Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
2000-04-06
2003-10-07
Weber, Jon P. (Department: 1651)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S219000
Reexamination Certificate
active
06630137
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of human medicine, particularly in the treatment of vascular disorders with activated protein C. More specifically, the present invention relates to formulations of activated human protein C.
BACKGROUND OF THE INVENTION
Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of homeostasis by inactivating Factors V
a
and VIII
a
in the coagulation cascade. Human protein C is made in vivo primarily in the liver as a single polypeptide of 461 amino acids. This single chain precursor molecule undergoes multiple post-translational modifications including 1) cleavage of a 42 amino acid signal sequence; 2) proteolytic removal from the one chain zymogen of the lysine residue at position 156 and the arginine residue at position 157 to make a 2-chain zymogen form of the molecule, (i.e., a light chain of 155 amino acid residues attached through a disulfide bridge to the serine protease-containing heavy chain of 262 amino acid residues); 3) vitamin K-dependent carboxylation of nine glutamic acid residues clustered in the first 42 amino acids of the light chain, resulting in nine gamma-carboxyglutamic acid residues; and 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain). The heavy chain contains the well established serine protease triad of Asp 257, His 211 and Ser 360. Finally, the circulating 2-chain zymogen is activated in vivo by thrombin at a phospholipid surface in the presence of calcium ion. Activation results from removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C (aPC) possessing enzymatic activity.
In addition to the enzymatic activities of aPC within the blood coagulation cascade, aPC also can autodegrade, leading to decreased functionality as an anticoagulant. Applicants have discovered an important degradation pathway. Autodegradation of the N-terminus of the light chain may result in a clip on either side of the histidine residue at position 10. Thus, this degradation pathway yields two inactive products: 1) des(1-9) activated protein C, wherein the first nine N-terminal residues of the light chain have been removed; and 2) des(1-10) activated protein C, wherein the first ten N-terminal residues of the light chain have been removed. This degradation pathway, which has not been previously reported, results in loss of anticoagulant activity due to the removal of the critical GLA residues at positions 6 and 7. Therefore, minimizing the level of the des(1-9) and des(1-10) activated Protein C autodegradation products is important in achieving a potent, high purity, activated protein C pharmaceutical formulation. These variants were previously unknown degradation products and are exceedingly difficult, if not impossible, to remove by conventional purification techniques. Applicants have further discovered that solid-state solubility is significantly enhanced in the presence of a select group of bulking agents.
It is clearly desirable to minimize such degradation of activated protein C in both the solution and lyophilized solid states. Accordingly, these discoveries allow the preparation of potent, high purity, activated protein C formulations which are pharmaceutically elegant to the health care provider.
The present invention provides improved formulations of activated protein C substantially free of such autodegradation products, particularly, des(1-9) and des(1-10) forms of the light chain of activated protein C. Therefore, said formulations are suitable for administration to a patient in need thereof.
SUMMARY OF THE INVENTION
The present invention provides a stable lyophilized formulation which may comprise, consist of, or consist essentially of activated protein C and a bulking agent selected from the group consisting of mannitol, trehalose, raffinose, sucrose, and mixtures thereof.
An aspect of the invention provides a lyophilized formulation consisting essentially of activated protein C; a salt; a bulking agent selected from mannitol, trehalose, raffinose, and sucrose, and mixtures thereof; and a buffer system such that upon reconstitution the resulting formulation has a pH between about 5.5 and about 6.1.
A preferred aspect of the invention provides a lyophilized formulation consisting of activated protein C; a salt; a bulking agent selected from mannitol, trehalose, raffinose, and sucrose, and mixtures thereof; and a buffer system such that upon reconstitution the resulting formulation has a pH between about 5.5 and about 6.0.
The present invention also provides a stable lyophilized formulation comprising, consisting of, or consisting essentially of about 2.5 mg/mL activated protein C, about 15 mg/mL sucrose, and about 20 mg/mL NaCl. Furthermore, the present invention provides a stable lyophilized formulation comprising, consisting of, or consisting essentially of about 5 mg/mL activated protein C, about 30 mg/mL sucrose, and about 38 mg/mL NaCl.
The present invention also provides a process for preparing a formulation comprising activated protein C and a bulking agent selected from the group consisting of. mannitol, trehalose, raffinose, and sucrose and mixtures thereof.
The invention also provides a unit dosage form comprising, consisting of, or consisting essentially of a unit dosage receptacle containing the formulation wherein the weight to weight ratio is about 1 part activated protein C, about 7.6 parts salt and about 6 parts bulking agent.
The invention further provides a method of treating disease states involving intravascular coagulation comprising the administration of a formulation of activated protein C described herein.
DETAILED DESCRIPTION OF THE INVENTION
For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below.
aPC or activated protein C refers to activated protein C whether recombinant or plasma derived. aPC includes and is preferably human activated protein C although aPC may also include other species or derivatives having protein C proteolytic, amidolytic, esterolytic, and biological (anticoagulant or pro-fibrinolytic) activities. Examples of protein C derivatives are described by Gerlitz, et al., U.S. Pat. No. 5,453,373, and Foster, et al., U.S. Pat. No. 5,516,650, the entire teachings of which are hereby incorporated by reference.
APTT—activated partial thromboplastin time.
r-hPC—recombinant human protein C zymogen.
r-aPC—recombinant activated protein C produced by activating protein C zymogen in vitro or in vivo or by direct secretion of the activated form of protein C from procaryotic cells, eukaryotic cells, or transgenic animals including, for example, secretion from human kidney 293 cells as a zymogen then purified and activated by techniques well known to the skilled artisan and demonstrated in Yan, U.S. Pat. No. 4,981,952, and Cottingham, WO 97/20043, the entire teachings of which are herein incorporated by reference.
Continuous infusion—continuing substantially uninterrupted the introduction of a solution into a blood vessel for a specified period of time.
Bolus injection—the injection of a drug in a defined quantity (called a bolus) at once.
Suitable for administration—a lyophilized formulation or solution that is appropriate to be given as a therapeutic agent.
Zymogen—protein C zymogen, as used herein, refers to secreted, inactive forms, whether one chain or two chains, of protein C.
Pharmaceutically acceptable buffer—a pharmaceutically acceptable buffer is known in the art. Pharmaceutically acceptable buffers include sodium phosphate, sodium citrate, sodium acetate, or TRIS.
Activated protein C is an antithrombotic agent with a wider therapeutic index than available anticoagulants, such as heparin and the oral hydroxycoumarin type anticoagulants. As an antithrombotic agent, aPC has a profound effect on the treatment of a wide variety of acquired disease states involving intravascular coagulation, including thrombotic stroke, deep vein thrombosis, pulmonary embolism, peripheral arterial thromb
Carlson Andrew David
Sheliga Theodore Arsay
Barrett Brian P.
Caltrider Steven P.
Eli Lilly and Company
Hostettler Danica
Weber Jon P.
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