Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
2001-02-22
2002-12-24
Eyler, Yvonne (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
C514S012200, C514S002600
Reexamination Certificate
active
06497869
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed toward aqueous compositions of granulocyte colony stimulating factor (G-CSF), particularly non-glycosylated G-CSF, having a pH of from about 5.0 to about 8.0, and a salt comprising sulfate ions at a concentration of from about 0.01M to about 1.0M.
The presence of the salt comprising sulfate ions unexpectedly stabilizes non-glycosylated G-CSF in an aqueous composition having a pH from about 5.0 to about 8.0.
BACKGROUND OF THE INVENTION
Colony stimulating factors induce proliferation, development and maturation of specific haematopoietic cells. G-CSF, in particular, leads to enhanced levels of circulating polymorphonuclear neutrophils (PMN), which play critical roles in the destruction of infectious agents. Human granulocyte colony stimulating factor (hG-CSF), for example, is used to stimulate haematopoiesis to protect patients undergoing bone marrow-suppressive chemotherapy against opportunistic infections.
G-CSF may also be used to afford similar protection for domesticated animals such as cattle, dogs, and cats. Infectious diseases in cattle and milk-producing cows, including shipping fever and bovine mastitis respectively, are a source of significant, persistent economic losses.
Shipping fever encompasses a collection of respiratory ailments afflicting cattle that are often detected in stressed animals within a population assembled from a number of different sources into a feed lot. The initial infections, generally caused by mycoplasma, chlamydia, bacteria, viruses, or mixtures thereof, are highly contagious and, although usually not lethal, they leave the animal in a debilitated state. Subsequent infection by another, opportunistic organism, especially
Pasteurella haemolytica
, is often the cause of mortality under these circumstances, rather than the initial infection. Bovine mastitis refers to an infection of the udders that may be caused by either gram negative or gram positive organisms. These infections are also highly contagious and may lead to a significant reduction in milk production by the affected cow, and, if scarring occurs, this loss may be permanent. Accordingly, methods and compositions that permit successful treatment of domesticated species with G-CSF could prevent or ameliorate the effects of such infectious diseases in such commercially important animals.
Mature bovine granulocyte colony stimulating factor, bG-CSF (GenBank Accession Number AF0925333), consists of 174 amino acids, of which 82% are identical with those in the corresponding human protein (GenBank Accession Number M17706). Both the human and bovine granulocyte colony stimulating factors are hydrophobic proteins, which include an odd number (five) of cysteine residues. Consequently, at least one of the cysteine side chains will present a free thiol moiety that may lead to the formation of untoward intramolecular and intermolecular disulfide linkages, resulting in the accumulation of insoluble, biologically inactive, dimeric or polymeric structures. This hypothesis has been proposed as one possible explanation for the observation that hG-CSF, bG-CSF and other G-CSF molecules, particularly recombinant, non-glycosylated forms produced in prokaryotic hosts, are difficult to formulate as stable, pharmaceutically acceptable compositions.
Glycosylated hG-CSF has been compared with de-glycosylated hG-CSF, prepared by in vitro enzymatic digestion with neuraminidase and endo-&agr;-N-acetylgalactosaminidase, with respect to its stability as a function of pH and temperature (Oh-eda et al., 1990,
J. Biol. Chem.
265 (20): 11432-35). The de-glycosylated hG-CSF, dissolved at a concentration of 1 &mgr;g/mL in 20 mM phosphate buffer containing 0.2 M NaCl and 0.01% Tween 20 was rapidly inactivated within the pH range of from about pH 7 to about pH 8 after a two-day incubation at 37° C. In contrast, glycosylated hG-CSF retained over 80% of its activity under the same conditions. Furthermore, evaluation of the thermal stability of both forms of hG-CSF, measured by biological assay and calorimetric analysis, indicated that de-glycosylated hG-CS F was less thermally stable than the native form of hG-CSF.
A number of approaches have been taken in order to provide stable, pharmaceutically acceptable G-CSF compositions. One approach to improving the composition stability of G-CSF involves the synthesis of derivatives of the protein. U.S. Pat. No. 5,665,863 to Yeh (the “'863 patent”) discloses the formation of recombinant chimeric proteins comprising G-CSF coupled with albumin, which have new pharmacokinetic properties. U.S. Pat. No. 5,824,784 to Kinstler et al. (the “'784 patent”) and U.S. Pat. No. 5,320,840 to Camble et al., (the “'840 patent”) disclose the chemical attachment of water-soluble polymers to proteins to improve stability and provide protection against proteolytic degradation. More specifically, the '784 patent discloses N-terminally modified G-CSF molecules carrying chemically attached polymers, including polyethylene glycol.
An alternative approach to increasing stability of G-CSF in composition involves alteration of the amino acid sequence of the protein. U.S. Pat. No. 5,416,195 to Camble et al. (the “'195 patent”) discloses genetically engineered analogues of G-CSF having improved composition stability, wherein the cysteine residue normally found at position 17 of the mature polypeptide chain, the aspartic acid residue found at position 27, and at least one of the tandem proline residues found at positions 65 and 66, are all replaced with a serine residue. Furthermore, U.S. Pat. No. 5,773,581 to Camble et al. (the “'581 patent”) discloses the genetically engineered G-CSF analogues of the '195 patent that have been covalently conjugated to a water soluble polymer.
Other approaches to improving the composition stability of G-CSF molecules have involved modification of the solvent in which the G-CSF is dissolved. U.S. Pat. No. 5,104,651 to Boone et al. (the “'651 patent”) discloses improved stability of G-CSF under conditions of low pH and minimal ionic strength. The '651 patent discloses a stabilized pharmaceutically acceptable composition consisting essentially of a pharmaceutically acceptable amount of G-CSF and acid, where the composition has a pH of 3.0 to 3.7 and a conductivity of less than 1000 &mgr;mhos/cm. In a preferred embodiment of this invention, no salt, other than a residual trace derived from the purification process, will be included in the composition.
U.S. Pat. No. 5,874,075 to Collins et al. (the “'075 patent”) discloses stable compositions of proteins, including G-CSF, comprising a liposome vesicle composed of negatively charged phospholipids, where only a portion of the protein is inserted into the lipid portion of the vesicle. The '075 patent also discloses compositions comprising liposome vesicles combined with G-CSF that has been covalently linked with polyethylene glycol.
A stable G-CSF containing composition is disclosed in GB 2193621 A (the “'621 application), which comprises at least one substance selected from the group consisting of a pharmaceutically acceptable surfactant, saccharide, protein and a high-molecular weight compound. Suitable high-molecular weight compounds include hydroxypropyl cellulose, hydroxymethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone. Proteins deemed useful in the compositions of the '621 application include human serum albumin, human serum globulin, gelatin, acid-treated gelatin, and alkali-treated gelatin.
U.S. Pat. No. 5,503,827 to Woog et al. (the “'827 patent”) discloses pharmaceutical preparations of G-CSF that include at least one bactericidal preservative selected from the group consisting of chlorobutanol, benzyl alcohol, benzalkonium chloride and mixtures thereof. Pharmaceutical preparations of the '827 patent may also include auxiliary substances, examples of which are stabilizing agents and organic hydrophilic polymers. Useful stabilize
Hay Josephine Nanette
Williams Kathleen Brimelow
Benson Gregg C.
Eyler Yvonne
Lee Christine S.
Li Ruixiang
Pfizer Inc.
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