Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-04-09
2001-10-09
Ungar, Susan (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387900, C530S389100, C530S389200
Reexamination Certificate
active
06300476
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an anti-peptide antibody raised against a portion of human cytochrome P450 3A4. A particular anti-peptide antibody was raised against a 21-amino acid peptide corresponding to residues 253-273 of human cytochrome P450 3A4, which is both specific to CYP3A4 and effectively inhibits CYP3A4 activity.
BACKGROUND OF THE INVENTION
The use of in vitro metabolism of therapeutic agents to address the potential in vivo induction, inhibition, drug-drug interaction and individual variability issues is known (for a recent review, see Rodrigues, 1994,
Biochem. Pharmacol.
48: 2147-2156). Central to these studies is the unambiguous identification of specific drug-metabolizing enzyme(s), particularly human cytochrome P450 isoform(s) responsible for the metabolism of drugs. This objective can be achieved by using selective cytochrome P450 inhibitors, antibodies, recombinant cytochrome P450s and correlation analysis (Rodrigues, 1994,
Biochem. Pharmacol.
48: 2147-2156).
Polyclonal or monoclonal antibodies produced against purified cytochrome P450 or specific peptide sequences unique to individual cytochrome P450 isoforms have been used to study the regulation, structure and function of cytochrome P450s.
Leeder et al. (1996,
Mol. Pharmacol.
49: 234-243) used epitope mapping studies to identify a minimum antibody binding sequence in CYP3A1 located in the K-helix of the protein.
Gelboin et al. (1995,
Biochemical Pharmacology
50(11):1841-1850) disclose a monoclonal antibody which is inhibitory to human cytochrome P450 3A3/4/5.
Cribb et al. (1995,
Drug Metabolism and Disposition
23(7): 671-675) disclose antipeptide antibodies against two overlapping synthetic peptides from human CYP2D6. Antipeptide antibodies against one of these synthetic peptides substantially inhibited recombinant human CYP2D6 activity.
Despite these advances, there remains a substantial need for specific and inhibitory antibodies to members of the human cytochrome P450 class of enzymes, especially to human CYP3A4. The present invention addresses and meets this need.
SUMMARY OF THE INVENTION
The present invention relates to antibodies against human CYP3A4 which are specific to human CYP3A4 in relation to other human P450 enzymes and which also substantially inhibit human CYP3A4 enzyme activity.
The present invention particularly relates to anti-peptide antibodies raised against a portion of the human CYP3A4 protein which are specific to human CYP3A4 in relation to other human P450 enzymes and which also substantially inhibit human CYP3A4 enzyme activity.
A preferred aspect of the present invention relates to anti-peptide antibodies raised against a 21 amino acid peptide corresponding to amino acid 253-273 of human CYP3A4, which is Val Lys Arg Met Lys Glu Ser Arg Leu Glu Asp Thr Gln Lys His Arg Val Asp Phe Leu Gln (SEQ ID NO:1).
Another embodiment of the present invention relates to antibodies raised against a peptide wherein a cysteine residue has been introduced at the amino terminus of SEQ ID NO:1, resulting in Cys Val Lys Arg Met Lys Glu Ser Arg Leu Glu Asp Thr Gln Lys His Arg Val Asp Phe Leu Gln (SEQ ID NO:2).
A preferred embodiment of the present invention relates to antibodies raised against the peptide of SEQ ID NO:2 which has been coupled to keyhole limpet haemocyanin.
Another embodiment of the present invention relates to antibodies raised against the inhibitory epitope which is contained within the 21 amino acid region of SEQ ID NO:1. A specific embodiment of this portion of the invention are antibodies raised against the inhibitory epitope within SEQ ID NO:1, which preferably is Arg Leu Glu Asp Thr Gln Lys His Arg (SEQ ID NO:3). It is also within the scope of this portion of the invention to raise antibodies against a peptide wherein a cysteine residue has been introduced at the amino terminus of SEQ ID NO:3, resulting in Cys Arg Leu Glu Asp Thr Gln Lys His Arg (SEQ ID NO:4) and to couple this peptide to keyhole limpet haemocyanin prior to immunization.
Another especially preferred embodiment of the present invention relates to antibodies raised against the inhibitory epitope contained within the 21 amino acid region of SEQ ID NO:1. This epitope comprises the middle portion of SEQ ID NO:1, preferably Leu Glu Asp Thr Gln Lys His (SEQ ID NO:9). As with all disclosed peptides within this specification, it is preferable to introduce a cysteine residue at the amino terminus (Cys Leu Glu Asp Thr Gln Lys His (SEQ ID NO:15) and to couple this peptide to keyhole limpet haemocyanin prior to immunization to generate anti-peptide antibodies of the present invention.
Another especially preferred embodiment of the present invention relates to antibodies raised against the inhibitory epitope contained within the 21 amino acid region of SEQ ID NO:1. This epitope comprises the middle portion of SEQ ID NO:1, preferably Glu Asp Thr Gln Lys His (SEQ ID NO:10). As with all disclosed peptides within this specification, it is preferable to introduce a cysteine residue at the amino terminus (Cys Glu Asp Thr Gln Lys His (SEQ ID NO: 16) and to couple this peptide to keyhole limpet haemocyanin prior to immunization to generate anti-peptide antibodies of the present invention.
An additional embodiment of the present invention relates to antibodies raised against peptides which comprise at least a portion of the peptide comprising the inhibitory epitope of SEQ ID NO:3. Such antibodies may be raised against peptides which include, but are not necessarily limited to, Val Lys Arg Met Lys Glu Ser Arg Leu Glu Asp Thr Gln Lys His Arg Val Asp (SEQ ID NO:5); Val Lys Arg Met Lys Glu Ser Arg Leu Glu Asp Thr Gln Lys His (SEQ ID NO:6); Leu Glu Asp Thr Gln Lys His Arg Val Asp (SEQ ID NO:7); and, Lys Glu Ser Arg Leu Glu Asp Thr Gln Lys His (SEQ ID NO:8). As disclosed above for antibodies raised against SEQ ID NOS: 1 and 2, it is preferable that the antibodies of this portion of the invention to raise antibodies against theses peptides wherein a cysteine residue has been introduced at the amino terminus (i.e., SEQ ID NOS: 11-14, respectively) of the synthetic peptide and this peptide has been coupled to keyhole limpet haemocyanin prior to immunization.
Especially preferred antibodies are anti-peptide antibodies raised against the synthetic peptide or protein fragments disclosed in SEQ ID NO:3, SEQ ID NO:9 and SEQ ID NO:10 and their respective cysteine-modified forms, disclosed as SEQ ID NOS:4, 15 and 16.
The anti-peptide antibodies of the present invention are specific to human CYP3A4 in relation to other human P450 enzymes and show an ability to inhibit at least about 80% of human CYP3A4 activity.
A preferred anti-peptide antibody of the present invention is specific to human CYP3A4 in relation to other human P450 enzymes and shows an ability to inhibit at least about 90% of human CYP3A4 activity.
An especially preferred anti-peptide antibody of the present invention is specific to human CYP3A4 in relation to other human P450 enzymes and shows an ability to inhibit at least about 90% to about 95% of human CYP3A4 activity.
The present invention relates to a synthetic peptide or protein fragment which is useful for generating antibodies against human CYP3A4 which are specific in relation to other human P450 enzymes and which also substantially inhibit human CYP3A4 enzyme activity.
A preferred embodiment of the present invention relates to a 21 amino acid synthetic peptide or protein fragment corresponding to amino acid 253-273 of human CYP3A4, which is Val Lys Arg Met Lys Glu Ser Arg Leu Glu Asp Thr Gln Lys His Arg Val Asp Phe Leu Gln (SEQ ID NO:1).
Another aspect of the present invention relates to a cysteine-modified version of SEQ ID NO:1, which is Cys Val Lys Arg Met Lys Glu Ser Arg Leu Glu Asp Thr Gln Lys His Arg Val Asp Phe Leu Gln (SEQ ID NO:2). Additionally, this portion of the invention relates to the peptide of SEQ ID NO:2 which has been coupled to keyhole limpet haemocyanin.
Another embodiment of the present invention relates to a synthetic peptide or protein fragment comprising the inhibit
Lu Anthony Y. H.
Wang Regina W.
Cocuzzo Anna L.
Merck & Co. , Inc.
Tribble Jack L.
Ungar Susan
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