Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-09-28
2003-09-09
Prouty, Rebecca E. (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C530S350000, C530S388900, C435S252300, C435S320100
Reexamination Certificate
active
06617450
ABSTRACT:
FIELD OF THE INVENTION
The invention pertains to P-glycoproteins of cynomologous monkey (
Macaca fascicularis
).
BACKGROUND OF THE INVENTION
P-glycoprotein (PGP; also known as multidrug transporter, MDR1) is a member of the ABC transporter superfamily and is expressed in the human intestine, liver and other tissues. This enzyme serves as an efflux pump exporting small molecules across the cell membrane. It has been known for several years that high level expression of PGP is a mechanism for tumor resistance to cancer chemotherapy. Intestinal expression of PGP may affect the oral bioavailability of drug molecules that are substrates for this transporter. PGP can efficiently efflux drugs back into the intestinal lumen and thus reduce the amount of drug that enters into circulation.
The measurement of interaction with PGP can provide a better understanding of the reasons why particular drugs demonstrate low or high bioavailability. Interaction with PGP can be studied using either direct assays of drug transport in polarized cell systems or with indirect assays such as drug-stimulated ATPase activity and inhibition of the transport of fluorescent substrates.
Therefore there is a need for additional PGP polypeptides, preferably which are closely related to the human PGP, for use in the foregoing drug assays.
SUMMARY OF THE INVENTION
Nucleic acids encoding the P-glycoprotein of cynomologous monkey (
Macaca fascicularis
) have now been identified, isolated, cloned and sequenced. This PGP is closely related (has a high degree of identity) to the human PGP. The invention provides isolated nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated polypeptides and inhibitors of the foregoing nucleic acids and polypeptides which reduce drug transport. The PGP nucleic acids and polypeptides are useful in assays for evaluating bioavailability of drugs, as well as for the optimization or discovery of drugs. In addition, the foregoing can be used in the diagnosis or treatment of conditions characterized by PGP activity and can be used in methods in which it is therapeutically useful to increase or decrease PGP activity.
According to one aspect of the invention, isolated nucleic acid molecules are provided selected from the group consisting of (a) nucleic acid molecules that code for the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, (b) allelic variants of (a), and (c) complements of (a) or (b). In certain embodiments, the isolated nucleic acid molecule codes for SEQ ID NO:2 or SEQ ID NO:4. In other embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4.
According to another aspect of the invention, isolated P-glycoprotein polypeptides or fragments thereof are provided which include at least one amino acid of a cynomologous P-glycoprotein selected from the group consisting of amino acids 12, 24, 30, 74, 78, 86, 89, 90, 91, 92, 95, 97, 99, 102, 103, 104, 185, 324, 363, 518, 635, 650, 656, 659, 677, 730, 738, 742, 745, 761, 765, 835, 851, 921, 967, 1003, 1027, 1038, 1048, 1103, 1128, 1168 and 1277 of SEQ ID NO:2 and amino acids 93, 94 and 95 of SEQ ID NO:4, wherein the P-glycoprotein is identical to a human P-glycoprotein except for the at least one amino acid of a cynomologous P-glycoprotein. In certain embodiments, the human P-glycoprotein is selected from the group of SEQ ID NO:5 and SEQ ID NO:6.
According to yet another aspect of the invention, isolated P-glycoprotein polypeptides or fragments thereof which include at least one amino acid of a cynomologous P-glycoprotein selected from the group consisting of amino acids 3, 6, 8, 10, 13, 17, 19, 20, 21, 26, 30, 36, 38, 48, 52, 56, 64, 74, 78, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 100, 101, 102, 103, 104, 105, 106, 110, 113, 145, 190, 197, 210, 231, 319, 324, 327, 345, 363, 395, 451, 455, 456, 468, 473, 494, 518, 530, 631, 641, 642, 648, 650, 655, 656, 664, 665, 672, 673, 674, 675, 683, 687, 689, 691, 692, 694, 701, 705, 715, 729, 730, 734, 742, 743, 745, 754, 757, 765, 835, 912, 918, 921, 940, 941, 944, 966, 967, 968, 970, 972, 981, 1008, 1015, 1023, 1024, 1048, 1093, 1096, 1103, 1128, 1142, 1146, 1147, 1156, 1160, 1163, 1166, 1250 and 1271 of SEQ ID NO:2 and amino acids 93 and 94 of SEQ ID NO:4, wherein the P-glycoprotein is identical to a dog P-glycoprotein except for the at least one amino acid of a cynomologous P-glycoprotein. In some embodiments, the dog P-glycoprotein is selected from the group of SEQ ID NO:7 and SEQ ID NO:8.
In preferred embodiments, the isolated P-glycoprotein polypeptides or fragments thereof include an amino acid sequence selected from the group consisting of SEQ ID NO:2, fragments of SEQ ID NO:2, SEQ ID NO:4 and fragments of SEQ ID NO:4. Yet other polypeptides include combinations of the foregoing dog, human and cynomologous PGP polypeptides.
According to still other embodiments of the invention, isolated nucleic acid molecules are provide which encode the foregoing isolated P-glycoprotein polypeptides or fragments thereof. Also included expression vectors comprising the foregoing isolated nucleic acid molecules operably linked to a promoter, as well as host cells transformed or transfected with the expression vectors.
In another aspect of the invention, agents which selectively binds the isolated PGP polypeptides are provided. Preferably the agent does not bind a human or dog P-glycoprotein, except those provided herein. In certain embodiments, the agent is a polypeptide preferably one selected from the group consisting of monoclonal antibodies, polyclonal antibodies, Fab antibody fragments, F(ab)
2
antibody fragments and antibody fragments including a CDR3 region. Also provided are agents which selectively binds the foregoing isolated nucleic acid molecules, preferably antisense nucleic acid molecules which selectively binds to the isolated nucleic acid molecule.
According to another aspect of the invention, methods for predicting the bioavailability of a compound are provided. The methods include measuring the transmembrane transport of a test compound by a first P-glycoprotein, comparing the transmembrane transport of the test compound by the first P-glycoprotein and a second P-glycoprotein to predict the bioavailability of the test compound, wherein the relative amount or rate of transport by the first P-glycoprotein and the second P-glycoprotein is predictive of bioavailability of the test compound. In certain embodiments the first P-glycoprotein is selected from the group consisting of dog P-glycoproteins and primate P-glycoproteins, preferably one of the foregoing polypeptides. In other embodiments the second P-glycoprotein is a human P-glycoprotein.
In still other aspects of the invention, methods for inhibiting P-glycoprotein transporter activity in a mammalian cell are provided. The methods include contacting the mammalian cell with an amount of one of the foregoing agents effective to inhibit P-glycoprotein transporter activity in the mammalian cell.
Also included in the invention are methods for increasing bioavailability of a drug in a subject. The methods include administering to a subject in need of such treatment one of the foregoing agents in an amount effective to increasing bioavailability of a drug. The inhibitor can be administered prior to administering the drug, or concurrently with the drug.
Also provided are methods for increasing P-glycoprotein transporter activity in a cell. These methods include contacting the cell with a molecule selected from the group consisting of the foregoing nucleic acid molecules, in an amount effective to increase P-glycoprotein transporter activity in the cell. The cell can be contacted under conditions whereby the P-glycoprotein is expressed.
According to yet another aspect of the invention, methods for identifying lead compounds for a pharmacological agent useful in the treatment of disease associated with P-glycoprotein transporter activity are
Crespi Charles L.
Steimel-Crespi Dorothy T.
Stocker Penny J.
Becton Dickinson and Company
Prouty Rebecca E.
Rao Manjunath N.
Wolf Greenfield & Sacks PC
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