Antigen derivatives and processes for their preparation

• US 4,446,128 A
• Filed: 05/18/1982
• Issued: 05/01/1984
• Est. Priority Date: 02/24/1978
• Status: Expired due to Term

• Current Assignee: Ciba-Geigy Corporation (Novartis Ag)
• Original Assignee: Ciba-Geigy Corporation (Novartis Ag)
• Inventors: Stanek, Jaroslav, Baschang, Gerhard, Tarcsay, Lajos, Hartmann, Albert, Dietrich, Felix M., Gisler, Roland
• Primary Examiner(s): Phillips, Delbert R.
• Application Number:
  US06/379,404
• Time in Patent Office: 714 Days
• Field of Search: 424/88, 424/177, 260/112.5 R, 260/112 R
• US Class Current: 424/194.1
• CPC Class Codes: A61K 2039/55583 Polysaccharides
• CPC Class Codes: A61K 2039/6062 Muramyl peptides
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/385 Haptens or antigens, bound to carriers
• CPC Class Codes: C07H 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C07H 9/04 Cyclic acetals
• CPC Class Codes: C07K 9/005 containing within the molecule the substructure with m, n > 0 and m+n > 0, A, B, D, E being heteroatoms; X being a bond or a chain, e.g. muramylpeptides
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y10S 530/806 Antigenic peptides or proteins
• CPC Class Codes: Y10S 530/807 Hapten conjugated with peptide or protein

Abstract

The invention relates to novel antigen derivatives comprising an antigen and at least one muramyl-peptide covalently bonded thereto, if appropriate via a spacer, to pharmaceutical preparations which contain such compounds and to their use as a vaccine.

The novel antigen derivatives produce a pronounced increase in the immuno-response to the antigen, and in particular also a cell-medicated immunity under clinically acceptable conditions of administration.

Claims

1. A novel antigen derivative, which comprises an antigen radical and one or more muramylpeptide radicals covalently bound thereto, directly or via a spacer, the antigen and muramylpeptide of those radicals each containing at least one functional group characterized in that upon reaction between antigen and muramylpeptide, antigen and spacer or muramylpeptide and spacer one of the below-mentioned covalent linking-structures is formed between the reactants, said muramylpeptide has the formula ##STR10## in which X is a carbonyl, carbonyloxy or sulfonyl group, R₁, R₄ and R₆ independently of one another represent hydrogen, alkyl having from 1 to 18 carbon atoms, benzyl which is unsubstituted or substituted in the aromatic nucleus by lower alkyl, hydroxyl, lower alkoxy, lower alkylenedioxy, mercapto, lower alkylmercapto, trifluoromethyl and/or halogen, or represent alkanoyl having from 2 to 18 carbon atoms, benzoyl or naphthoyl which are unsubstituted or substituted by halogen, lower alkyl, lower alkoxy, trifluoromethyl, hydroxyl or lower alkanoyloxy, or represent an acyl radical of a lower alkanesulfonic acid

or of phenylsulfonic acid, which is unsubstituted or substituted by lower alkyl or halogen, or represent carbamoyl, lower alkyl-carbamoyl or phenyl-carbamoyl, R₂ represents alkyl having 1 to 18 carbon atoms which is unsubstituted or substituted by hydroxy, lower alkoxy, lower alkanoyloxy, halogen, mercapto, lower alkylmercapto, lower alkanoylmercapto, carboxy, lower alkoxycarbonyl and/or carbamoyl, or represents phenyl which is unsubstituted or substituted by lower alkyl, hydroxy, lower alkoxy, lower alkanoyl, lower alkylenedioxy, halogen and/or trifluoromethyl, R₃, R₇, R₉ and R₁₃ are independently of one another hydrogen or lower alkyl, R₈ is hydrogen, lower alkyl which is unsubstituted or substituted by hydroxy, lower alkoxy, lower alkanoyloxy, mercapto, lower alkylmercapto, lower alkanoylmercapto, amino, lower alkanoylamino, carbamoylamino or cycloalkyl having 5 or 6 carbon atoms, or represents cycloalkyl-lower alkyl, the cycloalkyl radical of which contains 5 or 6 carbon atoms, phenyl or phenyl-lower alkyl, in which the phenyl radicals are unsubstituted or substituted as defined above, or heterocyclic radicals are 5- or 6-membered, contain one or two nitrogen atoms in the ring and may or may not contain a fused phenyl radical, R₇ and R₈ together can also represent alkylene having 3 or 4 carbon atoms, R₁₀, R₁₁ and R₁₂ independently of one another represent carboxyl, lower alkoxycarbonyl or carbamoyl which is unsubstituted or mono- or disubstituted at the nitrogen atom by unsubstituted or carboxy- or carbamoyl-substituted lower alkyl or by phenyl, benzyl, tetramethylene or pentamethylene and R₁₁ can also be hydrogen, with the proviso that the muramylpeptide and the antigen are bonded together via the covalent linking structures carbonyloxy ##STR11## carbonylimino ##STR12## wherein R is hydrogen or lower alkyl), carbonylthio ##STR13## thiocarbonyloxy ##STR14## and/or thiocarbonylimino ##STR15## wherein R'"'"' is hydrogen or lower alkyl) directly or via a spacer, and with the further proviso that the antigen-muramylpeptide derivative may or may not be fixed to a high molecular weight carrier and that the antigen is not a constituent of a vaccine against tumour cells.

2. A novel antigen derivative according to claim 1, wherein the antigen is the constituent of malaria merozoites, type-specific meningococcal polysaccharides A, B or C, M-proteins of streptococci, influenza virus haemagglutinins, partial sequences of human choriongonadotropin, grass pollen extracts, anti-specific T-cell lymphoblasts and their receptors for antigens, or antigen-specific immunoglobulins.

3. An antigen derivative according to claim 1 wherein R₁₃ represents hydrogen.

4. An antigen derivative according to claim 1, or 3, wherein the antigen is covalently bound to the carrier via the covalent linking structures carbonyloxy ##STR16## carbonylimino ##STR17## wherein R is hydrogen or lower alkyl) or carbonylthio ##STR18## directly or via a spacer, and the muramylpeptide is covalently bound to the antigen and may additionally also be bound to the carrier via the covalent linking structures mentioned in this claim, directly or via a spacer.

5. An antigen derivative according to claim 1 wherein a spacer is the radical or a α

,ω

-diamino-lower alkane bound via the nitrogen atoms, lower alkanedicarbonyl or α

-, β

- or γ

-aminoalkanecarbonyl.

6. An antigen derivative according to claim 1 whrein the antigen and the muramylpeptide are bound directly together.

7. An antigen derivative according to claim 6 wherein the muramylpeptide is bound via the substituent R₁₂ of formula II.

8. An antigen derivative according to claim 1, 5 or 7, wherein the antigen is a substance including ingredients of vaccines which brings about the specific immunisation of a living organism against infectious pathogens or undesired reactions, which are allergic sensitisation or rejection of transplanted foreign tissue.

9. An antigen derivative according to claim 1 wherein the antigen is the effective constituent, which represents or comprises the antigenic determinant, of vaccines against parasites, bacteria, viruses or physiological body-innate constituents, their modified forms or sub-units thereof.

10. An antigen derivative according to claim 7, wherein the antigen represents group C polysaccharide of Neisseria Meningitidis, merozoites of the malaria pathogen Plasmodium knowlesi, T-lymphoblasts, erythrocyte membranes, the active ingredient of food and mouth disease culture vaccine or of rabies vaccine, influenza virus antigen, tetanus toxoid, cholera toxoid, eicosapeptid which is identical with the C-terminal sequence of human choriongonadotropin, or Trypanosoma cruzi parasites, or components or parts of these antigens carrying the desired antigenic determinant.

11. An antigen derivative according to claim 10, which contains pro 1 mg or pro 10⁷ cells respectively of the antigen from 0.01 to 0.5 mg muramylpeptide, these values are derived from the results of the analytical determination of the product by means of the Morgan-Elson-reaction.

12. An antigen derivative according to claim 1 wherein the antigen and the muramylpeptide are bound together via the covalent linking structures carbonyloxy ##STR19## carbonylimino ##STR20## wherein R is hydrogen or lower alkyl) or carbonylthio ##STR21## directly or via a spacer.

13. An antigen derivative according to claim 1, 7, 9, 10, or 5 wherein the muramylpeptide is a compound of the formula II wherein R₁, R₃, R₄, R₆, R₇ and R₁₃ are hydrogen, X is carbonyl and R₂ is lower alkyl which is unsubstituted or substituted by hydroxyl or lower alkoxy, or is phenyl which is unsubstituted or substituted by hydroxyl, lower alkoxy, lower alkyl or halogen, and R₈, R₉, R₁₀, R₁₁ and R₁₂ are as defined in claim 4.

14. An antigen derivative according to claim 1, 7, 9, 10, or 5 wherein the muramylpeptide is a compound of the formula II wherein R₁, R₄, R₆, R₇ and R₁₃ are hydrogen, X is carbonyl, R₂ lower alkyl which is unsubstituted or substituted by hydroxyl or lower alkoxy or is phenyl which is unsubstituted or substituted by hydroxyl, lower alkoxy, lower alkyl or halogen, R₃ is methyl and R₈, R₉, R₁₀, R₁₁ and R₁₂ are as defined in claim 1.

15. An antigen derivative according to claim 1 wherein the muramylpeptide is a compound of the formula II wherein X is carbonyl, R₁, R₄, R₆, R₇, R₉ and R₁₃ are hydrogen, R₂ is lower alkyl which is unsubstituted or substituted by hydroxyl or methoxy or is phenyl which is unsubstituted or substituted by hydroxyl, methoxy, methyl, ethyl or halogen, R₃ is hydrogen or methyl, R₈ is benzyl, p-hydroxybenzyl, phenyl or lower alkyl which is unsubstituted or substituted by lower alkylmercapto or hydroxy, and R₁₀, R₁₁ and R₁₂ are carboxyl, lower alkoxycarbonyl or carbamoyl and R₁₁ can also be hydrogen.

16. An antigen derivative according to claim 1, 7, 9, 10, or 2 wherein the muramylpeptide is a compound of the formula II wherein X is carbonyl, R₁, R₄, R₆ and R₁₃ are hydrogen, R₂ is lower alkyl which is unsubstituted or substituted by hydroxyl or methoxy or is phenyl which is unsubstituted or substituted by hydroxyl, methoxy, methyl, ethyl or halogen, R₃, R₇ and R₉ are hydrogen or methyl, R₈ is methyl, ethyl, n-propyl, i-propyl, 2-methyl-propyl, methylmercaptomethyl, hydroxymethyl, hydroxyethyl, phenyl, benzyl, or p-hydroxybenzyl and R₁₀, R₁₁ and R₁₂ are carboxyl, lower alkoxycarbonyl or carbamoyl and R₁₁ can also be hydrogen.

17. An antigen derivative according to claim 1 wherein the muramylpeptide is a compound of the formula II wherein X is carbonyl, R₁, R₄ R₆ and R₁₃ are hydrogen, and R₇ and R₈ together are propylene or butylene.

18. An antigen derivative according to claim 1 containing a muramylpeptide of the formula II wherein R₁₁ and R₁₃ is hydrogen.

19. An antigen derivative according to claim 1 wherein wherein the muramylpeptide is a compound of the formula II wherein X is carbonyl, R₁, R₄, R₆, R₁₁ and R₁₃ hydrogen, R₂ is methyl, ethyl or phenyl, R₃ is hydrogen or methyl, R₇ is hydrogen, methyl, ethyl or n-propyl, R₈ is methyl, ethyl or isopropyl, R₉ is hydrogen or methyl, R₁₀ is carboxyl, carbamoyl, N-(carbamoylmethyl)-carbamoyl or N-(1-carboxy-ethyl)-carbamoyl and R₁₂ is carboxy or N-(1-carboxy-ethyl)-carbamoyl.

20. An antigen derivative according to claim 1 containing a muramylpeptide of the formula II wherein R₇ is methyl, ethyl or n-propyl.

21. An antigen derivative according to claim 1, 7, 9, 10, or 2 wherein the muramylpeptide is a compound of the formula II wherein the muramylpeptide is 2-acetamido-3-O-{[L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-methyl}-2-deoxy-D-glucose or 2-acetamido-3-O-{D-1-[L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-ethyl}-2-deoxy-D-glucose.

22. An antigen derivative according to claim 1 wherein the muramylpeptide is a compound of the formula II wherein the muramylpeptide is 2-acetamido-3-O-{D-1-[L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-propyl]-N-methyl-carbamoyl-ethyl}-2-deoxy-D-glucose.

23. A pharmaceutical preparation for enteral or parenteral administration for modulation of the immune response to warm-blooded animals including man containing an effective dose of an antigen derivative according to claim 1 together with a significant amount of a pharmaceutically acceptable carrier.

24. A method of using an antigen derivative according to claim 1 for modulating the immune response of warm-blooded animals including man by enterally or parenterally administrating an effective dose thereof.

25. An antigen derivative according to claim 1, wherein the antigen is the constituent of a vaccine against parasites, bacteria, viruses, or attenuated or killed forms or part-components thereof, constituent of a vaccine against structures innate in the body or immunological response structures, or the constituent of a vaccine suitable for specific desensitisation in the case of allergies.

26. An antigen derivative according to claim 1, wherein the antigen is the constituent of a vaccine against malaria, cholera, typhus, meningitis, rheumatic fever as a consequence of streptococcal infections, influenza, rabies or foot and mouth disease, for the induction of immunity against components of the reproductive system, for desensitisation or induction of specific immuno-tolerances to allergies or for the reinstatement of tolerance towards tissue constituents, and circulating molecules, innate in the body.

27. A method of immunizing warm-blooded animals comprising administering to said animals an effective amount of an antigen derivative according to claim 1.

28. A novel antigen derivative according to claim 3, wherein the antigen is fixed to a high molecular weight carrier.

29. A novel antigen derivative according to claim 28, wherein the carrier is a polymer of lactic acid and its derivatives, which carry a free carboxyl group at the chain end, alginic acid, polygalacturonic acid, pectic acid, carboxymethylcellulose, agarose or a basic, neutral or acidic polyaminoacid.

30. A novel antigen derivative according to claim 3, wherein the antigen is separated from the muramyl-peptide by a bridge member (spacer).

31. A novel antigen derivative according to claim 3, of the formula ##STR22## in which A is the radical of the antigen, T is the radical of the carrier, MP is the radical of the muramyl-peptide, Z'"'"' and Z"'"'"' are bivalent spacers and X'"'"', X", X"'"'"' and X"" are covalent linking structures, and m and n are integers greater than 0.

32. A pharmaceutical preparation according to claim 23 containing a solution or suspension of the antigen derivative together with carboxymethylcellulose.