Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
1999-06-22
2002-12-10
Wilson, James O. (Department: 1623)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C424S193100, C514S025000, C514S772400
Reexamination Certificate
active
06491919
ABSTRACT:
BACKGROUND OF THE INVENTION
The compounds monophosphoryl lipid A (MLA) and 3-O-deacylated monophosphoryl lipid A (3D-MLA) are attenuated derivatives of the lipid A component of bacterial lipopolysaccharide (LPS). LPS and lipid A are potent immunostimulants inducing both a humoral antibody response and a cell-mediated immune response in patients administered the compounds. Lipid A and LPS however can also display toxic side-effects such as pyrogenicity and local Shwarzman reactions. MLA and 3D-MLA are lipid A-like molecules that have been modified to attenuate the toxicity of LPS.
Like lipid A, the MLA and 3D-MLA molecules have a sugar backbone onto which long chain fatty acids are attached. The backbone is comprised of two six carbon sugar rings in glycosidic linkage. MLA and 3D-MLA are phosphorylated at the 4 position. Five to eight long chain fatty acids (12-14 carbons) are attached to the sugar backbone making MLA and 3D-MLA very hydrophobic molecules which are not readily water soluble.
The attenuated lipid A derivatives (ALDs) MLA and 3D-MLA are used as immunologic adjuvants in prophylactic vaccines for infectious disease and therapeutic vaccines for the treatment of cancerous tumors and chronic infections. Antigen preparations included in most vaccines are often complicated mixtures of water-soluble proteins making it difficult to formulate the water insoluble adjuvant in a water based vaccine. Therefore, MLA and 3D-MLA must be first mixed with solvents before they are added to the antigen preparation. However, the presence of solvents can further complicate the formulation of the vaccine, and in some cases can reduce the efficiency of its components. Further, solvents can irritate mucosal surfaces or cause inflammation at an injection site. A simple formulation of MLA or 3D-MLA containing no interfering co-solvents would allow maximum benefits to be derived from both the adjuvant and the antigen in a vaccine composition. The instant invention satisfies this need.
SUMMARY OF THE INVENTION
The subject invention involves an aqueous formulation of an attenuated lipid A derivative (ALD) and a surfactant and methods for its preparation and storage. Attenuated lipid A derivatives useful according to the subject invention include monophosphoryl lipid A (MLA) and 3-O-deacylated monophosphoryl lipid A (3D-MLA). Aqueous formulations of MLA (MLA/AF) or 3D-MLA (3D-MLA/AF) eliminate the need for undesirable solvents or a co-solvent system for vaccine preparation. The invention provides a stable aqueous composition of the ALD and a surfactant which when administered to mice with an antigen, enhances the cellular and humoral immune response of the animal to that antigen. Surprisingly, the aqueous formulation of the present invention induces high levels of serum and mucosal secreted IgA in immunized animals when administered intranasally. An embodiment of the claimed aqueous composition comprises a MLA or 3D-MLA to surfactant molar ratio of about 4:1 and has a particle size of approximately 50-70 nm. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is a preferred surfactant. Unexpectedly, when glycerol is added to the subject aqueous formulation before lyophilization, the composition is restored upon reconstitution with no additional sonication. Successful storage of the subject composition in a lyophilized state allows for convenient storage and transport of the aqueous formulation or vaccine compositions comprising the formulation.
A method of preparing the aqueous composition is disclosed. In one embodiment the ALD and the surfactant are dissolved and uniformly admixed in ethanol. The ethanol is then evaporated leaving a film. Water is added to the film. The ALD and surfactant are suspended in the water by sonication. The suspension is sonicated until clear. Animals administered the claimed composition with an antigen display enhanced humoral and cellular immune responses to that antigen. Methods for using the composition to enhance these responses are also disclosed and claimed.
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Corixa Corporation
Krishnan Ganapathy
Townsend and Townsend / and Crew LLP
Wilson James O.
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