Drug – bio-affecting and body treating compositions – Lymphokine – Interferon
Reexamination Certificate
1999-03-12
2001-01-30
Moezie, F. T. (Department: 1654)
Drug, bio-affecting and body treating compositions
Lymphokine
Interferon
C424S085100, C424S085700, C514S012200, C530S388230, C530S350000, C530S351000, C530S403000, C530S405000, C530S409000, C530S816000
Reexamination Certificate
active
06180096
ABSTRACT:
Throughout this disclosure, various publications, patents and patent applications are referenced. The disclosures of these publications, patents and patent applications are herein incorporated by reference.
FIELD OF THE INVENTION
The present invention pertains to formulations for the stabilization of PEG-interferon alpha conjugates during and after lyophilization, their production and use.
BACKGROUND OF THE INVENTION
Various natural and recombinant proteins have pharmaceutical utility. Once they have been purified, separated, and formulated, they can be parenterally administered for various therapeutic indications. However, parenterally administered proteins may be immunogenic, may be relatively water insoluble, and may have a short pharmacological half life. Consequently, it can be difficult to achieve therapeutically useful blood levels of the proteins in patients.
These problems can be overcome by conjugating the proteins to polymers, such as polyethylene glycol. Davis et al., U.S. Pat. No. 4,179,337 disclose conjugating polyethylene glycol (PEG) to proteins such as enzymes and insulin to obtain conjugates having less immunogenic effect than the original proteins and yet still retaining a substantial proportion of their physiological activity. Veronese et al., (Applied Biochem. and Biotech, 11:141-152 , 1985) disclose activating polyethylene glycols with phenyl chloroformates to modify a ribonuclease and a superoxide dimutase. Katre et al. U.S. Pat. Nos. 4,766,106 and 4,917,888 also disclose solubilizing proteins by polymer conjugation. Likewise, PEG and other polymers can be conjugated to recombinant proteins to reduce immunogenicity and increase half-life. See Nitecki, et al., U.S. Pat. No. 4,902,502, Enzon, Inc., International Application No. PCT/US90/02133, Nishimura et al., European Patent Application 154,316 and Tomasi, International Application Number PCT/US85/02572. For example, interferon alpha-2b is known to be effective for treatment of disease states such as renal cell carcinoma, AIDS-related Kaposi's sarcoma, chronic and acute hepatitis B, chronic and acute non-A, non-B/C hepatitis and hepatitis C. Improvement of the pharmacological half-life of interferon alpha-2b would improve treatment of these conditions.
While preparation of protein-polymer conjugates is beneficial, they cannot be used in a practical manner unless they can be stored for an extended period of time during manufacture and distribution to health care providers. Some protein-polymer conjugates, however, rapidly deteriorate, even in frozen solutions. Lyophilization (also known as freeze-drying) is a process that can render a pharmaceutical in a form that can overcome this deficiency.
Lyophilization is a process whereby water is sublimed from a composition after it is frozen. In this process, pharmaceuticals and biologicals that are relatively unstable in an aqueous solution over a period of time can be placed into dosage containers in an easily processed liquid state, dried without the use of damaging heat and stored in a dried state for extended periods.
Due to the low total mass of active substance in each dose, the formulations of most pharmaceuticals and biologicals, including protein-polymer conjugates, require additional ingredients to protect the active ingredient during the lyophilization process. For example, a pharmaceutical filled into a dosage container as a low-concentration aqueous solution can be susceptible to physical loss during the lyophilization vacuum process or adsorption to the container. A lyophilized formulation often contains bulking ingredients that increase the amount of solid material, as well as cryoprotectants, lyoprotectants and other stabilizers to protect the active component from damage. Which particular formulation will protect a given type of pharmaceutical, however, must be determined empirically.
There is a present need for a formulation suitable to protect protein-polymer conjugates, and in particular PEG-interferon alpha conjugates, from damage during lyophilization. Such a formulation should allow PEG-interferon alpha-polymer conjugates to maintain their biological activity, physical stability and chemical stability over extended periods of time.
SUMMARY OF THE INVENTION
The present invention provides formulations that permit stabilization of PEG-interferon alpha conjugates during and after lyophilization.
In one embodiment, the present invention provides aqueous formulations comprising PEG-interferon conjugates, a buffer, a stabilizer and a cryoprotectant. The present invention also contemplates processes for preparing stable, aqueous formulation solutions comprising admixing an effective amount of PEG-interferon alpha conjugates with a buffer, a stabilizer, a cryoprotectant and a solvent. In a preferred aspect of the process of the present invention, the formulation is prepared and maintained substantially free of dissolved oxygen and a head space of inert atmosphere above the formulation is maintained at a value of less than about 4% oxygen by volume.
The present invention is not limited to specific chemicals for the solution components. However, in a preferred embodiment, the buffer is sodium phosphate, the stabilizer is a poly(oxy-1,2-ethanediyl) derivative, the cryoprotectant is sucrose and the solvent is water. In such an embodiment, the sodium phosphate can comprise sodium phosphate dibasic anhydrous with sodium phosphate monobasic dihydrate.
The present invention is also not limited by the concentrations of the components of the formulations of the present invention. In one embodiment, the concentration of PEG-interferon alpha conjugates is preferably 0.03 to 2.0 mg interferon alpha per ml, while the concentration of sodium phosphate is preferably 0.005 to 0.1 molar, the concentration of poly(oxy-1,2-ethanediyl) derivative is preferably 0.01 to 1.0 mg/ml and the concentration of sucrose is preferably 20 to 100 mg/ml. In a particularly preferred embodiment, the mass of PEG-interferon conjugates is 0.1 mg of interferon alpha, the mass of sodium phosphate dibasic is 0.75 mg, the mass of sodium phosphate monobasic dihydrate is 0.75 mg, the mass of sucrose is 40 mg, the mass of poly(oxy-1,2-ethanediyl) derivative is 0.05 mg and the volume of water is 0.5 ml. Alternatively, the ratio of components is 0.08% of said PEG-interferon alpha conjugates as measured by the mass of the interferon alpha, 3.6% of sodium phosphate, 0.12% of poly(oxy-1,2-ethanediyl) derivative and 96.2% of sucrose, by weight.
While the present invention is not limited to a specific PEG-interferon alpha conjugate, in one embodiment, the PEG-interferon alpha conjugates comprise single PEG molecules conjugated to single interferon molecules. In such an embodiment, the interferon alpha molecules can be selected from the group consisting of interferon alpha-2a, interferon alpha-2b, interferon alpha-2c and consensus interferon. In a preferred embodiment, the interferon molecules are interferon alpha-2b. Likewise, while the present invention is not limited to a specific PEG molecule, in one embodiment, the polyethylene glycol is PEG
12000
. In a particularly preferred embodiment, the interferon alpha-2b molecules are linked to the PEG
12000
molecules with a urethane bond.
While not limited to a specific characterization, when single interferon alpha molecules are linked to single polymer molecules, the present invention contemplates that the resulting PEG-interferon alpha conjugates can comprise a mixture of positional isomers. In a preferred embodiment, one of the positional isomers is an interferon alpha-2b molecule linked to a PEG
12000
molecule at a histidine residue on the interferon alpha-2b molecule.
The present invention also contemplates a process of lyophilization, comprising lyophilization of the formulations described above to create a lyophilized powder. In a preferred embodiment, the process further comprises reconstitution of the lyophilized powder with water or other aqueous diluents, such as benzyl alcohol-containing bacteriostatic water for injection, to create a
Moezie F. T.
Schering Corporation
Wyatt Donald W.
LandOfFree
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