JP3001214B2 - Gunmainulin composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a composition comprising gamma polymorphic form of inulin that can be used as an immunoactive agent.

The properties and use of inulin in the novel gamma polymorphic form are described in International Patent Application No. PCT / AU86 / 00311. It is disclosed therein that this form of inulin may be used as an alternative pathway activator of complement and may be suitable as an anti-tumorigenic agent. The specification also discloses that the gamma polymorphic form of inulin has adjuvant activity.

Here, it has been found that the activity of the gamma polymorphic form of inulin as an immunoactive agent, such as an adjuvant, is enhanced when the antigen binding carrier material associates, and this association has a surplus effect.

According to the present invention there is provided a composition comprising particles comprising gamma polymorphic form of inulin or an inulin derivative and an antigen binding carrier substance.

The gamma polymorphic form of inulin or inulin derivative (hereinafter referred to as "gammaminulin") and the antigen-binding carrier material are capable of electrostatic or hydrophobic interactions, covalent bonding, co-precipitation, crystallization of one component onto another. , Or a combination thereof. Preferably, the particles are prepared by co-precipitating gamma mynulin and the antigen binding carrier material such that both components are contained in the same particle.

The antigen binding carrier material can comprise a low solubility material capable of binding proteinaceous, lipid, hydrocarbon and / or other antigens. For example, the antigen-binding carrier substance may be a metal-containing precipitate such as magnesium, calcium or aluminum phosphate, sulfate, hydroxide or a hydrate thereof, chitin (poly N-acetylglucosamine) or a deacetylated derivative thereof, or It may be selected from organic bases, such as basic cellulose derivatives, or organic acids, including sulfated or phosphorylated polysaccharides, such as heparin, dextran or cellulose derivatives.

The antigen binding carrier material is aluminum hydroxide (alu
m) It can also comprise poorly soluble particles of a substance such as a gel or a hydrated salt complex thereof. Conveniently, the particles of the antigen binding carrier material are smaller than 5 μm, more preferably 1 μm.
Smaller than −5 μm. Most preferably, the particles are 50-20
00 nanometer diameter. It is particularly preferred that it is endotoxin- and pyrogen-free and is a pharmaceutically acceptable antigen-binding carrier substance. Advantageously, the antigen-binding carrier has no tendency to aggregate or has not been subjected to anti-aggregation treatment.

In another aspect of the invention, there is provided a method of making a composition of the invention as broadly described above, the method comprising: providing a suspension of an antigen binding carrier material; and (i) said suspension. Is premixed with inulin or an inulin derivative and then the inulin or inulin derivative is converted to the gamma polymorphic form; or (ii) the suspension is premixed with the gamma polymorphic form of the inulin or inulin derivative; Associating the polymorphic form of inulin or inulin derivative with the antigen-binding carrier material.

Preferably, the suspension of microparticles of the antigen-binding carrier substance is added to a solution of inulin or an inulin derivative, and a controlled ratio of gamminulin to antigen-binding carrier substance, e.g., 200: 1-1: 1 and More preferably 50: 1-5:
Give 1 (w / w). The concentration of inulin is preferably about 5%
kept at w / v. A 6-7% w / v initial solution of inulin is convenient.

The resulting suspension is quenched to a temperature, such as 5 ° C., and cooled under the low temperature to crystallize the inulin before being placed at 37 ° C. for several days to facilitate the conversion of inulin to the gamma configuration. Stir for one day and continue washing, removing large particles.

Alternatively, particles of inulin, already in gamma polymorphic form, are precipitated on the antigen binding carrier material or combined intimately, or on particles either before or after conversion of inulin to the gamma configuration. Preferably, the antigen-binding carrier material is precipitated.

As mentioned above, it is particularly preferred to use pyrogen- and endotoxin-free inulin solutions or suspensions and antigen-binding carrier substance fractions. It is also preferred to use reaction conditions that minimize aggregation, depending on the antibody binding substance used. For example, when using alum as an antigen-binding substance, avoid using ammonia or water containing CO ₂ in the preparation of inulin or alum solution, and adsorb the arum gel suspension in the inulin solution in advance. It is appropriate to allow the suspension to then be removed.

The inulin fraction used in the present invention is typically prepared by the process disclosed in International Patent Application No.PUT / AU86 / 00311, which is incorporated herein by reference, however, as described above. It is desirable to avoid reagents or reaction conditions that cause aggregation. Therefore, when arum gel is used as the antigen-binding carrier substance, it is preferable to exclude ammonia in the preparation of inulin.

Broadly speaking, the gamma mayulin in the composition may comprise about
It has a molecular weight greater than 3000, more preferably greater than about 8000. Its molecular weight advantageously ranges from 8000 to about 16000, and after conversion to the gamma form is substantially insoluble in water at 37 ° C.

Inulin derivatives that can be used in the composition of the present invention include β-D- [2-1], such as obtained by enzymatically removing terminal glucose from inulin by using an invertase or inulase enzyme. Including derivatives of polyfructofuranosyl α-D-glycose.
Other derivatives include inulin that has been etherified or esterified in which the free hydroxy group has been chemically substituted in a known manner, for example by an alkyl, aryl or acyl group.

The compositions of the present invention produce an enhanced immune response as compared to only gamma myulin or an antigen binding carrier material.
This enhanced immune response is unexpected. Accordingly, the compositions of the present invention may be used as adjuvants or immunostimulants in vaccine preparations, similar to those described in specification No. PCT / AU86 / 00311, incorporated herein by reference.

Thus, in another aspect, the present invention provides an immunotherapy comprising gamma polymorphic form of inulin or an inulin derivative, particles as an active ingredient comprising an antigen binding carrier substance, and a pharmaceutically acceptable diluent or carrier. To provide a composition for use.

Any antigen or combination of antigens, such as one or more viruses, bacteria, molds or protozoan proteins, may be adsorbed on the compositions of the invention and combined with an acceptable carrier or excipient. May be dispensed into a vaccine preparation.
The compositions of the present invention have strong adsorption capacity for proteins in solution, but do not aggregate by themselves, unlike antigen-binding carriers such as alum gel.

In yet another aspect of the invention, a particle and antigen-binding carrier material comprising inulin or an inulin derivative in a gamma polymorphic form and one or more antigens adsorbed thereon are provided with a pharmaceutically acceptable carrier or diluent. There is provided a vaccine composition comprising

The present invention is illustrated with reference to the accompanying figures and the following non-limiting examples.

Example 1 Preparation of a Composition Containing Gamma Mayulin With Alum as an Antigen-Binding Carrier Aluminum hydroxide ("Arum gel") was prepared by purifying the purest aluminum chloride or sulfate available at pH 4 with analytical grade sodium hydroxide solution. ~ 10, preferably about 7.0
In addition, it is prepared by adjusting the conditions under which very fine arum gel particles are produced. First precipitate, ie pH
Up to four are discarded to remove pyrogens and materials that cause agglomeration of the alum. The second suspension is then washed by centrifugation or placed in pyrogen-free water to remove soluble material. After standing overnight, the excess supernatant is removed and the concentrated suspension is sterilized in an autoclave. Weight of the alum gel / ml
Is Al ³⁺ by standard chemical measurement or mass spectrometry
Calculated from the assay. All solutions, CO ₂ in order to minimize alum aggregation - preferably be prepared by free water, CO ₂ is eliminated from the process. Water is kept above the arum gel. The inulin solution was prepared as described in International Patent Application No. 1 except that the crude inulin was dissolved at 8-10% in freshly boiled water at 75 ° C. to remove CO ₂ and the ammonia solution was omitted. Prepared as described in PCT / AU86 / 00311. The solution is 1% v / v
Preparation of Arum gel suspension is easily slurried to remove large impurities, and the alum is removed by filtration or sedimentation. The inulin was cooled and the specification No.PCT
Filtered through DEAE-cellulose and Amberlite sulfonic acid resin (omitting ammonia) as described in / AU86 / 00311 or slurried repeatedly into alum gel until free of impurities. In the former case, the solution is subjected to a final treatment with alum gel after amberlite filtration and before zetapor filtration to ensure sterilization and endotoxin-free. Subsequently, the preparation is kept sterile and endotoxin-free. Where the inulin concentration is
Should be 6-7% w / v. The fine suspension of arum gel particles (prepared as described above, or an endotoxin-free preparation sterilized by "Alhydrogel" or another autoclave) was then brought to an inulin concentration of 5.0%. Provide a known w / w ratio of inulin: alm within the previously indicated range of 200: 1 to 1: 1, preferably 50: 1 to 5: 1 (w / w), while keeping it higher than w / v. To be added.
The suspension is quenched and stirred as before, ie at 5 ° C. for crystallization for several days and for 37 days shifted to 37 ° C. to transfer to the gamma configuration, then centrifuged and added to water. Resuspend, heat at 50-52 ° C for 1 hour, wash to zero refractive index of supernatant. The final suspension is differentially centrifuged or fractionated to remove particles larger than 1.5 μm as determined by hemocytometry. Alternatively, the preparation comprises particles of a convenient size, e.g.
In order to produce those in the range of ~ 1000 nanometers, the particles are made more brittle, especially by heat treatment, for example at 45-55 ° C (preferably 50-52 ° C) for 1 hour, and then disrupted by ultrasonication. You may. The fine suspension is, for example, 20μ
Adjust to 5% w / v inulin in 0.8% NaCl by adding a suitable preservative such as g / ml phenyl mercuric nitrate. The resulting composition (Gammamineulin / Alm hybrid preparation, or “Algammuli (Algammuli)
n) ") is described in specification No. PCT / AU8 for dissolved substances, insoluble, endotoxin content, sterility and complement activity.
Tested as described in 6/00311. In the process described above, inulin crystallizes around the alum particles to form porous capsules, which are then easily converted in situ to the gamma form. The particles have a high adsorption capacity for proteins in solution, similar to arum gel, but do not aggregate themselves, unlike arum gel.

Example 2 Protein Adsorption Capacity A suspension containing saline alone, a 50 mg g-IN / ml suspension of a gamma inulin (g-IN) / alm hybrid preparation, or a suspension containing the same amount of inulin or alum only or a mixture. the 0μl of Nigobutsu, was mixed with 10μl of saline containing 10 ^{5 125} hemocyanin 0.65μg keyhole limpet labeled with I in cpm, was adsorbed for 15 minutes at 23 ° C.. Each suspension was then mixed with 1 ml of saline, 20 μ of which was placed in a plastic test tube with an inner diameter of 6.5 mm in 600 μl.
Percoll colloids (density 1.126 g / ml) to form a 3 cm deep liquid column. The remainder of the suspension was centrifuged and washed, indicating that the hybrid preparation and the alum suspension adsorbed more than 95% of the label, while g-IN adsorbed less than 5% of the label. Indicated. Percoll tubes were centrifuged at 700 g for 4 minutes and processed immediately. In tubes containing free g-IN (with or without premixed alum) or the hybrid preparation, the turbid bulk representing g-IN remains in the top 50μ, while free g-IN In the test tubes containing the alum gels, all of the gels were found in or near the pellet. No pellet was seen in the tube containing the hybrid preparation. The tubes are then fractionated by copying off the top 50 μl, then 500 μl, while pelleting (bottom bottom).
50μ) was left in the test tube. All tubes were then counted in a gamma counter and the percentage of label in each fraction determined as illustrated in the following table: 95% for samples containing saline or g-IN only
The above label remained in the upper partial. The distribution of the label in the sample containing alum mixed with g-IN was the same as in the sample containing alum gel alone.

This type of experiment indicates that the adsorption capacity of the hybrid preparation is
It is closely related to gamma mynulin, indicating that it is distinguished from free arum gel in sedimentation.

Example 3 Demonstration of the Conjugation of Alm and Gamma Myulin by Electron Microscopy
Micron uniform ovals revealed that they were difficult to see unless previously stained with an electronically dense material such as phosphotungstate (FIG. 1B). In contrast, electron microscopy of particles of the hybrid preparation "algamulin" (FIG. 1A) revealed a similarly sized oval shape that is electronically dense, ie, clearly visible without staining. . This indicates that alum and gammaminulin are indeed contained in the same particle, and that electronically dense alum is contained and dispersed in the “algamulin” particles.

Example 4 Contrasting the Adjuvant Effect of a Gamma Maynulin / Alm Hybrid Preparation (Algamulin) With Any Component Only or a Simple Mixture Antigen prepared and adsorbed as described in Example 2 (eg keyhole phosphorus) When mice were injected with a hybrid preparation “algamulin” carrying (pet hemocyanin, KLH), the antibody response (in μg KLH-specific IgG / ml in serum) was determined by adsorbing the same antibody to arum gel, or Mixed with g-IN or adsorbed on alum gel and mixed with g-IN and increased several fold compared to what would occur in mice injected in parallel. In each case, the contents of g-IN and alum are equivalent by weight.

BALB / C female mice were mixed with 0.2 ml of endotoxin-free KLH in groups of 7 or 8 saline adsorbed on hybrid preparation "Algamulin", or Arumgel, or mixed with g-IN. Or adsorbed on alum gel and mixed with g-IN and injected intraperitoneally (ip). The dose of g-IN was always 1000 μg / mouse, regardless of free or hybrid form. To the mouse,
After 14 days, KLH without any adjuvant was injected ip, and after 7 days, serum was collected. KLH-specific I
gG / ml was measured by ELISA on the serum of each mouse and the geometric mean titer was calculated. Probability is expressed as Mann-Whitney
Were evaluated for geometric mean titers by the non-parametric rank test and for the mean difference of duplicate geometric mean titers by the Studentt test. FIG. 2A shows exemplary results and standard errors, where "algamulin" gave significantly higher specific serum antibody titers of 5.7-8.7 times the same dose of a simple mixture of arum and gamma inulin and the same It shows that the dose gives equally higher titers than either component alone. This experiment demonstrates the concept that a gamma mayulin / alum hybrid preparation is superior as a vaccine adjuvant compared to either component alone or a mixture, and that the conjugation of a given dose of arum to gamma mayulin is Have shown that the effect of this amount of alum is greatly enhanced by the additive mechanism. These tests show very low concentrations of KLH (1 mcg / m) that are barely possible to elicit a seropositive response on their own.
Mouse ip) and mimics conventional "weak antigens" such as can be found in commercial vaccines.

Example 5 Comparison of the Adjuvant Effect of High Dose Argamulin with Freund's Adjuvant Incomplete Freund's adjuvant (ICFA, water-in-oil emulsion of antigen) and Complete Freund's adjuvant (similar to CFA, ICFA but heat sterilized M. tuberculosis) Is considered a standard potent adjuvant, but is too toxic for clinical applications. CFA
Is even more powerful than ICFA. FIG. 2B shows the results of multiple comparisons of algamulin with CFA and ICFA using a protocol similar to Example 4. They show that 6 mg and 9 mg of argamulin / mouse give significantly greater specific serum antibody titers than those with ICFA and similar to those with CFA.

Example 6 Adjuvant effect over a wide range of algamulin doses and comparison with equivalent doses of alum alone FIG. 3 shows the algamulin dose using the protocol of Example 4 and expected to be in a physiologically acceptable range Is a summary of a number of the same tests, including FIG.3B shows that a given dose of arum conjugate as algamulin gives a specific serum antibody titer 6.4 to 12.6 times greater than an equivalent dose of alum alone, again showing the phase of the conjugate with gamma myulin. This shows the surplus effect.

Example 7 Comparison of the Seroconversion Enhancing Ability of Algamulin with Alum Alone at the Same Dose FIG. 4 shows that using 15-20 mice per data point,
Also, sub-seroconversion doses of KLH have been used (0.1 mc
g / mouse), except that seroconversion studies using the same protocol as in Example 4 are shown. Administration of KLH alone, or a mixture with only 200 mcg / mouse of gamma mynulin, resulted in a seroconversion of less than 10% of each serum-positive response (i.e., the mean plus two times the standard deviation of the mean of the KLH alone group). Specific serum antibody titers in excess of Such a positive seroconversion response was not obtained from KLH on Alm until the Alm dose reached 40 mcg / mouse. However, a very significant positive seroconversion response was achieved at 10 mcg / mouse when arum was conjugated to gamma myulin as algamulin, again showing the additive effect of conjugation.

Those skilled in the art will recognize that many modifications can be made to the compositions and techniques described herein without departing from the spirit and scope of the invention.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61P 37/00 A61K 31/00 637 37/08 637E A61K 31/715 31/715 33/06 31/725 38/00 33/06 38/21 37/02 37/66 G (58) Fields studied (Int.Cl. ⁷ , DB name) A61K 39/39 A61K 38/00-38/42 A61K 31/715-31/725 A61K 33/06 CA (STN) MEDLINE (STN) WPIDS (STN)

Claims (39)

(57) [Claims] 1. An immunoadjuvant composition comprising particles containing gamma polymorphic form of inulin or an inulin derivative in association with an antigen-binding carrier substance. 2. The composition according to claim 1, wherein the antigen-binding carrier substance is a low-soluble substance capable of binding proteins, lipids, carbohydrates and / or other antigenic substances. 3. The method according to claim 2, wherein the antigen-binding carrier substance is a magnesium, calcium or aluminum phosphate,
The composition according to claim 1 or 2, wherein the composition is selected from metal-containing precipitates such as sulfates, hydroxides or hydrates thereof. 4. The composition according to claim 3, wherein said antigen-binding carrier substance is an aluminum hydroxy gel. 5. The method according to claim 1, wherein the antigen-binding carrier substance is an organic base such as chitin or a deacetylated derivative thereof or a basic cellulose derivative, or a sulfated or phosphorylated polysaccharide such as heparin, dextran or a cellulose derivative. The composition according to claim 1 or 2, wherein the composition is selected from the group consisting of organic acids. 6. The composition according to claim 1, wherein the gamma polymorphic form of inulin or an inulin derivative has low solubility in an aqueous medium at 37 ° C. or higher. 7. The composition according to claim 1, wherein the gamma polymorphic form of inulin or inulin derivative has a molecular weight of more than 3000. 8. The method according to claim 8, wherein the inulin or inulin derivative is 80% or less.
3. A composition according to claim 1 or claim 2 having a molecular weight greater than 00. 9. The gamma polymorphic form of inulin is 8000-1600.
3. A composition according to claim 1 or 2 comprising particles which have a molecular weight in the range of 0 and are substantially insoluble in water at 37 ° C. 10. The composition according to claim 9, wherein said antigen-binding carrier substance is an aluminum hydroxy gel. 11. The composition according to claim 10, wherein said particles are in the form of a stable suspension of particles having a diameter of 50-2000 nanometers. 12. The inulin derivative of the gamma polymorphic form is β-D- [2-1] polyfructose, inulin in which free hydroxyl groups are etherified, or inulin in which free hydroxyl groups are esterified. The composition according to claim 1, wherein the composition is: 13. The composition according to claim 1, wherein the gamma polymorphic form of inulin or inulin derivative is co-crystallized in the same particles as the carrier substance. 14. An immunotherapeutic composition comprising as active ingredient gamma polymorphic form of inulin or an inulin derivative in association with an antigen-binding carrier substance, and a pharmaceutically acceptable diluent or carrier. Preparation. 15. The preparation according to claim 14, wherein said carrier or diluent is a sterile, aqueous medium. 16. The preparation according to claim 15, wherein said sterile, aqueous medium is an isotonic solution. 17. The preparation according to claim 14, which is in a form suitable for injection. 18. The preparation according to claim 15, which is in a form suitable for oral, rectal, vaginal, topical, nasal or ocular administration. 19. The preparation according to claim 14, further comprising a second active ingredient which is an immunomodulator. 20. The preparation according to claim 19, wherein said immunomodulator is a vaccinated antigen, an antigenic peptide sequence, or an anti-idiotype immunoglobulin. 21. The immunomodulator may be an interleukin or interferon or tumor necrosis factor or other cytokine, or a thymocyte stimulating factor or other thymic stimulating hormone, muramyl peptide or other microbial component or all microbial components or all. 20. The preparation according to claim 19, which is a microorganism or an endotoxin. 22. Particles comprising gamma polymorphic forms of inulin or inulin derivatives in association with an antigen-binding carrier substance, and a pharmaceutically acceptable carrier comprising one or more antigens adsorbed thereon. Or a vaccine composition comprising together with a diluent. 23. An activating or therapeutic agent comprising an immunotherapeutic preparation according to claim 14 in an amount effective for activating the alternative pathway of complement in the human or animal body. Drugs. 24. An activating or therapeutic agent comprising an immunotherapeutic preparation according to claim 14 in an amount effective for anti-tumor treatment in humans or animals. 25. A treatment comprising an effective amount of an immunotherapeutic preparation according to claim 14 or a vaccine composition according to claim 22 for treating a tumor in the human or animal body. For drugs. 26. An immunotherapeutic preparation or claim 22 according to claim 14 in an amount effective for the treatment of overt or latent preneoplastic symptoms in the human or animal body.
A therapeutic agent comprising the vaccine composition according to paragraph. 27. An immunotherapeutic preparation or claim 22 according to claim 14 in an amount effective for the human or animal body.
An agent for the treatment of infection by bacteria, mycoplasmas, molds, viruses, protists or other microorganisms, or the infestation of helminths or parasites, which comprises the vaccine composition according to the above item. 28. An agent for treating a condition associated with dysfunction of the immune system, comprising an immunotherapeutic preparation according to claim 14 in an amount effective for the human or animal body. 29. The therapeutic agent according to claim 28, wherein the condition associated with immune system dysfunction is an allergic condition. 30. The condition associated with immune system dysfunction is a primary condition.
29. The therapeutic agent according to claim 28, which is a secondary immunodeficiency disease. 31. The therapeutic agent according to claim 28, wherein the medical condition associated with immune system dysfunction is a rheumatic disease. 32. The therapeutic agent according to claim 28, wherein the condition associated with immune system dysfunction is a neurological condition. 33. The therapeutic agent according to claim 28, wherein the medical condition associated with dysfunction of the immune system is a gastrointestinal medical condition. 34. A human or animal body administered with an immunomodulator comprising an immunotherapeutic preparation according to claim 14 in an amount effective to enhance the immune response of the human or animal body. For enhancing an immune response in a human. 35. A vaccine antigen comprising the immunotherapeutic preparation according to claim 14 as an adjuvant.
An agent for promoting the effect of administering an antigenic peptide sequence or anti-idiotype immunoglobulin. 36. Step: providing a suspension of the antigen binding carrier material; and (i) mixing the suspension with inulin or an inulin derivative and then converting the inulin or inulin derivative to a gamma polymorphic form. Converting, or (ii) mixing the suspension with the gamma polymorphic form of inulin or inulin derivative, and associating the gamma polymorphic form of inulin or inulin derivative with the antigen binding carrier material. A method for producing the composition according to claim 1. 37. Step: (a) suspending the finely divided particles by recrystallizing crude inulin from an aqueous solution at a temperature substantially below 37 ° C. in the presence of a suspension of the carrier substance (B) obtaining said suspension at a temperature in the range of
(C) optionally further heating the suspension at a temperature in the range of 40-55 ° C for 0.5-1.5 hours; and (d) forming the formed ganmaynuline / carrier material eutectic particles. A method for producing a composition according to claim 1, comprising isolating from the suspension. 38. Step: (a) crude inulin in the presence of a suspension of a carrier substance
Recrystallizing from an aqueous solution at a temperature in the range of 25-45 ° C. to obtain the particles as a suspension; 2. A method for producing a composition according to claim 1, comprising heating and (c) isolating the formed ganmayulin / carrier material eutectic particles from the suspension. 39. A further step: (e) forming a suspension of the gamma mayulin / carrier material particles;
39. The method of claim 37 or claim 38, comprising treating the particles with an sonicator to reduce the diameter to a suitable size range.