Intramolecularly crosslinked (1.fwdarw.3)-.beta.-D-glucans

• US 5,574,023 A
• Filed: 01/06/1995
• Issued: 11/12/1996
• Est. Priority Date: 08/08/1991
• Status: Expired due to Term

• Current Assignee: Seikagaku Kogyo Kabushiki Kaisha
• Original Assignee: Seikagaku Kogyo Kabushiki Kaisha
• Inventors: Aketagawa, Jun, Tanaka, Shigenori, Shibata, Yuko
• Primary Examiner(s): Naff, David M.
• Assistant Examiner(s): Ware, Deborah K.
• Application Number:
  US08/369,321
• Time in Patent Office: 676 Days
• Field of Search: 514/54, 536/18.6, 435/101
• US Class Current: 514/54
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C08B 37/0024 beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Abstract

The invention stabilizes single helix conformation (1→3)-β-D-glucans by intramolecular crosslinking and provides (1→3)-β-D-glucans showing stable biological activities. Thus, intramolecularly crosslinked (1→3)-β-D-glucans having a stable single helix conformation are produced by introducing a functional group into hydroxyl groups of (1→3)-β-D-glucans, then causing the thus-obtained functional glucans to take a single helix conformation, and subjecting these, either as such or after binding them to a receptor therefor, to intramolecular crosslinking between the functional groups introduced, if necessary followed by releasing the receptor therefrom.

Claims

1. A method of producing an islolated (1→

3)-β

-D-glucan that is intramolecularly crosslinked to have a stable single helix conformation which method comprises;

(a) forming a single helix conformation of an isolated (1→

3)-β

-D-glucan having at least 20 D-glucoside units having hydroxyl groups, wherein the hydrogen atoms of at least part of the hydroxyl groups are substituted with functional groups that are reactive with a bifunctional crosslinking agent, and

(b) intramolecularly crosslinking the glucan between the functional groups described in said step (a) by using said crosslinking agent in the presence of polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone or water-soluble copolymers that contain at least one of polyethylene glycol, polyvinyl alcohol, or polyvinylpyrrolidone as principal constituents, which are not reactive with the functional groups described in said step (a) so as to prevent intermolecular crosslinking.

2. A method of producing an isolated (1→

3)-β

-D-glucan that is intramolecularly crosslinked to have a stable single helix conformation which method comprises;

(a) forming a single helix conformation of an isolated (1→

3)-β

-D-glucan having at least 20 D-glucoside units having hydroxyl groups wherein the hydrogen atoms of at least part of the hydroxyl groups are substituted with functional groups, that are reactive with a bifunctional crosslinking agent, and

(b) binding the glucan having a single helix conformation to a receptor for (1→

3)-β

-D-glucan, wherein said receptor is selected from the group consisting of horseshoe crab amebocyte lysate, factor G from horseshoe crab amebocyte lysate, arthropod phenol oxidase precursor, zymosan receptor from human lymphocytes, carbohydrate-receptive amino acids and tryptophan-containing peptides,

(c) intramolecularly crosslinking the glucan between the functional groups described in said step (a) by using said crosslinking agent in the presence of polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone or water-soluble copolymers that contain at least one of polyethylene glycol, polyvinyl alcohol, or polyvinylpyrrolidone as principal constituents, which are not reactive with the functional groups described in said step (a) so as to prevent intermolecular crosslinking, and

(d) eliminating said receptor.

3. An isolated (1→

3)-β

-D-glucan that is intramolecularly crosslinked to have a stable single helix conformation,

wherein said glucan that is intramolecularly crosslinked has the following characteristics;

(i) a single helix conformation content of at least 20% as determined in a hydrated solid state;

(ii) a single helix conformation retention of at least 50% as determined after one month storage at 4°

C. in the form of an aqueous solution or suspension;

(iii) retains horseshoe crab factor G activating ability, host-mediated antitumor activity, or both horseshoe crab factor G activating ability and host-mediated antitumor activity; and

(iv) has been intramolecularly crosslinked so as to prevent intermolecular crosslinking.

5. The isolated (1→

3)-β

-D-glucan that is intramolecularly crosslinked to have a stable single helix conformation according to claim 3 which is produced by a process comprising;

(a) forming a single helix conformation of a (1→

3)-β

-D-glucan having at least 20 D-glucoside units having hydroxyl groups, wherein the hydrogen atoms of at least part of the hydroxyl groups are substituted with functional groups that are reactive with a bifunctional crosslinking agent, and

(b) intramolecularly crosslinking the glucan between the functional groups described in said step (a) by using said crosslinking agent in the presence of polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone or water-soluble copolymers that contain at least one of polyethylene glycol, polyvinyl alcohol, or polyvinylpyrrolidone as principal constituents and which are not reactive with the functional groups described in said step (a) so as to prevent intermolecular crosslinking.

6. The isolated (1→

3)-β

-D-glucan that is intramolecularly crosslinked to have a stable single helix conformation according to claim 3 which is produced by a process comprising;

(a) forming a single helix conformation of a (1→

3)-β

-D-glucan having at least 20 D-glucoside units having hydroxyl groups, wherein the hydrogen atoms of at least part of the hydroxyl groups are substituted with functional groups that are reactive with a bifunctional crosslinking agent,

(b) binding the glucan having a single helix conformation to a receptor for said (1→

3)-β

-D-glucan, wherein said receptor is selected from the group consisting of horseshoe crab amebocyte lysate, factor G from horseshoe crab amebocyte lysate, arthropod phenol oxidase precursor, zymosan receptor from human lymphocytes, carbohydrate-receptive amino acids and tryptophan-containing peptides,

(c) intramolecularly crosslinking the glucan between the functional groups described in said step (a) by using said crosslinking agent in the presence of polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone or water-soluble copolymers that contain at least one of polyethylene glycol, polyvinyl alcohol, or polyvinylpyrrolidone as principal constituents, which are not reactive with the functional groups described in said step (a) so as to prevent intermolecular crosslinking, and

(d) eliminating the receptor.

4. A pharmaceutical composition comprising an isolated (1→

3)-β

-D-glucan that is intramolecularly crosslinked to have a stable single helix conformation and a pharmaceutically acceptable carrier,

wherein said glucan that is intramolecularly crosslinked has the following characteristics;

(i) a single helix conformation content of at least 20% as determined in a hydrated solid state;

(ii) a single helix conformation retention of at least 50% as determined after one month storage at 4°

C. in the form of an aqueous solution or suspension;

(iii) retains horseshoe crab factor G activating ability, host-mediated antitumor activity, or both horseshoe crab factor G activating ability and host-mediated antitumor activity; and

(iv) has been intramolecularly crosslinked so as to prevent intermolecular crosslinking.