Antitumor agent and process for manufacturing said agent

• US 4,645,667 A
• Filed: 12/27/1983
• Issued: 02/24/1987
• Est. Priority Date: 12/27/1982
• Status: Expired due to Fees

• Current Assignee: Morinaga Milk Industry Company Limited
• Original Assignee: Morinaga Milk Industry Company Limited
• Inventors: Sekine, Kazunori, Hashimoto, Yoshiyuki, Saito, Minoru, Toida, Tomohiro, Kawashima, Takuji, Kuboyama, Morio
• Primary Examiner(s): Goldberg, Jerome D.
• Assistant Examiner(s): Rollins, Jr., John W.
• Application Number:
  US06/566,074
• Time in Patent Office: 1,155 Days
• Field of Search: 424/195, 424/92
• US Class Current: 424/282.1
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12P 1/04 by using bacteria
• CPC Class Codes: C12R 2001/01 Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Abstract

This invention relates to an antitumor agent comprising as a main ingredient a chemically purified cell walls of microorganisms belonging to genus Bificobacterium having physical integrity of cell wall structure thereof, obtained without being subjected to chemical and/or physical destruction of said cell wall during removal of intracellular substances of said cell, and a process for manufacturing the same, which comprises the steps of; (a) treating said cells with surfactants, (b) treating said cells with proteases, nucleases, organic solvents and dilute acid, to remove intracellular substances out of said cells without destruction thereof, and (c) separating a chemically purified cell walls having physical integrity of cell wall structure thereof.

Claims

1. A process for preparing a substance having antitumor activity comprising the steps of;

(a) Culturing microorganisms selected from the group consisting of Bifidobacterium infantis, Bifidobacterium longum and mixtures thereof in a conventional medium for bididobacteria until a sufficient number of cells are obtained, and separating cells of said microorganisms from said medium,

(b) heating said cells, suspended in a physiological saline solution, at 60°

to 100°

C. for 10 to 30 minutes, and separating said cells from said suspension in physiological saline solution,

(c) suspending said separated cells of step (b) in 4-(2-hydroxethyl)-1-piperazineethanesulfonic acid buffer solution of about neutral pH containing 0.1 to 0.4% (w/v) of a surface active agent selected from the group consisting of polyethylene glycol p-isooctylphenyl ether, polyoxyethylene (20) Sorbitan mono-oleate, sodium dodecylsulfate and mixtures thereof, in a ratio of 15 to 20 ml per gram of said cells, maintaining said solution at 80°

to 90°

C. for 30 to 60 minutes to remove cellular substances, and separating said cells from said suspension in said buffer solution,

(d) suspending said separated cells of step (c) in a hydrophilic organic solvent selected from the group consisting of methanol, ethanol, propanol, acetone and mixtures thereof, at room temperature for 12 to 24 hours to remove said surface active agent, and separating said cells from said suspension in said organic solvent,

(e) suspending said separated cells of step (d) in 2-amino-hydroxymethyl-1,3-propanediol or phosphate buffer solution of about neutral pH containing 0.05 to 0.2% (w/v) protease selected from the group consisting of trypsin, chymotrypsin, papain and mixtures thereof and ribonuclease in a concentration of 1/100 to 1/200 of said protease and deoxyribonuclease in a concentration of 1/1000 to 1/2000 of said protease, hydrolizing the cellular protein and nucleic acid at about 37°

C. for 14 to 24 hours, and separating said cells from said suspension in the last said buffer solution,

(f) suspending said separated cells of step (e) in 0.01N HCl of pH 2 to 3 containing 0.05 to 0.2% (w/v) of pepsin, hydrolyzing the cellular protein at about 37°

C. for 14 to 24 hours, and separating said cells from said suspension in said 0.01N HCl solution,

(g) suspending said separated cells of step (f) in 2-amino-hydroxymethyl-1,3-propanediol or phosphate buffer solution of about neutral pH containing 0.05 to 0.2% (w/v) of pronase, hydrolyzing the proteinous substances at about 37°

C. for 14 to 24 hours, and separating said cells from said suspension in the last said buffer solution,

(h) removing cellular lipids from said separated cells of step (g) with an organic solvent selected from the group consisting of methanol, ethanol, acetone and mixtures thereof, with a methanol-chloroform mixture or an acetone-chloroform mixture, and then with hexane or chloroform in sequence,

(i) suspending said cells from which the lipids were substantially removed in a 2-amino-hydroxymethyl-1,3-propanediol or phosphate buffer solution of about neutral pH containing 0.05 to 0.14% (w/v) of pronase, hydrolyzing the proteinous substances at about 37°

C. for 14 to 24 hours, and separating said cells from said suspension in the last said buffer solution,

(j) suspending said separated cells from step (i) in a diluted sulfuric acid solution, heating from 10 to 20 minutes in boiling water, and separating said cells from said suspension in said diluted sulfuric acid solution,

(k) dialyzing said cells of step (j) using distilled water for 48 to 72 hours, and

(l) freeze-drying said dialyzed cells whereby cell walls having physical structural integrity without intracellular substances are obtained.

2. A process according to claim 1, wherein said heating in step (b) occurs at 60°

to 80°

C. for 20 to 30 minutes.

3. A process according to claim 1, wherein said heating in step (b) occurs in boiling water for about 10 to 15 minutes.

4. An antitumor agent made according to the process of claim 1.

5. A process for preparing a substance having antitumor activity comprising the steps of;

(a) culturing microorganisms selected from the group consisting of Bifodobacterium infantis, Bifidobacterium longun, and mixtures thereof in a conventional medium for bifidobacteria, and separating cells of said microorganisms from said medium,

(b) heating said cells, suspended in a physiological saline solution, at 60°

to 100°

C. for 10 to 30 minutes, and separating said cells from said suspension in physiological saline solution.

(c) suspending said separated cells of step (b) in 4-(2-hydroxethyl)-1-piperazineethanesulfonic acid buffer solution of about neutral pH containing 0.1 to 0.4% (w/v) of a surface active agent selected from the group consisting of polyethylene glycol p-isooctylphenyl ether, polyoxyethylene (20) sorbitan mono-oleate, sodium dodecylsulfate and mixtures thereof, in a ratio of 15 to 20 ml per gram of said cells, maintaining said solution at 80°

to 90°

C. for 30 to 60 minutes to remove cellular substances, and separating said cells from said suspension in said buffer solution,

(d) suspending said separated cells of step (c) in a hydrophilic organic solvent selected from the group consisting of methanol, ethanol, propanol, acetone and mixtures thereof, at room temperature for 12 to 24 hours to remove said surface active agent, and separating said cells from said suspension in said organic solvent,

(e) suspending said separated cells of step (d) in 2-amino-hydroxymethyl -1,3-propanediol or phosphate buffer solution of about neutral pH containing 0.05 to 0.2% (w/v) protease selected from the group consisting of trypsin, chymotrypsin, papain and mixtures thereof and ribonuclease in a concentration of 1/100 to 1/200 of said protease and deoxyribonuclease in a concentration of 1/1000 to 1/2000 of said protease, hydrolizing the cellular protein and nucleic acid at about 37°

C. for 14 to 24 hours, and separating said cells from said suspension in the last said buffer solution,

(f) suspending said separated cells of step (e) in 0.01N HCl of pH 2 to 3 containing 0.05 to 0.2% (w/v) of pepsin, hydrolyzing the cellular protein at about 37°

C. for 14 to 24 hours, and separating said cells from said suspension in said 0.01N HCl solution,

(g) suspending said separated cells of step (f) in 2-amino-hydroxymethyl -1,3-propanediol or phosphate buffer solution of about neutral pH containing 0.05 to 0.2% (w/v) of pronase, hydrolyzing the proteinous substances at about 37°

C. for 14 to 24 hours, and separating said cells from said suspension in the last said buffer solution,

(h) suspending said separated cells from step (g) in a diluted sulfuric acid solution, heating from 10 to 20 minutes in boiling water, and separating said cells from said suspension in said diluted sulfuric acid solution,

(i) removing cellular lipids from said separated cells of step (h) with an organic solvent selected from the group consisting of methanol, ethanol, acetone and mixtures thereof, with a methanol-chloroform mixture or an acetone-chloroform mixture, and then hexane or chloroform in sequence,

(j) suspending said cells from which the lipids were substantially removed in 2-amino-hydroxymethyl -1,3-propanediol buffer solution of about neutral pH containing 0.05 to 0.14% (w/v) of pronase, hydrolyzing the proteinous substances at about 37°

C. for 14 to 24 hours, and separating said cells from said suspension in the last said buffer solution,

(k) dialyzing said cells of step (j) using distilled water for 48 to 72 hours, and

(l) freeze-drying said dialyzed cells whereby cell walls having physical structural integrity without intracellular substances are obtained.