KS-2-A

• US 4,163,780 A
• Filed: 03/30/1978
• Issued: 08/07/1979
• Est. Priority Date: 03/30/1977
• Status: Expired due to Term

• Current Assignee: Kirin-Seagram Limited
• Original Assignee: Kirin-Seagram Limited
• Inventors: Maeda, Hiroshi, Suzuki, Fujio, Ishida, Nakao, Mizutani, Ituro, Fujii, Toshikatsu
• Primary Examiner(s): Goldberg, Jerome D.
• Application Number:
  US05/891,767
• Time in Patent Office: 495 Days
• Field of Search: 424/116, 195/81
• US Class Current: 424/116
• CPC Class Codes: A61K 36/07 Basidiomycota, e.g. Cryptococcus
• CPC Class Codes: C12N 1/145 Fungal isolates
• CPC Class Codes: C12P 1/02 by using fungi
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12R 2001/645 Fungi ; Processes using fungi
• CPC Class Codes: Y10S 435/811 Interferon

Abstract

KS-substance was obtained as raw material from the cultured mycelium of Daedalea dickinsii KSDD 6 (FERM-P No. 3993), Lentinus edodes KSLE 7 (FERM-P No. 3994) or Lentinus edodes KSLE 28 (FERM-P No. 4196), and by refining this substance a novel substance KS-2-A was obtained. This substance KS-2-A and also the KS-substance as the raw material both enhance the host defense function of the living body.

Claims

1. KS-2-A having the following physicochemical properties:

(1) Elemental analysis

C;

39.5%, h;

6.5%, n;

1.1%,

ash;

trace (0.4%);

(2) Molecular weight

9,000±

3,000 (by ultra-filtration),

7,000 to 9,000 (by equilibrium density gradient centrifugation),

8,000±

3,000 (by fluorescein polarization method);

(3) Appearance

amorphous white powder;

(4) Decomposition point

185°

C. (based upon measurement of the browning temperature by capillary method using "Silicone Oil WF-30");

(5) uv spectrum

No particular maximal absorption is observed as shown in FIG. 1;

(6) ir spectrum

The spectrum is shown in FIG. 2;

(7) the pH of the aqueous solution of this substance is 7.25;

(8) Solubility

Soluble in water, insoluble in ethanol, acetone, n-hexane, n-butanol, phenol and other organic solvents;

(9) Specific optical rotation

space="preserve" listing-type="equation">[α

].sub.D.sup.25 =+67.5°

±

2.0°

(in H.sub.2 O), (C=0.452%);

(10) homogeneity

a. The result of centrifugation in a linear gradient of 5 to 20% of CsCl in 0.1 M Tris-CHl buffer (pH 7.2) at 38,000 r.p.m., at 4°

C. for 15 hours is shown in FIG. 3, the substance being homogeneous as is apparent from the Figure;

comparative sedimentation experiments with viral nucleic acids show that the substance does not contain viral particles, RNA or DNA;

b. The substance is electrophoretically homogeneous as is shown in FIG. 4 which shows the results of electrophoresis on cellulose acetate using 0.1 M acetic acid/pyridine buffer (pH 3.5), chondroitin sulfate being used as control;

after 30 minutes of electrophoresis at 0.6 mA/cm and 160 V, chondroitin sulfate moves to the cathode, giving a mobility of 4 cm per 30 minutes, whereas KS-2-A moves slightly to the anode in 90 minutes (a mobility of 1 cm per 90 minutes);

the substance is also shown to be homogeneous by electrophoresis conducted under the same conditions as above except using 1 M pyridine/acetic acid buffer (pH 7.0);

(11) color reaction

Phenol-H₂ SO₄ reaction;

Positive

Anthrone reaction;

Positive

Molisch'"'"'s reaction;

Positive

Elson-Morgen reaction;

Negative

Carbazole-H₂ SO₄ reaction;

Positive

Reaction with Folin-Ciocarteu reagent;

Positive

Biuret reaction;

Positive

Reaction with FITC (fluorescein isothiocyanate);

Positive

Toluidin blue O staining;

Negative

Ninhydrin reaction;

Positive;

(12) Sugar composition

Fig. 5 shows the results of subjecting the substance to acid hydrolysis, followed by alditolation and then acetylation, and then determining the sugar composition by gas chromatography;

the substance is mainly composed of mannose and contains small quantities of glucose and galactose and also minute quantities of arabinose and xylose, the proportions of these components by weight being 74;

12;

12;

1;

1; and

(13) Amino acid composition

Amino acid analysis, made in an automatic amino acid analyzer after closing 10 mg of the substance in vacuo together with 3 ml of 6 N HCl, subjecting it to acid hydrolysis at 110°

C. for 22 hours and then removing by a rotary evaporator shows that the amino acids contained are mainly threonine, serine, glutamic acid, alanine and ammonia, and further slight quantities of aspartic acid, proline, glycine, valine and lysine are contained;

further, sometimes trace amounts of methionine, isoleucine leucine, tyrosine, phenylalanine, histidine, arginine and cystine are recognized.

2. A chemotherapeutic agent comprising an interferon titer increasing effective amount of KS-2-A in accordance with claim 1 as active component and a pharmaceutically acceptable carrier.

3. A method for manufacturing KS-2-A in accordance with claim 1 comprising culturing a strain selected from a group consisting of Daedalea dickinsii KSDD 6 (FERM-P No. 3993), Lentinus edodes KSLE 7 (FERM-P No. 3994) and Lentinus edodes KSLE 28 (FERM-P No. 4196) in a medium consisting essentially of distillers solubles under aerobic conditions until a substantial amount of KS-2-A is accumulated in the culture broth.

4. A method for increasing the interferon titer of a patient comprising administering to said patient an interferon titer increasing effective amount of KS-2-A as defined in claim 1.