 volumes of aqueous phase {OVA-containing aqueous solution (0.269-26.9 mg / ml)} and 40 volumes of oil phase {6.7% (w / w) SO-15 ( Nikko Chemical), 1.7% (w / w) HCO-60 (Nikko Chemical) -containing sesame oil at 50, sonicate (300 W, 26 kHz, 50:, 1 min) and cool on ice. 0VA containing W / 0 emulsion John was prepared. The final OVA concentration in the emulsion was between 0.04 and 4 mg / mi. The particle size distribution of the emulsion was measured by GALAI CIS-1 and had a particle size of 50-60 mm. Oral administration (0-1 mg OVA equivalent / mouse / day, day 0-4) of W / 0 emulsion containing OVA or OVA saline solution to BALB / c mice (6 W, male) for 5 consecutive days and complete OVA Freund Subcutaneous immunization (25 g / mouse, day 8) was performed on hind limbs and feet together with adjuvant (FCA (H37Ra), DIFCO), and serum antibody titers (total IgG, IgGK, IgGK) against OVA 21 days after immunization (day 29) IgG2a) was examined by enzyme-linked immunosorbent assay (ELISA). The basic procedure of EUSA was based on the previous report {cro '0 /. Immunol. 36, 873-884 (1992)}. A labeled anti-mouse IgG2a ゥ heron antibody was used. The serum antibody titer was expressed as the maximum dilution at which the absorbance at 450 nm in ELISA was 0.1 or more. The results are shown in FIGS.

(Figure 1 )

In the mice treated with 1 mg / mouse / day, the total IgG antibody titers of the W / 0 emulsion and the saline solution were almost the same. On the other hand, in the mice treated with 0.1 mg / mouse / day, only the W / 0 emulsion had the same total IgG antibody titer as the 1 mg / mouse / day group, and the W / 0 emulsion was consumed on a live basis. It was confirmed that the serum total IgG antibody titer against OVA was significantly lower than that of the solution (P <0.05). That is, this preparation induced immunological tolerance.

(Figure 2 )

The ratio of the IgGl antibody titer (average value of 4-5 cases) to the IgG2a antibody titer (average value of 4-5 cases) was higher in the mice administered with the OVA-containing W / 0 emulsion than in the mice administered the 0VA saline solution (IgGl / IgG2a). ) Was about twice as high. In the W / 0 emulsion group, the ratio of IgGlZIgG2a also increased as the OVA content increased. That is, this preparation promoted the transfer of helper T cells to Th2. Example 2 (oral administration of type II collagen-containing W / 0 emulsion)

Using type II collagen (CII) as antigen protein, 7 volumes of aqueous phase (CII-containing salt An acid (0.001N) solution (0.269-2.69 mg / ml and 40 volumes of oil phase {6.7% (w / w) SO-15, 1.7¾ (w / w) sesame oil containing HCO-60) are mixed at 50, C / I containing W / 0 emulsion was prepared by sonication (300 W, 26 kHz, 50:, 1 min) followed by ice-cooling The final concentration of CII in the emulsion was 0.04 to 0.4 mg / ml. The particle size distribution of the emulsion was measured by GALAI CIS-1 and had a particle diameter of 50 to 60 mm.Water / emulsion containing CII or a solution of CII hydrochloric acid (0.001N) was added to BALB / c mice (6W, Male) for 5 days orally (0-0.1 mg CII equivalent / mouse / day, day 0-4) and then subcutaneously immunize CII with FCA (H37Ra) on hind limb foot (100 g / mouse, day 8) Then, the serum antibody titer (total IgG, IgGK IgG2a) against CII on day 21 after immunization was examined by enzyme immunoassay (ELISA).

(Result)

In the mice treated with 0.1 mg / mouse / day, the total IgG antibody titer decreased by the same degree in both the W / 0 emulsion and the hydrochloric acid (0.001N) solution, but in the mice treated with 0.01 mg / mouse / day in emulsion. The total IgG antibody titer was as low as in the group administered with a 10-fold amount of hydrochloric acid (0.001N) solution. As shown in Fig. 3, the mice administered with the CII-containing W / 0 emulsion received a IgGl antibody titer (5 to 6 subjects) relative to the IgG2a antibody titer (average of 5 to 6 animals) compared to the mice administered the CII-containing hydrochloric acid (0.001N) solution. The ratio (IgGl / IgG2a) was about twice as high, indicating a Th2 dominant tendency. In the W / 0 emulsion group, the ratio of IgG1 to IgG2a increased as the CII content increased. Example 3 (oral administration of W / 0 microemulsion containing ovalbumin)

The composition of the OVA-containing W / 0 microemulsion is as follows: aqueous phase {OVA saline solution (0.755-75.5 mg / ml)}: oil phase {PDD [68! ¾ (w / w)], homotex PT [8¾ (w / w)], SLP—white SP [2¾ (/ w)], Cremophor EL [17% (w / w)]} = 5:95 (w / w), and the oil layer is mixed by shaking (rt, 10 min) ), The aqueous phase was added, and the mixture was further mixed by shaking (r., 3 min). The same operation as in Example 1 was performed, and a serum antibody titer against 0 VA (total IgG, IgGK IgG2a) on day 21 after immunization of BALB / c mice orally administered with a 0 VA-containing W / 0 microemulsion or a 0 VA saline solution was administered. The enzyme immunoassay (ELISA) Investigated by

(Result)

In the mice treated with 1 mg / mouse / day, both the W / 0 microemulsion and the saline solution reduced the total IgG antibody titer to the same extent. On the other hand, in the mice treated with 0.1 ing / mouse / day, only the W / 0 microemulsion reduced the total IgG antibody titer to the same extent as in the group treated with 1 mg / mouse / day. Also, as shown in FIG. 4, the mice administered with the OVA-containing W / 0 microemulsion had a 5-to 10-fold higher IgGl / IgG2a ratio than the mice administered with the OVA saline solution, and showed a tendency to be superior to Th2. This trend increased with increasing OVA content in the W / 0 microemulsion. Example 4 (oral administration of cytokeratin-containing W / 0 emulsion)

Cytokeratin (CK) purified from pig skin by anion exchange chromatography was used as the antigen protein. It was confirmed by immunoblotting that at least the cytokeratins Nos. 4, 5, 7, 8, 17, and 18 were contained in this purified product.

7 volumes of aqueous phase {CK-containing aqueous solution (0.269 mg / ml)} and 40 volumes of oil phase {6.7% (w / w) SO-15 (Nikko Chemical), 1.7S! (W / w) HCO-60 (Nikko Chemical) -containing sesame oil} at 50T :, mixed by sonication (300 W, 26 kHz, 50 "C, 1 min) and ice-cooled to prepare a CK-containing W / 0 emulsion. The final concentration of CK was 0.04 ing / ml The particle size distribution of the emulsion was measured by GALAI CIS-1 and had a particle size of 50 to 60 mm DS / Nh of this CK-containing W / 0 emulsion Therapeutic effects of spontaneous dermatitis in mice (Exp. Aniw. 46, 225-229 (1997)) were investigated.Experiment was conducted by rearing 4-week-old DS / Nh mice () in a conventional environment for 12 weeks. Dermatitis Divided into groups so that there was no deviation in the score, 2 mM Tris buffer (pH 8) administration group, CK-containing Tris buffer administration group (0.01 mg / mouse / day), W / 0 empty emulsion group And CK-containing W / 0 emulsion (0.01 mg / mouse / day) were administered in a total of four groups (n = 5 to 6), and oral administration for 5 days from the start of the experiment was performed once every three weeks at a time. The experiment was terminated 6 weeks after the start of administration, and the dermatitis score (0 to 5 according to the severity of dermatitis of each individual) and pruritus (observed for 5 minutes during observation) To the total number of seconds) About it.

(Result)

The results are shown in FIGS. In the W / 0 emulsion containing CK, the dermatitis score was suppressed and the pruritus associated with exacerbation of dermatitis was suppressed as compared with the groups administered with Tris buffer, empty emulsion and Tris buffer containing CK. Industrial applicability

This preparation is useful for treating various diseases related to the Tl / Th2 balance of helper T cells, such as autoimmune diseases and allergic diseases, and for suppressing rejection during organ transplantation.

Claims

The scope of the claims

I. An agent for promoting the transfer of helper T cells to Th2, comprising w / o emulsion containing an antigen.

2. The promoter according to claim 1, wherein the antigen is a protein or a peptide.

3. The promoter according to claim 1, which induces immune tolerance by transmucosal administration.

4. The promoter according to any one of claims 1 to 3, which is an agent for preventing or treating a disease associated with the balance of ThlZThZ of helper T cells.

5. The promoter according to claim 4, wherein the disease is a disease caused by a Thl-based immune reaction, an allergic disease, or a rejection reaction at the time of organ transplantation.

6. The promoter according to claim 5, wherein the disease caused by an immune reaction mainly based on Thl is an autoimmune disease.

7. The antibody titer ratio indicated by "lgGlZlgG2a" at the time of oral administration to a mouse is about twice or more as compared with the case where a control antigen-containing physiological saline is administered. An accelerator as described.

8. A method for promoting translocation of T cells into Th2, comprising orally administering an antigen in a W / 0 emulsion.

9. A method for promoting the transfer of helper T cells to Th2, which comprises administering the promoter according to any one of claims 1 to 7.

10. The use of W / 0 emulsion as a dosage form of antigenic protein to facilitate the transfer of helper T cells to Th2.

II. A method of using an antigen-containing W / 0 emulsion for producing a prophylactic or therapeutic agent for a disease associated with the Thl / Th2 balance of helper T cells.