Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-08-23
2001-08-28
Fonda, Kathleen K. (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S053000
Reexamination Certificate
active
06281202
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to compounds and methods associated with the epidermal growth factor (EGF) receptor. The invention is more particularly related to carbohydrates and carbohydrate analogs that bind to EGF receptors, and methods of use therefor (e.g., to kill or inhibit the growth of tumor cells with increased EGF receptor activity).
BACKGROUND OF THE INVENTION
Growth factors are substances that induce cell proliferation, typically by binding to specific receptors on cell surfaces. One such growth factor is epidermal growth factor (EGF). EGF induces proliferation of a variety of cells in vivo, and is required for the growth of most cultured cells.
The EGF receptor is a 170-180 kD membrane-spanning glycoprotein, which is detectable on a wide variety of cell types. The extracellular N-terminal domain of the receptor is highly glycosylated and binds EGF. The cytoplasmic C-terminal domain of the receptor contains an EGF-dependent tyrosine-specific protein kinase that is capable of both autophosphorylation and the phosphorylation of other protein substrates. The two domains are connected by a single 21 amino acid hydrophobic transmembrane region. The binding of EGF to its receptor activates the receptor tyrosine kinase, which phosphorylates a variety of cellular proteins (including the EGF receptor itself). This phosphorylation initiates a signal transduction pathway that ultimately leads to DNA replication, RNA and protein synthesis, and cell division. EGF also induces the concentration of the receptor into clathrin-coated pits, internalization into intracellular vesicles, and finally degradation in the lysosomes.
The amplification or overexpression of the EGF receptor is associated with the uncontrolled cell division of many cancers. Published studies support a role for the EGF receptor in cell transformation and maintenance of the transformed phenotype. There is also a high level of sequence homology between the EGF receptor and the avian v-erbB oncogene product. In addition, overexpression of the EGF receptor has been shown to result in the EGF-dependent transformation of NIH 3T3 cells.
Many tumors of mesodermal and ectodermal origin overexpress the EGF receptor. For example, the EGF receptor has been shown to be overexpressed in many gliomas, squamous cell carcinomas, breast carcinomas, melanomas, invasive bladder carcinomas and esophageal cancers. In addition, studies with primary human mammary tumors have shown a correlation between high EGF receptor expression and the presence of metastases, higher rates of proliferation, and shorter patient survival.
Attempts to exploit the EGF receptor system for anti-tumor therapy have generally involved the use of monoclonal antibodies against the EGF receptor. However, this approach has serious drawbacks. The monoclonal antibodies developed to date are effective inhibitors of cell growth in only some of the existing cancer cell lines. In addition, studies with athymic mouse xenografts suggest that therapeutic intervention with anti-receptor antibodies will require prolonged exposure, which may result in the generation of antibodies against the anti-receptor antibodies in the patient. Thus, to date, no successful anti-tumor therapies exploiting the EGF receptor system have been developed.
Accordingly, there is a need in the art for improved compounds and methods for treating EGF-receptor associated cancers. The present invention fulfills this need and provides further related advantages.
SUMMARY OF THE INVENTION
Briefly stated, this invention provides compounds and methods related to the epidermal growth factor (EGF) receptor. In one aspect of the invention, compounds are provided, having the formula X-Y-Z-R, wherein X is a sialic acid or a non-saccharide group that mimics the structure of a sialic acid group, Y and Z are independently a monosaccharide residue or a non-saccharide group that mimics the structure of a monosaccharide residue, and R is hydrogen or a carrier group, with the proviso that where X is a sialic acid group, Y and Z respectively are not the following: galactose and glucose, galactose and N-acetylglucosamine, galactose and galactose, galactose and N-acetylgalactosamine, sialic acid and sialic acid, sialic acid and galactose, N-acetylgalactosamine and galactose, N-acetylgalactosamine and N-acetylglucosamine, or N-acetylglucosamine and galactose. In an embodiment, X is a sialic acid group or a 2-hydroxyacetic acid group, Y is a galactose group or a cyclohexane group, Z is a glucose group, a cyclohexane group or an N-acetylglucosamine group, and R is hydrogen or a carrier group, with the proviso that where X is a sialic acid group and Y is a galactose group, then Z is not a glucose group or an N-acetylglucosamine group.
In a related embodiment, the present invention provides compositions comprising a liposome that includes a compound as described above or a carbohydrate compound having a type 2 saccharide chain without a sialic acid residue or an analog thereof, wherein said compound contains a carrier group. In an embodiment, the compound has the formula LNnT-C, wherein LNnT is a lacto-N-neotetraose group and C is a carrier group.
In another aspect, pharmaceutical compositions are provided, comprising: (a) a compound as described above or a carbohydrate compound having a type 2 saccharide chain without a sialic acid residue or an analog thereof; and (b) a pharmaceutically acceptable carrier or diluent. In an embodiment, the compound has the formula LNnT-C, wherein LNnT is a lacto-N-neotetraose group and C is a carrier group.
In another aspect of the invention, methods are provided for purifying an EGF receptor, comprising: (a) contacting a preparation containing an EGF receptor with a sialylated lactose carbohydrate compound, a carbohydrate compound having a type 2 saccharide chain without a sialic acid residue, or an analog of either, wherein said compound is immobilized on a solid support, and wherein said contacting takes place under conditions such that said EGF receptor binds to said immobilized compound; (b) separating the preparation from the immobilized compound, wherein the EGF receptor remains bound to the immobilized compound; and (c) isolating the bound EGF receptor from the immobilized compound.
In a further aspect of this invention, methods are provided for screening for a candidate molecule able to bind to an EGF receptor, comprising: (a) contacting an EGF receptor immobilized on a solid support with a candidate molecule and with a sialylated lactose carbohydrate compound, a carbohydrate compound having a type 2 saccharide chain without a sialic acid residue, or an analog of either; (b) separating unbound compound from the immobilized EGF receptor; and (c) detecting compound bound to the immobilized EGF receptor. In another embodiment, the method for screening comprises: (a) contacting a compound immobilized on a solid support with a candidate molecule and with an EGF receptor, wherein said compound is a sialylated lactose carbohydrate compound, a carbohydrate compound having a type 2 saccharide chain without a sialic acid residue, or an analog of either; (b) separating unbound EGF receptor from said immobilized compound; and (c) detecting EGF receptor bound to said immobilized compound, therefrom determining whether said candidate molecule binds to said EGF receptor.
In yet another aspect, this invention provides methods for inhibiting EGF receptor kinase activity comprising contacting an EGF receptor with a sialylated lactose carbohydrate compound, a carbohydrate compound having a type 2 saccharide chain without a sialic acid residue, or an analog of either. In an embodiment, the compound has the formula LNnT-R, wherein LNnT is a lacto-N-neotetraose group and R is hydrogen or a carrier group.
In a related aspect, methods are provided for inhibiting tumor cell growth within a biological preparation, comprising contacting a biological preparation with a sialylated lactose carbohydrate compound, a carbohydrate compound having a type 2 saccharide chain without a siali
Bremer Eric G.
Magnani John L.
Fonda Kathleen K.
GlycoTech Corp.
Seed Intellectual Property Law Group PLLC
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