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Iscom preparation and use thereof

a technology of iscom and preparation, applied in the field of iscom preparation, can solve the problems of prone to fever and local side effects, and achieve the effects of less toxic, less toxic, and easy tailoring of inmunogenicity or immune modulating properties

Inactive Publication Date: 2006-06-08
ISKONOVA AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0004] The present invention relates to the use of at least two purified peaks or defined fractions of quillaja saponin in iscom and iscom-matrix as separate entities (particles). I.e. these fractions are not combined in the very same iscom or iscom-matrix particles, and the particles with different loads are mixed together to constitute a formulation for immunisation. It has surprisingly turned out that a mixture of iscom or iscom matrixes each comprising a different fraction of Quillaja Saponaria Molina has lower toxicity than when these Quillaja Saponaria Molina fractions are integrated into the same iscom or iscom matrix particle. For example the mixture of fraction A-matrix and fraction C-matrix, or the use of fraction A-matrix or fraction C-matrix alone were considerably less toxic in mice than when the same fractions were integrated in the same iscom matrix (Example 4, table 1). Further, the inmunogenicity or immune modulating properties are easier to tailor, and the possibilities are considerably enhanced to make improved vaccine formulations optimised both for the target species and the needs / requirements of the vaccine antigens.
[0023] The use of saponin preparations according to this invention results in products with increased tolerability, increased immunogenicity. The preparations may be used in methods to tailor the immunogenicity including increased control of inflammatory, hypersensitivity and allergic reactions. This tailor making may be species dependent and may affect toxicity, tolerability and immunogenicity.
[0035] It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and / or diluent.

Problems solved by technology

Mice are particularly sensitive to quillaja saponins and overdosing leads to death within 4 days, often within 24 hours.
E.g. equines do not die from large doses of quillaja saponin, but they are prone to develop fever and local side effects after injection with free Quillaja Saponaria Molina, iscoms and iscom-matrix produced from Quillaja Saponaria Molina or mixed fractions of Quillaja Saponaria Molina.

Method used

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  • Iscom preparation and use thereof
  • Iscom preparation and use thereof
  • Iscom preparation and use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Quillaja Saponaria Molina subfragment saponins

[0051] Purification of crude Quillaja Saponaria Molina extract to fractions A, B and C. A solution (0.5 ml) of crude Quillaja bark extract in water (0.5 g / ml) is pre-treated on a sep-pak column (Waters Associates, Massachusetts.).

[0052] The pre-treatment involves washing of the loaded sep-pak column with 10% acetonitrile in acidic water in order to remove hydrophilic substances. Lipophilic substances including QH-A, QH-B and QH-C are then eluted by 70% acetonitrile in water.

[0053] The lipophilic fraction from the sep-pak column is then separated by a semipreparative HPLC column (CT-sil, C8, 10×250 mm, ChromTech, Sweden). The sample is eluted through the column by a gradient from 25% to 60% acetonitrile in acidic water. Three fractions are collected from the HPLC column during the separation. The residues after evaporation of these three fractions constitute QH-A, QH-B and QH-C.

[0054] The fractions designated QH-A, QH-B...

example 2

Prepartion of iscom matrix

[0055] Materials

[0056] Cholesterol, e.g., Sigma C 8503)

[0057] Phosphatidyl choline (egg derived) e.g., Sigma P 3556

[0058] MEGA-10 (Bachem AG, Switzerland)

[0059]Quillaja saponin fractions A and C (patent WO9611711)

[0060] 0.22 μm Sterile filters (Acrodisc)

[0061] PBS (10 mM phosphate buffered 150 mM saline, pH 6,8-7,4)

[0062] Slide-A-Lyzer casettes MW cut off 12-14.000 (Pierce)

[0063] MEGA-10 (stock solution)

[0064] Make a 20% (w / w) stock solution by adding 8 ml distilled water to 2.0 g of dry solid MEGA-10. Dissolve by gentle heating (30-50° C.). Filter through a 0.22 nm sterile filter, aliquot and store at −20° C.

[0065] Lipid mixture (15 mg / ml)

[0066] Dissolve 100 mg of each cholesterol and phosphatidyl choline in 10 ml 20% MEGA-10. The lipids dissolve slowly at 30-60° C. with slow stirring. Filter through a 0.22 nm sterile filter, aliquot and store at −20° C. After freezing, the lipid mixtures need to be heated up to 40° C. until clear. Temperate al...

example 3

Preparation of PR-8 protein micelles

[0080] 1 Dilute 12 mg of PR-8 monomers (1.5 mg / ml) with PBS to a final protein concentration of 1.0 mg / ml.

[0081] 2 Filter through 0.22 nm sterile filter

[0082] 3 Fill into Slide-A-lyzer

[0083] 4 Dialyse against 4 changes of 2 litres of PBS (24±1° C.) (for 48-60 hours)

[0084] 5 Aspirate from Slide-A-Lyzer

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Abstract

The invention relates to a composition comprising a mixture of at least two iscom complexes each complex comprising essentially one saponin fraction from Quillaja Saponaria Molina. The complexes may be iscom complexes or iscom matrix complexes. The invention also pertains to the use of such a mixture for the preparation of an immunomodulating pharmaceutical, and adjuvant, formulations for immunsation e.g. for production of monoclonal antibodies and a vaccine. Kits of pars comprising at least two parts, wherein each part comprises one iscom complex or one iscom matrix complex according to the invention are also embraced.

Description

FIELD OF INVENTION [0001] This invention relates to a composition comprising a mixture of at least two iscom complexes or iscom matrix complexes, each complex comprising one saponin fraction from Quillaja Saponaria Molina, and the use thereof as immunomodulators or adjuvants in formulations to be used for immunisations including vaccines. Especially the invention relates to the use of purified, semipurified or defined fractions of quillaja saponin in iscom and iscom-matrix adjuvanted vaccines. The use of saponin preparations according to this invention results in products with increased tolerability and increased immunogenicity. The preparations may be used in methods to tailor the immunogenicity with increased control of inflammatory, hypersensitivity and allergic reactions. PRIOR ART [0002] The immune stimulatory properties of quillaja saponins have been known for long (Ramon 1926) and quillaja saponins have been used in free form, sometimes in combination with Al(OH)3 in commerci...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K47/00A61K39/39
CPCA61K39/39A61K2039/55577A61P31/04A61P31/12A61P31/14A61P31/16A61P31/20A61P31/22A61P37/04A61P43/00
Inventor MOREIN, BRORLOVGREN BENGTSSON, KARIN
Owner ISKONOVA AB
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