Cell-associated glucosyltransferase, an antibody thereto, and a dental caries prophylactic composition containing said antibody as an effective component

• US 5,439,680 A
• Filed: 12/02/1994
• Issued: 08/08/1995
• Est. Priority Date: 01/22/1988
• Status: Expired due to Term

• Current Assignee: Ghen Corp.
• Original Assignee: Ghen Corp., Kanebo, Ltd. (HOYU Company Limited)
• Inventors: Yokoyama, Hideaki, Horikoshi, Toshio, Fujita, Isamu, Tokoro, Tohru, Kodama, Yoshikatsu, Hiraoka, Junichiro
• Primary Examiner(s): CHAN, Y CHRISTINA
• Application Number:
  US08/353,257
• Time in Patent Office: 249 Days
• Field of Search: 530/389.5, 530/853, 424/157.1, 424/165.1, 435/193, 435/885
• US Class Current: 424/157.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 8/66 Enzymes
• CPC Class Codes: A61P 1/02 Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61Q 11/00 Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
• CPC Class Codes: C07K 16/1275 from Streptococcus (G)
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 9/10 Transferases (2.) ribonucleases C12N9/22
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12R 2001/46 Streptococcus ; Enterococcus; Lactococcus
• CPC Class Codes: Y10S 435/885 Streptococcus
• CPC Class Codes: Y10S 530/853 Ovary; eggs; embryos

Abstract

Cell-associated glucosyltransferase of S. mutans serotypes c, e or f producing dental caries was isolated and purified and its characteristics as an enzyme were revealed. Furthermore, an antibody against said enzyme was prepared from eggs of the hens immunized with said enzyme. Since this antibody effectively prevents adherence of the aforementioned S. mutans to tooth surfaces, it is useful as an effective component for a dental caries prophylactic composition.

Claims

1. A composition for topical use to prevent adherence of Streptococcus mutans cells on tooth surfaces, which comprises:

(i) an effective amount of an egg yolk antibody obtained by the steps of;

(A) cultivating cells of Streptococcus mutans serotype c, e, or f in a nutrient-containing culture medium to obtain a culture containing cultured cells;

(B) removing the supernatant from the culture to recover the cultured cells;

(C) extracting a glucosyltransferase from the cultured cells using a urea or guanidine-HCl solution and purifying the glucosyltransferase, said purified glucosyltransferase having the following physicochemical characteristics;

(a) capable of catalyzing the synthesis of water-insoluble glucan from sucrose;

(b) having an optimum pH between 6.7 and 7.0 for catalyzing the synthesis of water-insoluble glucan from sucrose;

(c) losing its activity by heat treatment at 80°

C. for 5 minutes;

(d) having a molecular weight of 150 to 165 kilodaltons as determined by SDS-polyacrylamide gel electrophoresis; and

(e) antigenic in animals and capable of raising an antibody specifically binding to the glucosyltransferase;

(D) immunizing hens with the purified glucosyltransferase; and

(E) separating the egg yolk antibody, which specifically binds to the glucosyltransferase, from the eggs of the immunized hens; and

(ii) a physiologically acceptable diluent or carrier.

2. The composition of claim 1 in which the urea solution is a 6-10M urea solution and the guanidine-HCl solution is a 6M guanidine-HCl solution.

3. The composition of claim 2 in which the urea solution is an 8M urea. solution.

4. The compostion of claim 1 in which the glucosyltransferase is purified with an anion-exchange resin and hydroxylapatite.

5. A composition for topical use to prevent adherence of Streptococus mutans cells on tooth surfaces, which comprises;

(i) an effective amount of an egg yolk antibody obtained by the steps of;

(A) cultivating cells of Streptococcus mutans serotype c, e or f in a nutrient-containing culture medium to obtain a culture containing cultured cells;

(B) removing the supernatant from the culture to recover the cultured cells;

(C) contacting the cultured cells with a urea or guanidine-HCl solution to extract a glucosyltransferase from the cultured cells and thus form an extract containing the glucosyltransferase;

(D) removing urea or guanidine-HCl from the extract to obtain a glucosyltransferase preparation including the glucosyltransferase, which has the following physicochemical characteristics, when the glucosyltransferase is purified;

(a) capable of catalyzing the synthesis of water-insoluble glucan from sucrose;

(b) having an optimum pH between 6.7 and 7.0 for catalyzing the synthesis of water-insoluble glucan from sucrose;

(c) losing its activity by heat treatment at 80°

for 5 minutes;

(d) having a molecular weight of 150 to 165 kilodaltons as determined by SDS-polyacrylamide gel electrophoresis; and

(e) antigenic in animals and capable of raising an antibody specifically binding to the glucosyltransferase;

(E) immunizing hens with the glucosyltransferase preparation; and

(F) separating the egg yolk antibody, which specifically binds to the glucosyltransferase, from the eggs of the immunized hens; and

(ii) a physiologically acceptable diluent or carrier.

6. The composition of claim 5 in which the urea solution is a 6-10M urea solution and the guanidine-HCl solution is a 6M guanidine-HCl solution.

7. The composition of claim 6 in which the urea solution is an 8M urea solution.

8. The composition of claim 5, 6 or 7 in which urea or guanidine HCl is removed from the extract by dialysis.

9. A method of preventing adherence of Streptococcus mutans cells on tooth surfaces comprising topically applying an effective amount of an egg yolk antibody to the mouth of a person requiring the same, said egg yolk antibody being obtained by the steps of:

(A) cultivating cells of Streptococcus mutans serotype c, e or f in a nutrient-containing culture medium to obtain a culture containing cultured cells;

(B) removing the supernatant from the culture to recover the cultured cells;

(C) extracting a glucosyltransferase from the cultured cells using a urea or guanidine-HCl solution and purifying the glucosyltransferase, said glucosyltransferase having the following physicochemical characteristics;

(a) capable of catalyzing the synthesis of water-insoluble glucan from sucrose;

(b) having an optimum pH between glucan 6.7 and 7.0 for catalyzing the synthesis of water-insoluble glucan from sucrose;

(c) losing its activity by heat treatment at 80°

C. for 5 minutes;

(d) having a molecular weight of 150 to 165 kilodaltons as determined by SDS-polyacrylamide gel electrophoresis; and

(e) antigenic in animals and capable of raising an antibody specifically binding to the glucosyltransferase;

(D) immunizing hens with the purified glucosyltransferase; and

(E) separating the egg yolk antibody, which specifically binds to the glucosyltransferase, from the eggs of the immunized hens.

10. The method of claim 9 in which the urea solution is a 6-8M urea solution and the guanidine-HCl solution is a 6M guanidine-HCl solution.

11. The method of claim 10 in which the urea solution is an 8M urea solution.

12. The method of claim 9 in which glucosyltransferase is purified with an anion-exchange resin and hydroxylapatite.

13. A method of preventing adherence of Streptococcus mutans cells on tooth surfaces comprising topically applying an effective amount of an egg yolk antibody to the mouth of a person requiring the same, said egg yolk antibody being obtained by the steps of:

(A) cultivating cells of Streptococcus mutans serotyle c, e or f in a nutrient-containing culture medium to obtain a culture containing cultured cells;

(B) removing the supernatant from the culture to recover the cultured cells;

(C) contacting the cultured cells with a urea or guanidine-HCl solution to extract a glucosyltransferase from the cultured cells and thus form an extract containing the glucosyltransferase;

(D) removing urea or guanidine-HCl from the extract to obtain a glucosyltransferase preparation including the glucosyltransferase, which has the following physiochemical characteristics, when the glucosyltransferase is purified;

(a) capable of catalyzing the synthesis of water-insoluble glucan from sucrose;

(b) having an optimum pH between 6.7 and 7.0 for catalyzing the synthesis of water-insoluble glucan from sucrose;

(c) losing its activity by heat treatment at 80°

C. for 5 minutes;

(d) having a molecular weight of 150 to 165 kilodaltons as determined by SDS-polyacrylamide gel electrophoresis; and

(e) antigenic in animals and capable of raising an antibody specifically binding to the glucosyltransferase;

(E) immunizing hens with the glucosyltransferase preparation; and

(F) separating the egg yolk antibody, which specifically binds to the glucosyltransferase, from the eggs of the immunized hens.

14. The method of claim 13 in which the urea solution is a 6-10M urea solution or the guanidine-HCl solution is a 6M guanidine-HCl solution.

15. The method of claim 14 in which the urea solution is an 8M urea solution.

16. The method of claim 13, 14 or 15 in which urea or guanidine-HCl is removed from the extract by dialysis.