Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-05-19
2004-02-17
Ulm, John (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S091100, C435S320100, C536S023100, C536S023500, C530S350000, C436S504000
Reexamination Certificate
active
06692934
ABSTRACT:
FIELD OF THE INVENTION
The invention claims isolated nucleic acid encoding all or a portion of novel members of the organic anion transport protein (“OATP”) designated OATP2, OATP-RP1, OATP-RP2, OATP-RP3, OATP-RP4 and OATP-RP5. Also claimed are vectors containing the nucleic acid sequences, host cells containing the vectors and polypeptides having all or part of the amino acid sequence of OATP2, OATP-RP1, OATP-RP2, OATP-RP3, OATP-RP4 and OATP-RP5. Tissue expression of the transporter is described as well as some of its substrates. Also claimed are uses for these novel OATPs, including for targeting drugs to specific tissues, for modulating the concentration of endogenous substrates, and for identifying a substrate capable of being transported by a novel OATP of the invention.
BACKGROUND OF THE INVENTION
The liver functions in the clearance of a large variety of metabolic products, drugs and other xenobiotics by transporting them across the sinusoidal membrane into the hepatocyte. Several classes of transport systems have been described that mediate these processes including the Na+/taurocholate cotransporter polypeptide, NTCP, in rat and human liver (Hagenbuch, B., et al. (1991)
Proc. Natl. Acad. Sci. USA
88:10629-33; Hagenbuch, B. et al., (1994)
J. Clin. Invest
. 93:1326-31) and a family of organic anion transporting polypeptides (OATPs) that are principally expressed in liver, kidney and brain, and transport a broad spectrum of substrates in a sodium-independent manner (Meier, P. J., et al., (1997)
Hepatology
26:1667-77; Wolkoff, A. W., (1996)
Semin. Liver Dis
. 16:121-127). The distribution of this latter family of transporters in liver, kidney and choroid plexus in the brain is thought to reflect common physiological requirements of these organs for the clearance of a multitide of organic anions. There are three OATP isoforms in the rat: roatp1 (Jacquemin, E., et al., (1994)
Proc. Natl. Acad. Sci. USA
91:133-37); roatp2 (Noe, B. A., et al., (1997)
Proc. Natl. Acad. Sci. USA
94:10346-50; and roatp3 (Abe, T., et al., (1998)
J. Biol. Chem
. 273:11395-401). In addition to bile acids, OATPs are known to transport a variety of other compounds. These include, depending on the transporter, unconjugated and conjugated steroids such as estrone sulfate, estradiol-17B-glucuronide, aldosterone, and cardiac glycosides (Boussuyt, X., et al., (1996)
J. Pharmacol. Exp. Ther
. 276:891-6; Boussuyt, X. (1996)
J. Hepatol
. 25:733-8; Kanai, N., et al., (1996)
Am. J. Physiol
. 270:F319-F325; Kanai, N., et al., (1996)
Am. J. Physiol
. 270:F326-F331; Noe, B. A., et al., (1997)
Proc. Natl. Acad. Sci. USA
94:10346-50). Bromosulfophthalien (Jacquemin, E., et al., (1994)
Proc. Natl. Acad. Sci. USA
91:133-7); mycotoxin (Kontaxi, M., et al., (1996)
J. Pharmacol. Exp. Ther
. 279:1507-13); leukotriene C
4
(Li, L., et al., (1 998)
J. Biol. Chem
. 273:16184-91); and thyroid hormone (Abe, T., et al., (1998)
J. Biol. Chem
. 273:11395) are additional substrates.
Several proteins have been identified. Jacquemin,E., et al., (1994)
Proc. Natl. Acad. Sci. U.S.A
., 91:133-137 reported the first cloning and identification of a member of the OATP transporter family, namely the rat oatp1. The first cloning and identification of a human OATP was reported in Kullak-Ublick, G. A., et al., (1995)
Gastroenterology
, 109:1274-1282. Its expression was found in liver, kidney brain and other organs. The authors concluded, based on substrate specificities, that it was not the human orthologue of rat oatp1.
Substrate specificities of rat oatp1 are discussed in Kullak-Ublick, G. A. et al., (1994)
Hepatology
, 20:411-416, while substrate specificities of human OATP are discussed in Bossuyt, X., et al., (1996)
J. Hepatol
., 25:733-738.
Data was later discovered showing that rat oatp1 is involved in the transport of steroids (Bossuyt, X., et al., (1996)
J. Pharmacol. Exp. Ther
., 276:891-896), and that human OATP acts as a transporter for the psychoactive hormone DHEAS (Kullak-Ublick, G. A., et al., (1998)
FEBS Lett
., 424:173-176). For a review of the OATP family and organic anoin transport in the liver, see Wolkoff, A. W., (1996)
Semin. Liver Dis
., 16:121-127.
A third rat OATP isoform that was shown to transport thyroid hormones T3 and T4 was cloned and reported in Abe,T., et al., (1998)
J. Biol. Chem
., 273:22395-22401.
All references cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
The present invention encompasses novel organic anion transport proteins (“OATP”) and polynucleotides encoding said OATPs. The OATPs disclosed herein are designated OATP2, OATP-RP2, OATP-RP3, OATP-RP4, OATP-RP5 and OATP-RP1. A polynucleotide sequence of each OATP is disclosed herein, along with the deduced amino acid sequence. The cDNAs encoding the OATPs of the present invention have been deposited with the American Type Culture Collection and given Accession Numbers ATCC 207213 (OATP2), ATCC 207212 (OATP-RP2), ATCC 207209 (OATP-RP3), ATCC 207210 (OATP-RP4), ATCC 207211 (OATP-RP5), and ATCC 207214 (OATP-RP1).
The present inventors sequenced the cDNAs encoding the novel OATPs and determined the primary sequence of the deduced proteins. Disclosed herein are the nucleic acid sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of OATP2; the nucleic acid sequence (SEQ ID NO:3) and amino acid sequence (SEQ ID NO:4) of OATP-RP2; the nucleic acid sequence (SEQ ID NO:5) and amino acid sequence (SEQ ID NO:6) of OATP-RP3; the nucleic acid sequence (SEQ ID NO:7) and amino acid sequence (SEQ ID NO:8) of OATP-RP4; the nucleic acid sequence (SEQ ID NO:9) and amino acid sequence (SEQ ID NO:10) of OATP-RP5; and the nucleic acid sequence (SEQ ID NO:11) and amino acid sequence (SEQ ID NO:12) of OATP-RP1.
The OATPs of the present invention can be produced by: (1) inserting the cDNA of a disclosed OATP into an appropriate expression vector; (2) transfecting the expression vector into an appropriate transfection host(s); (3) growing the transfected host(s) in appropriate culture media; and (4) assaying the transport activity in the transfected cells.
The present invention therefore provides a purified and isolated nucleic acid molecule, preferably a DNA molecule, having a sequence which codes for an OATP, or an oligonucleotide fragment of the nucleic acid molecule which is unique to an OATP of the invention. In a preferred embodiment of the invention, the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:1 (OATP2). In another preferred embodiment, the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:3 (OATP-RP2). In still another preferred embodiment the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:5 (OATP-RP3). In still another preferred embodiment of the present invention the purified and isolated nucleic acid molecule has the nucleotide sequence as shown in SEQ ID NO:7 (OATP-RP4). In still another preferred embodiment the purified and isolated nucleic acid molecule has the sequence as shown in SEQ ID NO:9 (OATP-RP5). In still another preferred embodiment of the present invention the purified and isolated nucleic acid molecule has the nucleotide sequence as shown in SEQ ID NO:11 (OATP-RP1).
The invention also contemplates a double stranded nucleic acid molecule comprising a nucleic acid molecule of the invention or an oligonucleotide fragment thereof hydrogen bonded to a complementary nucleotide base sequence.
The terms “isolated and purified nucleic acid”, “isolated and purified polynucleotide”, “substantially pure nucleic acid”, and “substantially pure polynucleotide”, e.g., substantially pure DNA, refer to a nucleic acid molecule which is one or both of the following: (1) not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (i.e., one at the 5′ end and one at the 3 end) in the naturally occurring genome of the organism from which the nucleic acid is derived; or (2
Hsiang Bonnie
Huang Xin
Kirchgessner Todd G.
Lynch Jean S.
Wang Zhaoqing
Bristol-Myers Squibb Co.
Chernyshev Olga N.
Sher Audrey F.
Ulm John
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