Aminoalkyl glucosaminide phosphate compounds and their use...

Details Aminoalkyl glucosaminide phosphate compounds and their use... Aminoalkyl glucosaminide phosphate compounds and their use...

1999-11-12

2001-10-16

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C424S278100, C536S001110, C536S018400, C536S117000, C536S119000

Reexamination Certificate

active

06303347

ABSTRACT:

TECHNICAL FIELD
This invention relates generally to aminoalkyl glucosaminide phosphate (AGP) compounds which have activity as adjuvants and immunoeffectors, and methods and compositions related thereto.
BACKGROUND OF THE INVENTION
Humoral immunity and cell-mediated immunity are the two major branches of the mammalian immune response. Humoral immunity involves the generation of antibodies to foreign antigens. Antibodies are produced by B-lymphocytes. Cell-mediated immunity involves the activation of T-lymphocytes which either act upon infected cells bearing foreign antigens or stimulate other cells to act upon infected cells. Both branches of the mammalian immune system are important in fighting disease. Humoral immunity is the major line of defense against bacterial pathogens. In the case of viral disease, the induction of cytotoxic T lymphocytes (CTLs) appears to be crucial for protective immunity. An effective vaccine stimulates both branches of the immune system to protect against disease.
Vaccines present foreign antigens from disease causing agents to a host so that the host can mount a protective immune response. Often vaccine antigens are killed or attenuated forms of the microbes which cause the disease. The presence of non-essential components and antigens in these killed or attenuated vaccines has encouraged considerable efforts to refine vaccine components including developing well-defined synthetic antigens using chemical and recombinant techniques. The refinement and simplification of microbial vaccines, however, has led to a concomitant loss in potency. Low-molecular weight synthetic antigens, though devoid of potentially harmful contaminants, are themselves not very immunogenic. These observations have led investigators to add adjuvants to vaccine compositions to potentiate the activity of the refined vaccine components.
Presently, the only adjuvant licensed for human use in the United States is alum, a group of aluminum salts (e.g., aluminum hydroxide, aluminum phosphate) in which vaccine antigens are formulated. Particulate carriers like alum serve to promote the uptake, processing and presentation of soluble antigens by macrophages. Alum, however, is not without side-effects and enhances humoral (antibody) immunity only.
An effective adjuvant potentiates both a humoral and cellular immune response in vaccinated animals. Further, an adjuvant must enhance a host's natural immune response and not aggravate the host system. A well-defined synthetic adjuvant free from extraneous matter which is stable and easy to manufacture would provide these qualities. Compounds that have been prepared and tested for adjuvanticity (Shimizu et al. 1985, Bulusu et al. 1992, Ikeda et al. 1993, Shimizu et al. 1994, Shimizu et al. 1995, Miyajima et al. 1996), however, often display toxic properties, are unstable and/or have unsubstantial immunostimulatory effects.
The discovery and development of effective adjuvants is essential for improving the efficacy and safety of existing vaccines. Adjuvants impart synthetic peptides and carbohydrate antigens with sufficient immunogenicity to insure the success of the synthetic vaccine approach. There remains a need for new compounds having potent immunomodulating effects.
SUMMARY OF THE INVENTION
The compounds of the subject invention are aminoalkyl glucosaminide phosphate compounds (AGPs) which are adjuvants and immunoeffectors. An aminoalkyl (aglycon) group is glycosidically linked to a 2-deoxy-2-amino-&agr;-D-glucopyranose (glucosaminide) to form the basic structure of the claimed molecules. The compounds are phosphorylated at the 4 or 6 carbon on the glucosaminide ring. Further, the compounds possess three 3-alkanoyloxyalkanoyl residues.
The compounds of the subject invention are immunoeffector molecules augmenting antibody production in immunized animals, stimulating cytokine production and activating macrophages. In accordance with the -subject invention, methods for using these compounds as adjuvants and immunoeffectors are disclosed.
DETAILED DESCRIPTION OF THE INVENTION
The compounds of the subject invention are adjuvant and immunoeffector molecules which are aminoalkyl glucosaminide phosphates (AGPs). The compounds comprise a 2-deoxy-2-amino-&agr;-D-glucopyranose (glucosaminide) in glycosidic linkage with an aminoalkyl (aglycon) group. Compounds are phosphorylated at the 4 or 6 carbon on the glucosaminide ring and have three alkanoyloxyalkanoyl residues. The compounds of the subject invention are described generally by Formula I,
wherein X represents an oxygen or sulfur atom in either the axial or equitorial position, Y represents an oxygen atom or NH group, “n”, “m”, “p” and “q” are integers from 0 to 6, R
1
, R
2
, and R
3
represent normal fatty acyl residues having 1 to 20 carbon atoms and where one of R
1
, R
2
or R
3
is optionally hydrogen, R
4
and R
5
are hydrogen or methyl, R
6
and R
7
are hydrogen, hydroxy, alkoxy, phosphono, phosphonooxy, sulfo, sulfooxy, amino, mercapto, cyano, nitro, formyl or carboxy and esters and amides thereof; R
8
and R
9
are phosphono or hydrogen. The configuration of the 3′ stereogenic centers to which the normal fatty acyl residues are attached is R or S, but preferably R. The stereochemistry of the carbon atoms to which R
4
or R
5
are attached can be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to fall within the scope of the subject invention.
The heteroatom X of the compounds of the subject invention can be oxygen or sulfur. In a preferred embodiment, X is oxygen and typically in the equitorial position. Although the stability of the molecules could be effected by a substitution at X, the immunomodulating activity of molecules with these substitutions is not expected to change.
The number of carbon atoms between heteroatom X and the aglycon nitrogen atom is determined by variables “n” and “m”. Variables “n” and “m” can be integers from 0 to 6. In a preferred embodiment, the total number of carbon atoms between heteroatom X and the aglycon nitrogen atom is from about 2 to about 6 and most preferably from about 2 to about 4.
The compounds of the subject invention are aminoalkyl glucosaminide compounds which are phosphorylated. Compounds can be phosphorylated at position 4 or 6 (R
8
or R
9
) on the glucosaminide ring and are most effective if phosphorylated on at least one of these positions. In a preferred embodiment, R
8
is phosphono and R
9
is hydrogen.
In one embodiment, the compounds of the subject invention are hexaacylated, that is they contain a total of six fatty acid residues. The aminoalkyl glucosaminide moiety is acylated at the 2-amino and 3-hydroxyl groups of the glucosaminide unit and at the amino group of the aglycon unit with 3-hydroxyalkanoyl residues. In Formula I, these three positions are acylated with 3-hydroxytetradecanoyl moieties. The 3-hydroxytetradecanoyl residues are, in turn, substituted with normal fatty acids (R
1
-R
3
), providing three 3-n-alkanoyloxytetradecanoyl residues or six fatty acid groups in total.
In another embodiment, the compounds of the subject invention are pentaacylated, that is they contain a total of five fatty acid residues. More specifically, the 3-hydroxytetradecanoyl residues of Formula I are substituted with normal fatty acids at two of the three R
1
, R
2
and R
3
positions, with the third R
1
, R
2
or R
3
position being hydrogen. In other words, at least one of —OR
1
, —OR
2
or —OR
3
is hydroxyl.
The chain length of normal fatty acids R
1
-R
3
can be from 1 to about 20, and typically from about 7 to about 16 carbons. Preferably, R
1
-R
3
are from about 9 to about 14 carbons. The chain lengths of these normal fatty acids can be the same or different. Although, only normal fatty acids are described, it is expected that unsaturated fatty acids (i.e. fatty acid moieties having double or triple bonds) substituted at R
1
-R
3
on the compounds of the subject invention would produce biologically active molecules. Further, slight modifications in the chain leng

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