Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-03-07
2004-07-06
Mertz, Prema (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S069100, C435S071200, C435S471000, C435S252300, C435S320100, C435S325000
Reexamination Certificate
active
06759514
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to transporters, proteins involved in transport of substances from the outside to the inside of cells or vice versa.
BACKGROUND OF THE INVENTION
Recently, the involvement of various transporters localized on the plasma membrane in the uptake system for nutrients and endogenous substances into cells and their transport mechanisms have been clarified (Tsuji, A. and Tamai, I., Pharm. Res., 13, 963-977, 1996). These transporters recognize the structures of substances to be transported to selectively transport specific substances. Transporters that recognize the relatively wide range of structures may possibly recognize foreign substances such as drugs by mistake, and actively take in them into cells. It is believed that drugs permeate through the plasma membrane fundamentally by simple diffusion depending on their physicochemical properties such as molecular size, hydrophobicity, and hydrogen-binding capacity. Particularly, in the case of ionic drugs, only molecules in the non-dissociated form can permeate through the plasma membrane according to the pH partition hypothesis. However, it has become evident that a number of drugs penetrate through the cell membrane by a specific mechanism other than simple diffusion, that is, an active transport mediated by transporters, in organs that require efficient exchange of intracellular and extracellular substances, including small intestine, uriniferous tubule, placenta, epithelial cells of choroid plexus, hepatocytes, and blood-brain barrier (Tamai, I. and Tsuji, A., Pharmacia, 31, 493-497, 1995; Saito, H. and Inui, K., Igaku no Ayumi, 179, 393-397, 1996; Tamai, I., Yakubutsu Dotai (Pharmacokinetics), 11, 642-650, 1996). For example, it is known that although oral &bgr;-lactam antibiotics of the non-esterified type are amphoteric or negatively charged in physiological pHs and sparingly soluble in fat, they are readily absorbed through the intestine. The transport study using the isolated membrane-vesicles system demonstrated that an H
+
-driven peptide transporter localized on the brush-border is involved in the absorption process of these drugs (Tsuji, A. et al., J. Pharmacol. Exp. Ther., 241, 594-601, 1987). Although the specificity of a peptide transport system in terms of the peptide size is so strict as to recognize di- or tri-peptides but not tetrapeptides or larger peptides, it has a rather broad substrate specificity to recognize peptides comprising non-natural amino acids. The peptide transporter mediates transport of &bgr;-lactam antibiotics mistakenly due to its broad substrate specificity. This property has been unexpectedly utilized in the clinical field (Tsuji, A., American Chemical Society (eds. Taylor, M. D., Amidon, G. L.), Washington, D.C., 101-134, 1995). Furthermore, a possibility that a transporter is also involved in permeation of substances with a high hydrophobicity such as fatty acids through the plasma membrane has been reported (Schaffer, J. and Lodish, H., Cell, 79, 427-436, 1994).
Since various transporters are supposed to be distributed in organs and cells based on the physiological roles of the organs and cells, their distribution and functions may be specific to organs. Therefore, transporters are expected to be used to impart an organ specificity to pharmacokinetics. In other words, an organ-specific drug delivery system (DDS) can be constructed utilizing transporters. If drug absorption solely relied on simple diffusion is improved by elevating its hydrophobicity, an effect of the drug obtained in the initial transport in the liver can be enhanced and the drug can non-specifically translocates into any organ. In addition, it would also be possible to increase the drug absorption independently of its fat-solubility by designing the drug utilizing the substrate specificity of transporters (Hayashi, K. et al., Drug Delivery System, 11, 205-213, 1996). For this purpose, it is necessary to identify various transporters at the molecular level and analyze their properties in detail. However, their molecular level identification are greatly behind studies on their membrane physiology because they are difficult to handle biochemically and require complicated processes in their functional assays.
Recently, cDNAs of several transporters have been cloned by the expression cloning method using Xenopus oocytes, a foreign gene expression system, and structural homology among them has been revealed (Fei, Y.-J. et al., Nature, 368, 563-566, 1994). For example, Koepsell et al. cloned an organic cation transporter, OCT1, which is assumed to be localized on a basement membrane, using the expression cloning method in 1994 (Grundemann, D. et al., Nature, 372, 549-552, 1994). Subsequently, OCT2 was identified by homology cloning based on the sequence of OCT1 (Okuda, M. et al., Biochem. Biophys. Res. Commun., 224, 500-507, 1996). OCT1 and OCT2 show homology as high as 67% to each other (Grundemann, D. et al., J. Biol. Chem., 272, 10408-10413, 1997). Both of them are intensely expressed in the kidney, but differ in the organ distribution; OCT1 is also expressed in the liver, colon, and small intestine, while OCT2 expression is specific to the kidney.
Only a few reports on identification of transporters at the molecular level, including the reports, are available, and there would be many unknown transporters that may be clinically useful.
SUMMARY OF THE INVENTION
An object of this invention is to provide a family of novel transporter genes, proteins encoded by these genes, and their use.
The present inventors have screened a fetal gene library constructed using the subtractive method by random sequencing based on a working hypothesis that fetal genes include those which are involved in various disorders including cancer and are specifically or intensely expressed in fetal tissues. The inventors discovered an unknown gene showing a significant homology with those for organic cation transporters, OCT1 and OCT2, and attempted to isolate this gene, which was assumed to encode a novel transporter. Thus, the inventors succeeded in isolating the desired gene by screening a cDNA library derived from human fetus. Furthermore, the inventors studied the transporter activity of a protein encoded by the isolated human gene and found that the protein, in fact, functioned as a transporter for various organic cations. The inventors also succeeded in isolating a mouse gene corresponding to the isolated human gene.
This invention relates to a family of novel transporter genes, proteins encoded by these genes, and their use, and more specifically to:
(1) a protein comprising an amino acid sequence set forth in SEQ ID NOs: 1, 3, 22, or 27, or a protein comprising said amino acid sequence in which one or more amino acid residues are substituted, deleted, or added, and having an activity to transport an organic cation;
(2) a protein encoded by a DNA hybridizing to a DNA comprising nucleotide sequence according to SEQ ID NOs: 2, 4, 23, or 28, and having an activity to transport an organic cation;
(3) a DNA encoding the protein according to (1) or (2);
(4) a vector comprising the DNA according to (3);
(5) a transformant expressibly carrying the DNA according to (3);
(6) a method for producing the protein according to (1) or (2), the method comprising culturing the transformant according to (5);
(7) an antibody that binds to the protein according to (1) or (2); and
(8) a DNA specifically hybridizing to a DNA comprising a nucleotide sequence set forth in SEQ ID NOs: 2, 4, 23, or 28, and consisting of at least 15 nucleotides.
Nucleotide sequences of cDNAs of novel human transporters isolated by the present inventors are shown in SEQ ID NO: 2 (designated as “human OCTN1”) and SEQ ID NO: 4 (designated as “human OCTN2”), respectively. Amino acid sequences of proteins encoded by these cDNAs are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. Amino acid sequences of these two proteins included in the transporter proteins of this invention showed such a high overall homology as about 76%, and both o
Nezu Jun-ichi
Oku Asuka
Chugai Seiyaku Kabushiki Kaisha
Fish & Richardson PC
Mertz Prema
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