MXPA00007209A - Formulations for protection of peg-interferon alpha conjugates - Google Patents

Abstract

The present invention provides formulations that prevent loss and damage of PEG-interferon alpha conjugates during and following lyophilization. The formulations of the present invention protect PEG-interferon alpha conjugates from loss and degradation during the lyophilization process, as well as degradation during subsequent storage. The formulations of the present invention are suitable for protection of PEG-interferon alpha conjugates from various types of degradation, including, but not limited to loss of biological activity and changes in the degree and/or nature of conjugation. A preferred PEG-interferon alpha conjugate protectable in the formulations of the present invention is an interferon alpha-2b-polyethylene glycol (12,000) conjugate.

Description

FORMULATIONS FOR THE PROTECTION OF CONJUGATES OF PEG- INTERFERON ALPHA Throughout this description, reference is made to various publications, patents and patent applications. The description of these publications, patents and patent applications are incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates to formulations for the stabilization of PEG-alpha interferon conjugates during and after lyophilization, their production and use.

BACKGROUND OF THE INVENTION Several natural and recombinant proteins possess pharmaceutical utility. Once they have been purified, separated and formulated, they can be administered parenterally for various therapeutic indications. However, proteins administered parenterally may be immunogenic, may be relatively insoluble in water, and may have a short pharmacological lifespan. Consequently, it may be difficult to achieve therapeutically useful blood levels of the proteins in patients. These problems can be overcome by conjugating the proteins with polymers, such as polyethylene glycol. Davis et al., US Pat. No. 4,179,337 describes the conjugation of polyethylene glycol (PEG) with proteins such as enzymes and insulin, to obtain conjugates that have less immunogenic effect than the original proteins, but nevertheless still retain a substantial proportion of their physiological activity. Veronese et al., (Applied Biochem. And Biotech., 11: 141-152, 1985) describe the activation of polyethylene glycols with phenyl chloroformates, to modify a ribonuclease and a superoxide dimutase. Katre et al., U.S. Patent Nos. 4,766,106 and 4,917,888 also describe the solubilization of proteins by polymer conjugation. Likewise, PEG and other polymers can be conjugated with recombinant proteins to reduce immunogenicity and increase the life span. See Nitecki et al., U.S. Patent No. 4,902,502, Enzon, Inc., International Application No. PCT / US90 / 02133, Nishimura et al., European Patent Application 154,316 and Tomasi, International Application No. PCT / US85 / 02572. For example, it is known that interferon alfa-2b is effective for the treatment of disease states such as renal cell carcinoma, AIDS-related Kaposi's sarcoma, chronic and acute hepatitis B, chronic and acute hepatitis A, not B / C, and hepatitis C. The improvement of the pharmacological lifespan of interferon alfa-2b would improve the treatment of these conditions. While the preparation of protein-polymer conjugates is beneficial, they can not be used in a practical way unless they can be stored for a prolonged period of time during manufacture and distribution to health care providers. Some protein-polymer conjugates, however, deteriorate easily, even in frozen solutions. Lyophilization (also known as freeze drying) is a process that can turn a pharmacist into a form that can overcome this deficiency. Lyophilization is a process by which water is sublimated from a composition after it is frozen. In this process, pharmaceuticals and biologics that are relatively unstable in an aqueous solution over a period of time can be placed in dosing containers in an easily processed liquid state, dried without the use of harmful heat and stored in a dried state for periods of time. prolonged Due to the low total mass of active substance in each dose, most pharmaceutical and biological formulations including protein-polymer conjugates require additional ingredients to protect the active ingredient during the lyophilization process. For example, a pharmaceutical contained in a dosing container as an aqueous solution of low concentration may be susceptible to physical loss during the vacuum process of lyophilization or adsorption to the container. A lyophilized formulation often contains dough ingredients that increase the amount of solid material, as well as cryoprotectants, lyoprotectants and other stabilizers to protect the active component against damage. However, the determination of which particular formulation will protect a particular type of pharmacist must be empirical. There is a current need for a suitable formulation to protect protein-polymer conjugates, and in particular PEG-interferon alpha conjugates, against damage during lyophilization. Such a formulation should allow the PEG-interferon alpha polymer conjugates to maintain their biological activity, physical stability and chemical stability for extended periods of time.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides formulations that allow the 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 further contemplates a process for preparing stable, aqueous formulation solutions comprising mixing 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 top space of inert atmosphere is maintained over the formulation at a value 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 derivative of poly (oxy-1,2-ethanediyl), the cryoprotectant is sucrose and the solvent is water. In such an embodiment, the sodium phosphate may comprise anhydrous dibasic sodium phosphate with monobasic sodium phosphate dihydrate. The present invention is furthermore not limited to 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) derivative. 1, 2-ethanediyl) is preferably 0.01 to 1.0 mg / ml, and the sucrose concentration 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 dibasic sodium phosphate 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 derivative of poIi (oxy-1,2-ethanediyl) is 0.05 mg, and the volume of water is 0.5 ml. Alternatively, the component ratio is 0.08% of said conjugates of PEG-interferon alpha, as measured by the mass of interferon alpha, 3.6% of sodium phosphate, 0.12% of derivative of poly (oxy-1,2-ethanediyl), and 96.2% 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 simple PEG molecules conjugated with simple interferon molecules. In such an embodiment, interferon alpha molecules can be selected from the group consisting of interferon alpha-2, interferon alpha-2b, interferon alpha-2c and consensus interferon. In a preferred embodiment, the interferon molecules are interferon alpha-2b. Also, while the present invention is not limited to a specific PEG molecule, in one embodiment, polyethylene glycol is PEG? 20- In a particularly preferred embodiment, the interferon alpha-2b molecules are linked to the PEG molecules. - | 2ooo with a urethane bond. While not limited to a specific characterization, when linking simple alpha interferon molecules to simple polymer molecules, the present invention contemplates that the resulting PEG-interferon alpha conjugates may comprise a mixture of positional isomers. In a preferred embodiment, one of the positional isomers is an interferon alpha-2b molecule linked to a PEG12000 molecule in a histidine residue in the interferon alpha-2b molecule. The present invention further contemplates a lyophilization process, comprising the lyophilization of the formulations described above, to create a lyophilized powder. In a preferred embodiment, the process further comprises reconstituting the lyophilized powder with water or other aqueous diluents, such as bacteriostatic water with benzyl alcohol for injection, to create a reconstituted solution (bacteriostatic water for injection, Abbott Laboratories, Abbott Park, IL). The present invention further contemplates lyophilized powders produced by lyophilization of the formulations described above. In a preferred embodiment, the lyophilized powder comprises 0.08% of said PEG-interferon alpha conjugates, 3.6% of said sodium phosphate, 0.12% of said poly (oxy-1,2-ethanediol) derivative and 96.2% of said sucrose. , in weigh. Also, articles of manufacture comprising a syringe or a container containing an effective amount of said lyophilized powders are contemplated. In a preferred embodiment, the article of manufacture further comprises a volume of water for reconstitution of the powder. In a particularly preferred embodiment, the powder is reconstituted with bacteriostatic water. In another preferred embodiment, the lyophilized powder is reconstituted with the same volume of water that was removed from the lyophilization solution during lyophilization. The present invention also contemplates processes for the treatment of diseases in animals. In one embodiment, this process comprises introducing the reconstituted solution into an animal that has a disease. In one modality, the animal is human. In a preferred embodiment, the human being is infected with a hepatitis virus, such as hepatitis C virus. In a preferred embodiment alternatively, the human being has cancer.

DETAILED DESCRIPTION OF THE INVENTION "PEG-interferon alpha conjugates" are alpha interferon molecules covalently adhered to a PEG molecule. In preferred embodiments, the PEG-interferon alpha conjugates of the present invention comprise interferon alpha-2a (Roferon, Hoffman La-Roche, Nutley, NJ), interferon alpha-2b (Intron, Schering-Plow, Madison, NJ), interferon alfa-2c (Berofor Alpha, Boehringer Ingelheim, Ingelheim, Germany), or consensus interferon as defined by the determination of a natural interferon alpha consensus sequence (Infergen, Amgen, Thousand Oaks, CA). Polymers, on the other hand, are molecules that have repeating chemical units covalently adhered. Often, the approximate molecular weight of the polymer is designated by a number following the name of the chemical unit repeated. For example, "PEG12000" or "polyethylene glycol (12,000)" refers to a polyethylene glycol polymer having an average molecular weight of about 12,000. In a PEG-2ooo polymer > the number of polyethylene glycol units repeated in the polymer is approximately 273. It is understood that these designations are approximate, since the polymers are manufactured in the form of a mixture having a distribution of chain lengths, providing an average molecular weight, and often it is impossible to manufacture a polymer having a precise and uniform molecular weight or number of repeating units. Various other polymers and their production methods are well known in the art. Methods for creating protein-polymer conjugates are well known in the art. For example, U.S. Patent 5,691, 154, issued to Callstrom et al., U.S. Patent No. 5,686,071, issued to Subramanian et al., U.S. Patent No. 5,639,633, issued to Callstrom et al., U.S. Patent No. 5,492,821, issued to Callstrom et al., U.S. Patent No. 5,447,722 issued to Lang et al., And U.S. Patent No. 5,091,176, issued to Braatz et al., All provide methods for the production of protein conjugates. polymer. The conjugation of polymers with proteins can produce a simple polymer molecule conjugated with a protein, or multiple such conjugations with a simple protein. The degree of conjugation depends on the reaction conditions and the desired result. In a preferred embodiment, the PEG-interferon alpha conjugate in the formulations of the present invention comprises a simple alpha2-b interferon conjugated to a simple PEG12000. In a particularly preferred embodiment, the interferon alpha-2b molecule is linked to the PEG-2ooo molecule with a urethane linkage. Reagents and methods for the production of this protein-polymer conjugate can be found in U.S. Patent No. 5,612,460, issued to Zalipsky, and in U.S. Patent No. 5,711, 944, issued to Gilbert et al. When said protein-polymer conjugate is used in the formulations solutions of the present invention, the preferred concentration of PEG-interferon alpha conjugate is 0.03 to 2.0 mg interferon alpha per ml. When a simple interferon alpha molecule is linked to a simple polymer molecule, the resultant PEG-interferon alpha conjugates can be in the form of a single positional isomer, or in a mixture of positional isomers. A "mixture of positional isomers" indicates that the individual PEG-interferon alpha conjugates may be linked to different sites on different alpha interferon molecules. For example, in one embodiment of the present invention, the PEG-interferon alpha mixture contains at least one PEG-interferon-alpha conjugate linked to a histidine residue of the interferon-alpha molecule, while another conjugate of PEG-interferon alpha is bound in another site of the interferon alpha molecule (for example, the term amino). As described above, the preservation of PEG-interferon alpha conjugates can be achieved by lyophilization. Lyophilization is a freeze drying process of a composition wherein a frozen aqueous mixture is treated to remove water. Commonly, the process involves the sublimation of water from frozen aqueous solutions, usually under reduced pressure conditions. After lyophilization, the PEG-interferon alpha conjugate can be stored for extended periods of time. However, the PEG-interferon alpha conjugates are subject to damage during and after lyophilization. Damage to PEG-interferon alpha conjugates can be characterized by protein loss, loss of biological activity, or by the change in the degree and / or nature of alpha interferon conjugation. For example, a PEG-interferon alpha conjugate can be degraded into interferon alpha and free PEG, resulting in a decrease in the degree of conjugation. Likewise, the resulting free PEG may become available for conjugation with another interferon alpha, potentially producing an increase in the degree of conjugation in this target molecule. Likewise, a PEG-interferon alpha conjugate can undergo an intramolecular change of PEG from one conjugation site to another within the same molecule, thus changing the nature of alpha interferon conjugation. The present invention protects PEG-interferon alpha conjugates against damage, including them in formulations that prevent damage during and after lyophilization. While the present invention is not limited to a particular formulation, in a preferred embodiment, the method uses a buffer, stabilizer, cryoprotectant and solvent, in addition to the PEG-interferon alpha conjugate. The buffers are suitable for maintaining the pH of the formulation in a range of 4.5 to 7.1, preferably 6.5-7.1, and more preferably 6.8.

The use of a sodium phosphate dibasic and monobasic phosphate buffer system is preferred. When an anhydrous / monobasic dibasic sodium phosphate dihydrate system is used, it is preferably in mass quantities of dibasic to monobasic, at a preferred total concentration of 0.005 to 0.1 molar. Other buffer systems suitable for maintaining the desired pH range include sodium citrate / citric acid and sodium acetate / acetic acid. A stabilizing agent is useful to prevent adsorption of the PEG-interferon alpha conjugate to the stainless steel and glass surfaces of the equipment using to make and store the formulations containing the PEG-interferon alpha conjugate. As an example, the poly (oxy-1,2-ethanediyl) derivatives are useful as stabilizing agents. The mono-9-octadecenoate poly (oxy-1,2-ethanediyl) derivatives (Polysorbate 80) constitute a preferred stabilizing agent. When polysorbate 80 is used, the preferred construction is 0.01 to 1 mg / ml. Cryoprotectants, also known as cryoprotective agents or compounds, are agents that protect chemical compounds, cells or tissues against the deleterious effects of freezing, such as those that usually accompany lyophilization. In the case of PEG-interferon alpha conjugates, cryoprotectants can protect them against damage, adsorption and loss of vacuum used in lyophilization. While the present invention is not limited to a specific cryoprotectant, examples include, but are not limited to, carbohydrates such as saccharides, sucrose, sugar alcohols such as mannitol, surface active agents such as Tweens, as well as glycerol. and dimethylsulfoxide. A preferred cryoprotectant is a carbohydrate. A preferred carbohydrate is a saccharide or disaccharide. A preferred disaccharide is sucrose. Also, the present invention is not limited to any particular amount of cryoprotectant used. In one embodiment, the cryoprotectants are present in an amount sufficient to allow the PEG-interferon alpha conjugate to be lyophilized. In such embodiment, the cryoprotectants may be present in an amount of 0.05% to 90%, preferably 0.05% -50%, and more preferably in an amount of about 0.15% to about 10%, based on the total weight of the PEG-interferon alpha solution. When sucrose is used, the preferred concentration is 20 to 100 mg / ml. Formulations that include an effective amount of biologically active PEG-interferon alpha conjugates are useful in the treatment of disease states, preferably as aqueous injectable solutions. An effective amount means that the formulation or powder has an adequate concentration of biologically active component to treat a disease state in an animal. For example, the preferred interferon a! Fa-2b-PEG? 2ooo conjugates are suitable for the treatment of disease states such as renal cell carcinoma, AIDS-related Kaposi's sarcoma, chronic and acute hepatitis B, chronic hepatitis and water. not A, not B / C, and hepatitis C. A solution containing an effective amount of this PEG-interferon alpha conjugate contains 0.03 to 2.0 mg / ml of PEG-2ooo-interferon-alpha-2b conjugate, measured by mass protein EXAMPLE This example provides a description of a formulation of the present invention and the protection of a PEG-interferon alpha conjugate during lyophilization and storage. The PEG-interferon alpha conjugate is introduced into a lyophilization formulation, is lyophilized and stored as a dry powder. The components of the formulation are the following: TABLE 1 Formulation for lyophilization and storage Component Mq / vial * Interferon alfa-2b-PEG? 200o 0.1 / *** Dibasic sodium phosphate anhydrous 0.75 Sodium phosphate monobasic dihydrate 0.75 Sucrose 40 Polysorbate 80 0.05 Water for injection (qs ad) 0.5 ml ** Quantity contained in volume of label of 0.5 ml. * Water is sublimated during lyophilization. ** Based on the protein mass After lyophilization, the resulting powder is stored, and after a period of six months, the samples are reconstituted with water for analysis. The reconstituted solution is analyzed for determination of protein mass content, conjugate degree of PEG-interferon alpha conjugate, bioactivity and visual clarity. The results are presented in table 2.

TABLE 2 Stability Information Stability information in vial of 100 μg s \ * Label filling is 0.5 ml / vial ** CCS: White powder; after reconstruction, a clear, colorless solution, essentially free of visible particles.

The results show that the content of total protein mass is relatively stable during the nine-month period. Additionally, the change in the degree of monopegylated interferon alpha-2b (ie, degradation to free and free interferon polymer, or creation of dipegylated interferon) is negligible. The bioactivity measured by a cell-based antiviral assay remains essentially unchanged. The reconstituted solutions remain clear, colorless, and free of visible particles throughout the six-month period. This demonstrates surprisingly high stability during lyophilization and subsequent storage. From the foregoing, it is clear that the present invention provides suitable formulations to protect conjugates of PEG-interferon alpha against one year during lyophilization and during subsequent storage.

Claims (23)

NOVELTY OF THE INVENTION CLAIMS

1. - An aqueous formulation comprising conjugates of

PEG-interferon alfa, a buffer, a stabilizer, a cryoprotectant and a solvent. 2. The formulation of claim 1, wherein said buffer is sodium phosphate, said stabilizer is a derivative of po! I (oxy-1,2-ethanediyl), said cryoprotectant is sucrose and said solvent is water.

3. The formulation of claim 2, wherein said sodium phosphate comprises anhydrous dibasic sodium phosphate and monobasic sodium phosphate dihydrate.

4. The formulation of claim 2, wherein the concentration of said PEG-interferon alpha conjugates is 0.03 to 2.0 mg interferon alpha per ml, the concentration of said sodium phosphate is 0.005 a 0. 1 molar, the concentration of said poly (oxy-1,2-ethanediyl) derivative is 0. 01 to 1.0 mg / ml, and the concentration of said sucrose is 20 to 100 mg / ml.

5. The formulation of claim 3, wherein the mass of said PEG-interferon alpha conjugates is 0.1 mg of interferon alpha, the mass of said dibasic sodium phosphate anhydrous is 0.75 mg, the mass of said monobasic sodium phosphate. Dihydrate is 0.75 mg, the mass of said sucrose is 40 mg, the mass of said poly (oxy-1, 2-ethanediol) derivative is 0.05 mg, and the volume of said water is 0.5 ml.

6. The formulation of claim 5, wherein said PEG-interferon alpha conjugates comprise simple PEG molecules conjugated with simple alpha interferon molecules.

7. The formulation of claim 6, wherein said alpha interferon molecules are selected from the group consisting of interferon alfa-2a, interferon alfa-2b, interferon alfa-c and consensus interferon.

8. The formulation of claim 7, wherein said polyethylene glycol is PEG12000.

9. The formulation of claim 8, wherein said interferon alpha molecules are interferon alpha-2b.

10. The formulation of claim 9, wherein said interferon alpha-2b molecules are linked to said PEG? 2ooo molecules with a urethane linkage.

11. The formulation of claim 10, wherein said PEG-interferon alpha conjugates comprise a mixture of positional isomers.

12. The formulation of claim 11, wherein one of said positional isomers comprises said interferon alpha-2b molecule linked to said PEG200o molecule in a histidine residue in said interferon alpha-2b molecule.

13. A lyophilization process, comprising freeze-drying the formulation of claim 12 to create a lyophilized powder.

14. - The process of claim 13, further comprising reconstituting the lyophilized powder with water, to create a reconstituted solution.

15. The process of claim 14, wherein said water comprises bacteriostatic water.

16. A lyophilized powder, produced by lyophilization of the formulation of claim 12.

17. The lyophilized powder of claim 16, wherein said powder comprises 0.08% of said PEG-interferon alpha conjugates, as measured by the mass of alpha interferon, 3.6% of said sodium phosphate, 0.12% of said poly (oxy-1,2-ethanediyl) derivative, and 96.2% of said sucrose, by weight.

18. An article of manufacture, comprising a syringe containing an effective amount of the powder of claim 16.

19. The article of manufacture of claim 18, further comprising a volume of water for reconstitution of said powder.

20. The article of manufacture of claim 19, wherein said water comprises bacteriostatic water.

21. An article of manufacture comprising a vial containing an effective amount of the powder of claim 16.

22. The article of manufacture of claim 21, further comprising a volume of water for reconstitution of said powder.

23. - The article of manufacture wherein said water comprises bacteriostatic water.