WO2015039961A2 - Modulateurs de slc22a13 - Google Patents

Modulateurs de slc22a13 Download PDF

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WO2015039961A2
WO2015039961A2 PCT/EP2014/069467 EP2014069467W WO2015039961A2 WO 2015039961 A2 WO2015039961 A2 WO 2015039961A2 EP 2014069467 W EP2014069467 W EP 2014069467W WO 2015039961 A2 WO2015039961 A2 WO 2015039961A2
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slc22a13
cells
kidney
cell
expression
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Stefan Golz
Dirk GRÜNDEMANN
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Bayer Pharma Aktiengesellschaft
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention is in the field of molecular biology, more particularly, the present invention relates to screening methods identifying modulators of SLC22A13 and the use of those.
  • the solute carrier (SLC) group of membrane transport proteins includes over 300 members organized into 47 families.
  • SLC gene nomenclature system was originally proposed by the Human Genome Organization (HUGO) and is the basis for the official HUGO names of the genes that encode these transporters.
  • HUGO Human Genome Organization
  • a more general transmembrane transporter classification can be found in TCDB database.
  • Solutes that are transported by the various SLC group members are extraordinarily diverse and include both charged and uncharged organic molecules as well as inorganic ions.
  • SLCs contain a number of hydrophobic transmembrane alpha helices connected to each other by hydrophilic intra- and extra-cellular loops. Depending on the SLC, these transporters are functional as either monomers or obligate homo- or hetero-oligomers.
  • the SLC group includes examples of transport proteins that are:
  • secondary active transporters allow solutes to flow uphill against their electrochemical gradient by coupling to transport of a second solute that flows downhill with its gradient such that the overall free energy change is still favorable
  • the SLC series does not include members of transport protein families which have previously been classified by other widely accepted nomenclature systems including:
  • primary active transporters allow flow uphill against electrochemical gradients
  • ABC ATP Binding Cassette
  • energy releasing event such as ATP hydrolysis • ion channels
  • Solute carrier family 22 member 13 is a protein that in humans is encoded by the SLC22A13 gene.
  • SLC22A13 is an evolutionary conserved integral membrane protein, since highly similar orthologues have been found in many mammalian and vertebrate species. Rigorous conservation suggests an essential function.
  • OCTL1, ORCTL3, OAT10 Several functional names have been proposed for this carrier (OCTL1, ORCTL3, OAT10), but it is designated SLC22A13 in this application.
  • SLC22A13 is described as a urate, p-aminohippurate and nicotinate transpoter [3].
  • Converting a drug candidate with good in vitro properties into a drug with sufficient in vivo properties is the third stage of the drug design process.
  • the approaches used in this stage include: the introduction of bioisosters; the design of prodrugs transforming themselves into an active form in the body; twin drugs carrying two pharmacophore groups that bind to one molecule; and soft drugs, which have a pharmacological action localized in specific organs (their distribution in other organs gives rise to metabolic destruction or inactivation).
  • the technical problem underlying the present invention is the provision of a SLC22A13 transport assay system reflecting the physiological activity of SLC22A13 which allows therapeutic intervention for disorders that are related to the malfunction or the lack of this transporter.
  • the solution is the provision of the natural substrates i.e. aspartate (Asp), glutamate (Glu), or taurine (Tau) for SLC22A13 enabling transport systems for the identification of modulators.
  • SLC22A13 is an evolutionary conserved integral membrane protein, since highly similar orthologues have been found in many mammalian and vertebrate species. Rigorous conservation suggests an essential function. Several functional names have been proposed for this carrier (OCTL1, ORCTL3, OAT10), but it is designated simply SLC22A13 here.
  • the present invention is directed to the function of SLC22A13 transporter and therapeutic uses thereof.
  • the present invention relates to a method for identifying and/or obtaining a compound capable of modulating transport, comprising contacting a test compound with a system for measuring those transport activity, which system comprises an SLC22A13 polypeptide or a functional fragment thereof, and a substrate for measuring transport by the system; and detecting an altered level of the those transport activity of the SLC22A13 polypeptide or functional fragment in the presence of the test compound compared to the described transport activity in the absence of the test compound and/or presence of a control.
  • This method is useful to identify and obtain drugs for the treatment of disorders related to SLC22A13 transporter function or the lack of it as well as for determining the toxicity of a given compound, for example whether it blocks the transporter activity.
  • the present invention relates to the use of a compound capable of modulating transport activity of the SLC22A13 for the manufacture of a medicament for the treatment and/or prophylaxis of a disease related to SLC22A13 transport activity.
  • therapeutic intervention through SLC22A17 is envisaged for the treatment of kidney or CNS disease or cancer.
  • the present invention relates to the use of a compound capable of modulating SLC22A13 transport activity or expression of the SLC22A13 so as to reduce the intracellular level of the substrates in a target cell for the manufacture of a medicament for inducing cell death in a target cell,
  • This embodiment is particularly suited for the treatment of malignant diseases, in particular cancer.
  • the finding of the SLC22A13 enables diagnostic methods for determining the presence of or a susceptibility to a disease or a disorder the SLC22A13 is involved in, which therefore is also subject of the present invention.
  • Fig. 1 shows the nucleotide sequence of human SLC22A13 polynucleotide (SEQ ID NO: 1).
  • Fig. 2 shows the amino acid sequence of human SLC22A13 polypeptide (SEQ ID NO: 2).
  • Fig. 3 shows nucleotide sequence of SEQ ID NO: 3 (SLC22A13; forward primer)
  • Fig. 4 shows nucleotide sequence of SEQ ID NO: 4 (SLC22A13; reverse primer)
  • Fig. 5 shows nucleotide sequence of SEQ ID NO: 5 (SLC22A13; probe)
  • Fig. 6 shows nucleotide sequence of SEQ ID NO: 6 (RPL32; forward primer)
  • Fig. 7 shows nucleotide sequence of SEQ ID NO: 7 (RPL32; reverse primer)
  • Fig. 8 shows nucleotide sequence of SEQ ID NO: 8 (RPL32; probe)
  • Fig. 9 shows: Tissue distribution of SLC22A13 from human. Human tissues and cells were analyzed for SLC22A13h mRNA by real-time RT-PCR (see Methods for details). Results are given relative to the mRNA level of kidney.
  • the quality of zones was verified with amplicons for 8 proteins of known expression pattern (the order of kidney zone samples is as for SLC22A13): SGLT2 (human gene symbol SLC5A2), EAAT3 (SLC1A1), PEPT2 (SLC15A2), UT2 (SLC14A2 iso 2), UT3 (SLC14A1), UT1 (SLC14A2 iso 1), UPK1B (UPK1B, Uroplakin IB), and GLAST (SLC1A3).
  • Fig. 11 shows: Immunofluorescent detection of SLC22A13 in rat kidney sections.
  • A Overview with cortex (top), outer medulla, and inner medulla (bottom).
  • B, C Outer stripe of outer medulla (top) and inner stripe of outer medulla (bottom).
  • D Inner medulla with collecting ducts. Arrowheads indicate lateral cell views with intense basolateral signal.
  • Fig. 12 shows: SLC22A13 colocalizes with the anion exchanger AE1 in rat kidney sections. Paraffin slices (7 ⁇ ) were incubated with antibodies against SLC22A13 (red signal) and AE1 (green). Overlay (addition) of both images reveals a perfect match (yellow).
  • Fig. 13 shows: Difference image of cell lysates.
  • 293 cells stably transfected with pEBTetD/SLC22A13h were cultivated for 20 h in the presence (to express the transporter) or absence (control) of 1 ⁇ g/ml doxycycline in growth medium.
  • Cells were washed with uptake buffer, incubated 1 h with 1 mmol/1 orotic acid in MES uptake buffer (pH 5.0), washed again and then lysed with methanol.
  • Lysates were analyzed by full scan LC-MS (zic-pHILIC column, methanol solvent, negative ionization mode, scan time 2 s). Arrows indicate increase (red) or decrease (cyan) of some identified compounds caused by transporter expression; numbers indicate m/z.
  • Fig. 14 shows: Expression of SLC22A13h in 293 cells diminishes uptake of 3H-aspartate and 3H-glutamate.
  • 293 cells stably transfected with pEBTetD/SLC22A 13h or pEBTetD/OAT2h as indicated were cultivated in dishes for 20 h in the presence (to express the transporter) or absence (control) of 1 ⁇ g/ml doxycycline in growth medium. Cells were washed, incubated at 37 °C with 0.1 ⁇ / ⁇ 3H-aspartic acid (Asp), 3H-glutamic acid (Glu), or 3H-orotic acid in uptake buffer for the indicated periods, washed and lysed.
  • Asp 3H-aspartic acid
  • Glu 3H-glutamic acid
  • Fig. 15 shows: Efflux of Asp, Tau, and Glu from 293 cells without or with expression of SLC22A13h.
  • Cells were washed twice with prewarmed sodium- and phosphate-free uptake buffer, and then incubated at 37 °C with 2 ml buffer; at 10, 20, and 30 min, buffer samples (0.2 ml) were collected successively. Finally, cells were washed and lysed. Buffer samples and cell lysates were frozen and later analyzed for amino acid content by LC-MS/MS (positive ionization). A straight line was fitted to each efflux data set.
  • the present invention generally relates to the SLC22A13 transporter and to various uses thereof, for example in therapeutic and diagnostic applications as well as research tool.
  • SLC22A13 polypeptide refers to those forms, fragments, or domains of a SLC22A13 polypeptide which retain the biological and/or antigenic activity of a SLC22A13 polypeptide.
  • Naturally occurring SLC22A13 polypeptide refers to a polypeptide produced by cells which have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including but not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Derivative refer to polypeptides which have been chemically modified by techniques such as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.
  • Constant amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
  • a “signal sequence” or “leader sequence” can be used, when desired, to direct the polypeptide through a membrane of a cell.
  • Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.
  • Oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide. Oligopeptides comprise a stretch of amino acid residues of at least 3, 5, 10 amino acids and at most 10, 15, 25 amino acids, typically of at least 9 to 13 amino acids, and of sufficient length to display biological and/or antigenic activity.
  • inhibitor is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists. The term antagonist can be used interchangeably.
  • Activator is any substance which e.g. enhances, stimulates or activates a chemical or physiological reaction or response, e.g. a transport activity.
  • agonist can be used interchangeably.
  • Fusion proteins are useful for generating antibodies against SLC22A13 polypeptides and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of SLC22A13 polypeptides. Protein affinity chromatography or library- based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a SLC22A13 fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment can comprise at least 54, 75, 100, 125, 139, 150, 175, 200, 225, 250, or 275 contiguous amino acids of SEQ ID NO: 2 or of a biologically active variant, such as those described above.
  • the first polypeptide segment also can comprise full- length SLC22A13.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include, but are not limited to ⁇ galactosidase, ⁇ - glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione- S -transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, herpes simplex virus (HSV) BP16 protein fusions and G- protein fusions (for example G(alpha)16, Gs, Gi).
  • a fusion protein also can be engineered to contain a cleavage site located adjacent to the SLC22A13.
  • a naturally occurring SLC22A13 polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated SLC22A13 polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise SLC22A13 nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.
  • SLC22A13 polynucleotide sequence is provided by NCBI Reference Sequence NM_004256.3 or in Gene ID 9390, wherein the nucleotides coding for SLC22A13 are represented by SEQ ID NO: 1.
  • SLC22A13 cDNA molecules can be made with standard molecular biology techniques, using SLC22A13 mRNA as a template. SLC22A13 cDNA molecules can thereafter be replicated using molecular biology techniques known in the art. An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • SLC22A13 polynucleotides can be synthesized using synthetic chemistry techniques.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode SLC22A13 having an amino acid sequence shown in SEQ ID NO: 2 or a biologically active variant thereof.
  • SLC22A13 can be obtained, for example, by purification from human cells, by expression of SLC22A13 polynucleotides, or by direct chemical synthesis.
  • SLC22A13 can be purified from any human cell which expresses the receptor, including those which have been transfected with expression constructs which express SLC22A13.
  • a purified SLC22A13 is separated from other compounds which normally associate with SLC22A13 in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • SLC22A13 polynucleotides can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding SLC22A13 and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding SLC22A13. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g. , baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g. , Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g. , baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV
  • control elements or regulatory sequences are those non-translated regions of the vector - enhancers, promoters, 5' and 3' untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO, and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding SLC22A13, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected.
  • vectors which direct high level expression of fusion proteins that are readily purified can be used.
  • Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene).
  • BLUESCRIPT a sequence encoding SLC22A13 can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ - galactosidase so that a hybrid protein is produced.
  • pIN vectors or pGEX vectors also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • sequences encoding SLC22A13 can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used. These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
  • An insect system also can be used to express SLC22A13.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • Sequences encoding SLC22A13 can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • Successful insertion of SLC22A13 will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which SLC22A13 can be expressed.
  • a number of viral-based expression systems can be used to express SLC22A13 in mammalian host cells.
  • sequences encoding SLC22A13 can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing SLC22A13 in infected host cells [Engelhard, 1994)].
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • HACs also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding SLC22A13. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding SLC22A13, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including the ATG initiation codon
  • the initiation codon should be in the correct reading frame to ensure translation of the entire insert.
  • Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic.
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed SLC22A13 in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express SLC22A13 can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1 -2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced SLC22A13 sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems can be used to recover transformed cell lines.
  • herpes simplex virus thymidine kinase [Logan, (1984)] and adenine phosphoribosyltransferase [Wigler, (1977)] genes which can be employed in ti or aprV cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate [Lowy, (1980)]
  • npt confers resistance to the aminoglycosides, neomycin and G- 418 [Wigler, (1980)]
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively [Colbere-Garapin, 1981]. Additional selectable genes have been described.
  • trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine.
  • Visible markers such as anthocyanins, ⁇ - glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system
  • SLC22A13 (human gene symbol SLC22A13) is a member of the SLC22 family of transport proteins. The sequences are described under gene ID 9390. We have used the strategy of LC-MS difference shading to search for specific and cross-species substrates of SLC22A13.
  • SLC22A13 has unique functional features. Uptake of organic anions such as p-aminohippuric acid (PAH), orotic acid, and nicotinic acid (not shown) can be detected, but is markedly slower than with other family members. With the amino acids Asp and Glu, there is no indication of uptake. It is obvious from the time courses of uptake that efflux is the predominant direction of transport (Fig. 14): SLC22A13 counteracts the EAAT-catalyzed uptake of 3H-Asp and 3H-Glu. By contrast, in the same experimental setting OAT2, which catalyses both uptake and efflux of glutamate [11], increases accumulation of 3H- Glu.
  • PHA p-aminohippuric acid
  • nicotinic acid not shown
  • GSA guanidinosuccinic acid probably has no physiological significance; it is generated non- enzymatically from cytosolic argininosuccinate, a urea cycle intermediate [20, 21]. However, it can be considered as a useful Asp-related model substrate: there was marked reduction (factor 2- 5) of intracellular GSA concentration by SLC22A13 expression; as with Asp, we could not detect any short-time uptake of GSA in our standard assay (10 ⁇ ). Accumulation was observed, however, after 24 h incubation with 1 mM GSA. Thus, with sufficient force, the direction of transport can be reversed from efflux to uptake.
  • SLC22A13 is primarily expressed in kidney (Fig. 9).
  • Our antibody reveals expression of SLC22A13 in the basolateral domain of type A intercalated cells of rat kidney.
  • Type A intercalated cells are expressed within the distal convoluted tubule (DCT), connecting tubule (CNT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD) and the initial inner medullary collecting duct (IMCD) [22].
  • DCT distal convoluted tubule
  • CNT connecting tubule
  • CCD cortical collecting duct
  • OMCD outer medullary collecting duct
  • IMCD initial inner medullary collecting duct
  • Type B cells are rare in the outer stripe of the outer medulla, and absent from the inner stripe and the inner medullary collecting duct [24]. Stained cells, in morphological accordance [23], protrude into the lumen (Fig. 3).
  • Type A intercalated cells are defined by a proton efflux pump in the luminal cell membrane and AE1 (a splice variant of the erythrocyte bicarbonate/chloride exchanger) in the basolateral membrane domain [25]. These transporters are fuelled by cytosolic carboanhydrase II which generates H+ and HC03- from CO2 and H2O.
  • type A cells a major purpose of type A cells is to generate and secrete protons into urine to counter acidosis.
  • Type B intercalated cells serve to create and secrete bicarbonate into the lumen in alkalosis.
  • the presence of AE1 in the basolateral membrane is conclusive for diagnosis of type A intercalated cells [23].
  • Our double-staining (Fig. 4) establishes that SLC22A13 is a close companion of AE1 in the basolateral membrane of type A intercalated cells.
  • the main osmolytes in salt-loaded rat kidney outer medulla are inositol (estimated intracellular concentration, 34 mM), betaine (26 mM), Tau (26 mM), glycerophosphocholine (20 mM), and several amino acids: Gly (11 mM), Glu (8 mM), Gin (5 mM), Asp (4 mM), Ala (4 mM), and Ser (3 mM) [30]. These numbers suggest that Tau, Asp, and Glu in sum (38 mM) have only limited impact on total osmolyte content (>141 mM) in outer medulla, the main expression site of SLC22A13.
  • AE1 catalyzes antiport of bicarbonate and chloride; this electroneutral exchange [25] does not create "free” protons.
  • Asp and Glu can be synthesized directly and in large quantity from citric acid cycle intermediates oxaloacetate and 2-oxoglutarate in the cytosol or in mitochondria (which are abundant in type A cells). Ammonia, the required cosubstrate, is available in type A cells [25, 32].
  • Type B intercalated cells by contrast to type A cells, have to create and secrete bicarbonate (HC03-); here, absence of SLC22A13 makes sense, since generation of free protons would be counterproductive.
  • Tau we consider a concomitant substrate, with sufficient structural similarity to principal substrates Asp and Glu to trigger transport (albeit at relatively low efficiency, see Fig. 9). Indeed, it has been shown that EAAT2 from Drosophila transports both aspartate and taurine [33], and that in rat primary astrocyte cultures an unidentified channel catalyzes efflux of Asp, Glu, and Tau [34].
  • SLC22A13 catalyzes unidirectional efflux of aspartate and glutamate. Expression in rat kidney is restricted to the basolateral plasma membrane of type A intercalated cells.
  • SLC22A13 is a key component for producing electrostatically untied protons and is involved in distal renal tubular acidosis.
  • the renal tubular acidosis (RTA) syndromes encompass a disparate group of tubular transport defects that have in common the inability to secrete hydrogen ions (H + ), a defect that is disproportionately large in relation to any reduction in the glomerular filtration rate (GFR) [1-3].
  • H + hydrogen ions
  • GFR glomerular filtration rate
  • This inability results in failure to excrete acid in the form of ammonium (NH4 + ) ion and titratable acids or to reabsorb some of the filtered bicarbonate (HCO3 " ) load. In either situation, there is a fall in plasma bicarbonate leading to chronic metabolic acidosis.
  • Much of the morbidity in the RTA syndromes is attributable to the systemic consequences of chronic metabolic acidosis namely growth retardation, bone disease and kidney stones [37].
  • dRTA was the first RTA recognized, and thus, the terms 'Type or 'classic RTA' have been used to describe this form of RTA. dRTA is characterized by an inability to lower urine pH despite severe acidemia and minimal HCO3 " wastage.
  • Proximal RTA (Type 2), by contrast, is characterized by marked HCO3 " wastage, but preserved ability to lower urine pH when plasma HCO3 " (and therefore filtered HCO3 " ) is below a certain level.
  • the term 'Type 3 RTA' is used to describe patients, in whom HCO3 " wastage coexists with failure to lower urine pH despite profound acidemia, thus demonstrating a mixed pattern of tubular dysfunction [4].
  • Patients with RTA are often growth retarded because of the chronic metabolic acidosis unless alkaline therapy is initiated earlier in life.
  • Associated features are nephrocalcinosis, nephrolithiasis, hypercalciuria and hypocitraturia.
  • Polyuria is often encountered, and may be related, at least in part, to the associated hypokalemia. Other extrarenal manifestations depend on the gene mutated and the type of mutation. Hemolytic anemia may be seen in some types of hereditary RTA associated with AE1 mutations, whereas deafness is an important feature in some H + -ATPase mutations.
  • dRTA distal renal tubular acidosis
  • Acid-base transport within the distal nephron is primarily effected by specific transporters located in intercalated cells.
  • Distal RTA can be attributed to failure of the kidney-intercalated cells to acidify the urine normally as a result of dysfunction in any of the transporters involved in the overall process of acidifying the urine maximally.
  • distal H + -ion secretion there is a failure to lower urine pH maximally and excrete acid as ammonium and other titrable buffers which with time results in the development of hyperchloremic metabolic acidosis, the hallmark of classic or Type 1 dRTA.
  • the incomplete form of distal RTA like complete dRTA, presents with failure to maximally lower urine pH, but blood pH and plasma bicarbonate are normal. Acid load with ammonium chloride reveals the defect by showing that urine pH could not be maximally lowered, and remains above 5.3.
  • the currently known mutations resulting in inherited dRTA have been identified in transporters present in intercalated cells such as the anion exchanger 1 (AE1), the Bl and a4 subunits of H + -ATPase and the cytosolic carbonic anhydrase II (CA II).
  • AE1 anion exchanger 1
  • CA II cytosolic carbonic anhydrase II
  • the rate of H+-ion secretion by a-intercalated cells is importantly influenced by the rate of Na + transport in the neighboring principal cells that are involved in Na + reabsorption and K + secretion, but not in H + secretion.
  • the various mutations in the acid-base transporters involved in the causation of dRTA involve complex mechanisms that are specific for each transporter and some of the specific mutations involved. It has been long recognized that cytosolic CA, by catalyzing the hydration of CO2 to H + and HCO3 " , plays a key role in the intracellular generation of these ions from CO2 that enters the tubular cells [37].
  • codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences referred to herein can be engineered using methods generally known in the art to alter SLC22A13 polynucleotides for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site- directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • Any type of antibody known in the art can be generated to bind specifically to an epitope of SLC22A13.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of SLC22A13.
  • Fab fragment antigen binding protein
  • F(ab') 2 fragment antigen binding protein
  • Fv fragment antigen binding protein
  • An antibody which specifically binds to an epitope of SLC22A13 can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the SLC22A13 immunogen.
  • an antibody which specifically binds to SLC22A13 provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to SLC22A13 do not detect other proteins in immunochemical assays and can immunoprecipitate SLC22A13 from solution.
  • SLC22A13 can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, SLC22A13 can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response.
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • mineral gels e.g., aluminum hydroxide
  • surface active substances e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies which specifically bind to SLC22A13 can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B- cell hybridoma technique, and the EBV-hybridoma technique.
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to SLC22A13.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries.
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template.
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single- chain antibodies can be produced directly using, for example, filamentous phage technology.
  • Antibodies which specifically bind to SLC22A13 also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents. Other types of antibodies can be constructed and used therapeutically in methods of the invention. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies", also can be prepared.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which SLC22A13 is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of SLC22A13 gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non- phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phos- phorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • Modifications of SLC22A13 gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the SLC22A13 gene. Oligonucleotides derived from the transcription initiation site, e.g. , between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature. An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a SLC22A13 polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent SLC22A13 nucleotides, can provide sufficient targeting specificity for SLC22A13 mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular SLC22A13 polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a SLC22A13 polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5'-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • These modified oligonucleotides can be prepared by methods well known in the art.
  • Ribozymes are RNA molecules with catalytic activity. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences. The coding sequence of a SLC22A13 polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from a SLC22A13 polynucleotide.
  • ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art.
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.
  • Specific ribozyme cleavage sites within a SLC22A13 RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate SLC22A13 RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ ID NO: 1 and its complement provide sources of suitable hybridization region sequences.
  • hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease SLC22A13 expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme- encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • the test compound is preferably a small molecule which binds to and occupies the active site of SLC22A13 polypeptide, thereby making the ligand binding site inaccessible to substrate such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • compositions suitable for administration typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • the invention includes pharmaceutical compositions comprising a regulator of SLC22A13 expression or activity (and/or a regulator of the activity or expression of a protein in the SLC22A13 signaling pathway) as well as methods for preparing such compositions by combining one or more such regulators and a pharmaceutically acceptable carrier. Also within the invention are pharmaceutical compositions comprising a regulator identified using the screening assays of the invention packaged with instructions for use. For regulators that are inhibitors of SLC22A13 activity or which reduce SLC22A13 expression, the instructions specify use of the pharmaceutical composition for treatment of disorders of the central nervous system. For regulators that are activators of SLC22A13 activity or increase of SLC22A13 expression, the instructions specify use of the pharmaceutical composition for treatment of cardiovascular diseases, kidney and liver diseases.
  • An antagonist of SLC22A13 may be produced using methods which are generally known in the art.
  • purified SLC22A13 may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind SLC22A13.
  • Antibodies to SLC22A13 may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Antibodies like those which bind to an epitope comprising Glutamate at position 441 of SLC22A13 are preferred.
  • the polynucleotides encoding SLC22A13, or any fragment or complement thereof may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding SLC22A13 may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding SLC22A13.
  • complementary molecules or fragments may be used to modulate SLC22A13 activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding SLC22A13.
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence complementary to the polynucleotides of the gene encoding SLC22A13.
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition containing SLC22A13 in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of SLC22A13, antibodies to SLC22A13, and mimetics, agonists, antagonists, or inhibitors of SLC22A13.
  • the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EMTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases SLC22A13 activity relative to SLC22A13 activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50 ED50.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation. Normal dosage amounts can vary from 0.1 micrograms to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation- mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun", and DEAE- or calcium phosphate-mediated transfection.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of SLC22A13 gene or the activity of SLC22A13 by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of SLC22A13 gene or the activity of SLC22A13 can be assessed using methods well known in the art, such as hybridization of nucleotide probes to SLC22A 13 -specific mRNA, quantitative RT-PCR, immunologic detection of SLC22A13, or measurement of SLC22A13 activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • a method of screening for identifying and/or obtaining a compound capable of modulating SLC22A13 transport activity comprising:
  • a method of screening for therapeutic agents useful in the treatment of a SLC22A13 associated disease comprising
  • the system comprises a SLC22A13 transporter polypeptide or a functional fragment thereof, and one or more of the identified substrates and wherein the transport activity is measured using one or more of the aforementioned substrates
  • a method of screening for therapeutic agents useful in the treatment of a SLC22A13 associated disease comprising
  • kidney disease preferably renal tubular acidosis, acidosis or CNS disease or cancer
  • the system comprises a SLC22A13 transporter polypeptide or a functional fragment thereof, and one or more of the identified substrates and wherein the transport activity is measured using one or more of the aforementioned substrates
  • Identified substrates are selected from the group consisting of glutamate, aspartate, and taurine and their salts and acids, derivatives thereof and isotope labeled compounds.
  • a preferred salt is a soluble salt e.g. a sodium salt.
  • a more preferred substrate is glutamate or aspartate and salts or acids thereof.
  • a preferred SLC22A13 associated disease is a disease comprised in the group of diseases consisting of kidney disease, CNS disease, acidosis or cancer.
  • a method of screening for identifying and/or obtaining a compound for the treatment and/or prophylaxis of a disease comprised in the group of diseases consisting of kidney disease, CNS disease, acidosis or cancer contacting a test compound with a system for measuring SLC22A13 transport activity, which system comprises an SLC22A13 polypeptide or a functional fragment thereof, and an identified substrate for measuring SLC22A13 transport activity by the system; and
  • a method of screening for identifying and/or obtaining a compound for treating a disease related to SLC22A13 activity comprises: providing a transgenic animal or a mutant animal, which animal expresses a variant SLC22A13 gene, due to the deregulation of the SLC22A13 activity in cells or tissue of said animal compared to cells or tissue of a corresponding wild type or control animal; contacting the animal with a test compound; and detecting an improvement in a condition of the animal in response to the test compound, wherein the condition is a symptom of a disorder of the, CNS disease or cancer.
  • said contacting step further includes contacting said system or animal with at least one second test substance in the presence of said first test substance.
  • a compound known to activate or inhibit SLC22A13 activity is added to the system or administered to the animal.
  • test substance is a therapeutic agent.
  • test substance is a mixture of therapeutic agents.
  • test substance is comprised in and subjected as a collection of test substances.
  • a method of obtaining and manufacturing a drug comprising the steps of the method of any one of the foregoing embodiments.
  • a method of determining toxicity of a compound comprising the steps of the method of any one of the foregoing embodiments, wherein a reduced or enhanced level or activity of the SLC22A13 is indicative for the efficacy of the compound.
  • any one of the foregoing embodiments further comprising modifying said substance to alter, eliminate and/or derivatize a portion thereof suspected causing toxicity, increasing bioavailability, solubility and/or half-life.
  • SLC22A13 substrates or a derivative or analog thereof, an SLC22A13 polypeptide or functional fragment thereof, a nucleic acid molecule encoding said SLC22A13 polypeptide or functional fragment thereof for use in a method of any one of the foregoing embodiments.
  • a compound which inhibits SLC22A13 transport activity of an SLC22A13 polypeptide for as a medicament for the treatment and/or prophylaxis of a disease related to the CNS A compound which activates transport activity of an SLC22A13 polypeptide for as a medicament for the treatment and/or prophylaxis of a disease related to the kidney preferably renal tubular acidosis or acidosis.
  • a compound identified by the one of the aforementioned screening method which activates transport activity of an SLC22A13 polypeptide as a medicament for the treatment and/or prophylaxis of a disease related to the kidney preferably renal tubular acidosis or acidosis.
  • a compound which activates transport activity of an SLC22A13 polypeptide as a medicament for the modulation of blood acidity is provided.
  • a compound which inhibits transport activity of an SLC22A13 polypeptide as a medicament for the modulation of blood acidity is a compound which inhibits transport activity of an SLC22A13 polypeptide as a medicament for the modulation of blood acidity.
  • a compound which activates transport activity of an SLC22A13 polypeptide as a medicament for the treatment and/or prophylaxis of a disease related to cancer is provided.
  • a preferred SLC22A13 kidney disease is renal tubular acidosis.
  • the renal tubular acidosis (RTA) syndromes encompass a disparate group of tubular transport defects that have in common the inability to secrete hydrogen ions (H+), a defect that is disproportionately large in relation to any reduction in the glomerular filtration rate (GFR) [1-3].
  • H+ hydrogen ions
  • GFR glomerular filtration rate
  • This inability results in failure to excrete acid in the form of ammonium (NH4+) ion and titratable acids or to reabsorb some of the filtered bicarbonate (HC03-) load. In either situation, there is a fall in plasma bicarbonate leading to chronic metabolic acidosis.
  • Much of the morbidity in the RTA syndromes is attributable to the systemic consequences of chronic metabolic acidosis namely growth retardation, bone disease and kidney stones [37].
  • dRTA was the first RTA recognized, and thus, the terms 'Type or 'classic RTA' have been used to describe this form of RTA. dRTA is characterized by an inability to lower urine pH despite severe acidemia and minimal HCO3 " wastage.
  • Proximal RTA (Type 2), by contrast, is characterized by marked HC03- wastage, but preserved ability to lower urine pH when plasma HC0 3 ⁇ (and therefore filtered HC0 3 " ) is below a certain level.
  • the term 'Type 3 RTA' is used to describe patients, in whom HC0 3 " wastage coexists with failure to lower urine pH despite profound acidemia, thus demonstrating a mixed pattern of tubular dysfunction [4].
  • Patients with RTA are often growth retarded because of the chronic metabolic acidosis unless alkaline therapy is initiated earlier in life.
  • Associated features are nephrocalcinosis, nephrolithiasis, hypercalciuria and hypocitraturia.
  • Polyuria is often encountered, and may be related, at least in part, to the associated hypokalemia.
  • Other extrarenal manifestations depend on the gene mutated and the type of mutation. Hemolytic anemia may be seen in some types of hereditary RTA associated with AE1 mutations, whereas deafness is an important feature in some H+-ATPase mutations. Hypokalemia is a striking feature of dRTA or Type 1 RTA, but it is also frequently seen with proximal RTA or Type 2 RTA [9]. Hyperkalemic forms of distal RTA, however, were later recognized. The first type described was attributable to aldosterone deficiency, and the term Type 4 RTA was coined to describe it. In addition, hyperkalemic distal renal tubular acidosis (dRTA) due to a combined tubular defect in hydrogen (H + ) and potassium (K + ) ion secretion possibly resulting from a voltage-dependent defect.
  • dRTA hyperkalemic
  • Acid-base transport within the distal nephron is primarily effected by specific transporters located in intercalated cells.
  • Distal RTA can be attributed to failure of the kidney-intercalated cells to acidify the urine normally as a result of dysfunction in any of the transporters involved in the overall process of acidifying the urine maximally.
  • distal H+-ion secretion there is a failure to lower urine pH maximally and excrete acid as ammonium and other titrable buffers which with time results in the development of hyperchloremic metabolic acidosis, the hallmark of classic or Type 1 dRTA.
  • the incomplete form of distal RTA like complete dRTA, presents with failure to maximally lower urine pH, but blood pH and plasma bicarbonate are normal.
  • Acid load with ammonium chloride reveals the defect by showing that urine pH could not be maximally lowered, and remains above 5.3.
  • the currently known mutations resulting in inherited dRTA have been identified in transporters present in intercalated cells such as the anion exchanger 1 (AE1), the Bl and a4 subunits of H + -ATPase and the cytosolic carbonic anhydrase II (CA II). It should be noted, however, that the rate of H + -ion secretion by a-intercalated cells is importantly influenced by the rate of Na + transport in the neighboring principal cells that are involved in Na+ reabsorption and K + secretion, but not in H + secretion.
  • AE1 anion exchanger 1
  • CA II cytosolic carbonic anhydrase II
  • the various mutations in the acid-base transporters involved in the causation of dRTA involve complex mechanisms that are specific for each transporter and some of the specific mutations involved. It has been long recognized that cytosolic CA, by catalyzing the hydration of CO2 to H + and HCO3 " , plays a key role in the intracellular generation of these ions from CO2 that enters the tubular cells [37]. A more preferred kidney disease is distal renal tubular acidosis.
  • Acidosis is an increased acidity in the blood and other body tissue (i.e., an increased hydrogen ion concentration).
  • Acidosis is said to occur when arterial pH falls below 7.35 (except in the fetus), while its counterpart (alkalosis) occurs at a pH over 7.45.
  • the term acidemia describes the state of low blood pH, while acidosis is used to describe the processes leading to these states.
  • the rate of cellular metabolic activity affects and, at the same time, is affected by the pH of the body fluids.
  • the normal pH of arterial blood lies between 7.35 and 7.50 depending on the species (e.g., healthy human-arterial blood pH varies between 7.35 and 7.45).
  • Blood pH values compatible with life in mammals are limited to a pH range between 6.8 and 7.8. Changes in the pH of arterial blood (and therefore the extracellular fluid) outside this range result in irreversible cell damage.
  • Associated diseases are metabolic acidosis and respiratory acidosis.
  • Kidney disorders may lead to hypertension or hypotension. Examples for kidney problems possibly leading to hypertension are renal artery stenosis, pyelonephritis, glomerulonephritis, kidney tumors, polycistic kidney disease, injury to the kidney, or radiation therapy affecting the kidney. Excessive urination may lead to hypotension.
  • kidney disease is selected from the group consisting of kidney disorders including acute and chronic kidney diseases as (but not limited to): acute kidney failure, acute nephritic syndrome, analgesic nephropathy, atheroembolic renal disease, chronic kidney failure, chronic nephritis, congenital nephrotic syndrome, end-stage renal disease, goodpasture syndrome, interstitial nephritis, kidney cancer, kidney damage, kidney infection, kidney injury, kidney stones, lupus nephritis, membranoproliferative GN I, membranoproliferative GN II, membranous nephropathy, minimal change disease, necrotizing glomerulonephritis, nephroblastoma, nephrocalcinosis, nephrogenic diabetes insipidus, nephropathy - IgA, nephrosis (nephrotic syndrome), polycystic kidney disease
  • acute kidney failure acute nephriti
  • CNS disease is selected from the group consisting of CNS disorders include disorders of the central nervous system as well as disorders of the peripheral nervous system.
  • CNS disorders include, but are not limited to brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal degeneration, motor neuron disease, dementia, including ALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke, posttraumatic brain injury, and small-vessel cerebrovascular disease.
  • Dementias such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia and Parkinsonism linked to chromosome 17, frontotemporal dementias, including Pick's disease, progressive nuclear palsy, corticobasal degeneration, Huntington's disease, thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia, schizophrenia with dementia, and Korsakoff's psychosis, within the meaning of the definition are also considered to be CNS disorders.
  • CNS disorders such as mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, vascular cognitive impairment, attention deficit disorders, attention deficit hyperactivity disorders, and memory disturbances in children with learning disabilities are also considered to be CNS disorders.
  • Pain within the meaning of this definition, is also considered to be a CNS disorder. Pain can be associated with CNS disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post- stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
  • CNS disorders such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post- stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
  • Non-central neuropathic pain includes that associated with post mastectomy pain, phantom feeling, reflex sympathetic dystrophy (RSD), trigeminal neuralgiara-idioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with peripheral nerve damage, central pain (i.e.
  • Headache pain for example, migraine with aura, migraine without aura, and other migraine disorders
  • episodic and chronic tension- type headache tension-type like headache, cluster headache, and chronic paroxysmal hemicrania are also CNS disorders.
  • Visceral pain such as pancreatits, intestinal cystitis, dysmenorrhea, irritable Bowel syndrome, Crohn's disease, biliary colic, ureteral colic, myocardial infarction and pain syndromes of the pelvic cavity, e.g., vulvodynia, orchialgia, urethral syndrome and protatodynia are also CNS disorders.
  • vulvodynia, orchialgia, urethral syndrome and protatodynia are also CNS disorders.
  • a disorder of the nervous system are acute pain, for example postoperative pain, and pain after trauma.
  • Cancer disorders within the scope of the invention comprise any disease of an organ or tissue in mammals characterized by poorly controlled or uncontrolled multiplication of normal or abnormal cells in that tissue and its effect on the body as a whole.
  • Cancer diseases within the scope of the invention comprise benign neoplasms, dysplasias, hyperplasias as well as neoplasms showing metastatic growth or any other transformations like e.g. leukoplakias which often precede a breakout of cancer.
  • Cells and tissues are cancerous when they grow more rapidly than normal cells, displacing or spreading into the surrounding healthy tissue or any other tissues of the body described as metastatic growth, assume abnormal shapes and sizes, show changes in their nucleocytoplasmatic ratio, nuclear polychromasia, and finally may cease.
  • Cancerous cells and tissues may affect the body as a whole when causing paraneoplastic syndromes or if cancer occurs within a vital organ or tissue, normal function will be impaired or halted, with possible fatal results.
  • the ultimate involvement of a vital organ by cancer, either primary or metastatic, may lead to the death of the mammal affected. Cancer tends to spread, and the extent of its spread is usually related to an individual's chances of surviving the disease.
  • Cancers are generally said to be in one of three stages of growth: early, or localized, when a tumor is still confined to the tissue of origin, or primary site; direct extension, where cancer cells from the tumour have invaded adjacent tissue or have spread only to regional lymph nodes; or metastasis, in which cancer cells have migrated to distant parts of the body from the primary site, via the blood or lymph systems, and have established secondary sites of infection.
  • Cancer is said to be malignant because of its tendency to cause death if not treated. Benign tumors usually do not cause death, although they may if they interfere with a normal body function by virtue of their location, size, or paraneoplastic side effects. Hence benign tumors fall under the definition of cancer within the scope of the invention as well.
  • cancer cells divide at a higher rate than do normal cells, but the distinction between the growth of cancerous and normal tissues is not so much the rapidity of cell division in the former as it is the partial or complete loss of growth restraint in cancer cells and their failure to differentiate into a useful, limited tissue of the type that characterizes the functional equilibrium of growth of normal tissue.
  • Cancer tissues may express certain molecular receptors and probably are influenced by the host's susceptibility and immunity and it is known that certain cancers of the breast and prostate, for example, are considered dependent on specific hormones for their existence.
  • cancer under the scope of the invention is not limited to simple benign neoplasia but comprises any other benign and malign neoplasia like 1) Carcinoma, 2) Sarcoma, 3) Carcinosarcoma, 4) Cancers of the blood forming tissues, 5) tumors of nerve tissues including the brain, 6) cancer of skin cells.
  • Cancer according to 1) occurs in epithelial tissues, which cover the outer body (the skin) and line mucous membranes and the inner cavitary structures of organs e.g. such as the breast, lung, the respiratory and gastrointestinal tracts, the endocrine glands, and the genitourinary system.
  • Ductal or glandular elements may persist in epithelial tumors, as in adenocarcinomas like e.g. thyroid adenocarcinoma, gastric adenocarcinoma, uterine adenocarcinoma.
  • adenocarcinomas like e.g. thyroid adenocarcinoma, gastric adenocarcinoma, uterine adenocarcinoma.
  • Cancers of the pavement cell epithelium of the skin and of certain mucous membranes, such as e.g. cancers of the tongue, lip, larynx, urinary bladder, uterine cervix, or penis, may be termed epidermoid or squamous cell carcinomas of the respective tissues and and are in the scope of the definition of cancer as well.
  • Cancer according to 2) develops in connective tissues, including fibrous tissues, adipose (fat) tissues, muscle, blood vessels, bone, and cartilage like e.g. osteogenic sarcoma; liposarcoma, fibrosarcoma, synovial sarcoma.
  • Cancer according to 3) is cancer that develops in both epithelial and connective tissue.
  • Cancer disease within the scope of this definition may be primary or secondary, whereby primary indicates that the cancer originated in the tissue where it is found rather than was established as a secondary site through metastasis from another lesion.
  • Cancers and tumor diseases within the scope of this definition may be benign or malign and may affect all anatomical structures of the body of a mammal.
  • cancers and tumor diseases of I) the bone marrow and bone marrow derived cells (leukemias), II) the endocrine and exocrine glands like e.g. thyroid, parathyroid, pituitary, adrenal glands, salivary glands, pancreas III) the breast, like e.g.
  • the mammary glands of either a male or a female the mammary ducts, adenocarcinoma, medullary carcinoma, comedo carcinoma, Paget's disease of the nipple, inflammatory carcinoma of the young woman, IV) the lung, V) the stomach, VI) the liver and spleen, VII) the small intestine, VIII) the colon, IX) the bone and its supportive and connective tissues like malignant or benign bone tumour, e.g.
  • malignant osteogenic sarcoma benign osteoma, cartilage tumors; like malignant chondrosarcoma or benign chondroma; bone marrow tumors like malignant myeloma or benign eosinophilic granuloma, as well as metastatic tumors from bone tissues at other locations of the body;
  • X) the mouth, throat, larynx, and the esophagus XI) the urinary bladder and the internal and external organs and structures of the urogenital system of male and female like ovaries, uterus, cervix of the uterus, testes, and prostate gland, XII) the prostate, XIII) the pancreas, like ductal carcinoma of the pancreas;
  • XIV) the lymphatic tissue like lymphomas and other tumors of lymphoid origin, XV) the skin, XVI) cancers and tumor diseases of all anatomical structures belonging to the the respiration and respiratory systems including thoracal
  • the cDNAs coded by the SLC22A13 genes from human and rat were generated by RT-PCR, cloned into pUC19, fully sequenced, and inserted into expression vector pEBTetD.
  • pEBTetD is an episomal Epstein-Barr plasmid vector for doxycycline-inducible protein expression in human cell lines based on the simple tetracycline repressor [13].
  • the amino acid sequence of SLC22A13h corresponds to GenBank entry NM_004256.
  • the 5'-interface between pEBTetD and cDNA is GTTTAAACTT AAGCTT GCCACC ATGGCTCAGTTTGTC (polylinker in bold, cDNA underlined); the 3'-interface is AAGAGCTGGACC CTCGAG CGATCGC.
  • the amino acid sequence of SLC22A13r corresponds to GenBank entry NM_001126285 except for Ser (AGC) at position 352.
  • the 5'-interface is GTTTAAACTT AAGCTT GCCACC ATGGCCC AGTTTGC A ; the 3'-interface is TCCTACTTCTGA CTCGAG CGATCGCGGCCGC. Plasmid pEB TetD/O AT2h has been described previously [11].
  • 293 cells ATCC CRL-1573; also known as HEK-293 cells
  • HEK-293 cells a transformed cell line derived from human embryonic kidney
  • the growth medium was Dulbecco's Modified Eagle Medium (Life Technologies 31885-023, Invitrogen, Düsseldorf, Germany) supplemented with 10% fetal calf serum (PAA Laboratories, Colbe, Germany).
  • PAA Laboratories, Colbe, Germany fetal calf serum
  • Uptake buffer contains 125 mmol/1 NaCl, 25 mmol/1 HEPES-NaOH pH 7.4, 5.6 mmol/1 (+)glucose, 4.8 mmol/1 KCl, 1.2 mmol/1 KH2PO4, 1.2 mmol/1 CaCl 2 , and 1.2 mmol/1 MgS0 4 .
  • uptake buffer without KH2PO4 was used to avoid MS interference. After preincubation at 37 °C for at least 20 minutes in 4 ml of uptake buffer, the buffer was replaced with 2 ml of substrate in uptake buffer. The total substrate concentration if not indicated otherwise was 0.1 ⁇ / ⁇ for radiotracer assays and 10 ⁇ / ⁇ for unlabeled compounds (LC-MS/MS quantitation). Incubation at 37 °C was stopped after 1 min by rinsing the cells four times each with 4 ml ice-cold uptake buffer. In efflux experiments, 200 ⁇ of uptake buffer were repeatedly collected from the same dish.
  • Radioactivity was determined, after cell lysis with 0.1% v/v Triton X-100 in 5 mmol/1 TRIS-HCl pH 7.4, by liquid scintillation counting.
  • LC-electrospray ionization-MS/MS analysis cells were lysed with methanol and stored at - 20 °C. After centrifugation (1 min, 16000 x g, 20 °C) of thawed lysates, 20 ⁇ samples were analyzed by LC-MS/MS on a triple quadrupole mass spectrometer (4000 Q TRAP, Applied Biosystems, Darmstadt, Germany).
  • Atlantis HILIC Silica column (5 ⁇ , 3.0 x 50 mm; Waters); 10 mM ammoniumacetate pH 4.3, 0.1% formic acid in methanol, gradient: 0.3 ml/min, 90% B at 0 min, 10% B at 5 min, 90% B at 8 min, stop at 10 min.
  • the other amino acids were analyzed like Asp and Glu.
  • Atmospheric pressure ionization with positive or negative electrospray was used.
  • the optimal collision energy for argon-induced fragmentation in the second quadrupole was determined for each analyte.
  • solute content of cell lysates was determined for 4 conditions (paired dishes, incubation time 1 min): a) transporter expression on, uptake buffer; b) expression off, uptake buffer; c) expression on, substrate in uptake buffer; d) expression off, substrate in uptake buffer.
  • Acute uptake mediated by heterologously expressed carrier was then calculated as (c - a) - (d - b). This approach takes into account endogenous solute content and non-specific uptake.
  • Protein was measured by the BCA assay (Pierce) with bovine serum albumin as standard. The protein content of MS samples was estimated from 3 matched cell dishes.
  • lysates of cells with or without transporter expression are analyzed by fullscan LC-MS. From these data sets, gray scale images with axes of m/z and time are generated in which low intensities are rendered black and high intensities are rendered white. Finally, a difference image is created based on RGB pixel information, combining the red channel from the transporter active image with the green and blue channels from the transporter inactive image. Thus, compounds only present in the active or inactive data set can be spotted as red or cyan signals, respectively, while compounds present in equal amounts in both sets remain scales of gray.
  • LC-MS Difference Shading [11, 12] was employed to search for substrates.
  • guanidinosuccinic acid (GSA) by LC-MS/MS fragmentation analysis; a commercial sample displayed a matching product ion spectrum.
  • a difference image (Fig. 5) illustrates several further intracellular changes after incubation for 1 h with 1 mmol/1 orotic acid, a slow-uptake substrate introduced in a previous study [11].
  • Expression of SLC22A13h caused an increase of orotic acid and MES (a congener of taurine, present as buffer at 25 mmol/1 extracellular), but a decrease of GSA, glutamate (Glu), aspartate (Asp), and taurine (Tau).
  • a TaqManTM realtime PCR assay was employed on a 7900 HT Sequence Detection system (Applied Biosystems, Darmstadt, Germany) according to the manufacturer's protocols.
  • a TaqManTM realtime PCR assay 85 ⁇ g of total RNA was incubated for 1.5 h at 37 °C with 2 U/ ⁇ Omniscript reverse transcriptase (Qiagen, Hilden, Germany) in the supplied buffer plus 9.5 ⁇ random hexamer primer, 0.5 mM per dNTP, and 3000 U RNaseOUTTM (Invitrogen) in a final volume of 680 ⁇ .
  • the resulting cDNA was diluted 1 :10 with water and directly used as template in PCR.
  • a PCR reaction (20 ⁇ ) contained, in addition to 5 ⁇ cDNA and 10 ⁇ qPCR MasterMix Plus (Eurogentec, Seraing, Belgium), 0.2 ⁇ per SLC22A13 amplification primer (forward: SEQ ID NO 3; reverse: SEQ ID NO 4), and 0.2 ⁇ FAMTM/TAMRATM-labeled SLC22A13 probe (SEQ ID NO 5; designed to cross ex on boundaries).
  • the thermal protocol was set to 2 min at 50 °C, followed by 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C.
  • a sample which can be a chemical compound or an antibody acting as channel blocker or transport inhibitor is reacted in a reaction mixture simultaneously or in succession with an adhesive cell culture expressing the transporter of interest.
  • a part of the experiment is also a compound or peptide labeled radiochemically either with a tritium or 125- iodine label known to be specifically transported by the transporter of interest through the cell membrane.
  • a cell line expressing the transporter is cultured in an appropriate container (eg. 96 well plate for scintillation counting) and with an appropriate growth medium at an optimal cell density and temperature. Then, to determine the transport blocking or inhibiting properties of compounds, the growth medium is replaced by a buffer, eg. PBS containing the compounds or antibodies at a fixed or varying concentration.
  • an appropriate container eg. 96 well plate for scintillation counting
  • the buffer is replaced by a buffer containing the radiolabeled compound and incubated again for a specific time. Only if the transporter has not been blocked by the compounds, the radiolabeled compound is transported into the cells.
  • the buffer is removed and the cells are washed several times with a buffer without the radiolabeled compound. Finally the buffer is removed and replaced by cell lysis buffer and a scintillation fluid. The container is then counted in an appropriate scintillation counter.
  • a sample which can be a chemical compound acting as an agonist or antagonist or an antibody acting as an antagonist is reacted in a reaction mixture simultaneously or in succession with a receptor membrane preparation.
  • a part of the reaction mix is also a compound or peptide labelled radiochemically either with a tritium or 125-iodine label known to bind specifically to the transporter.
  • the receptor membrane preparation is mixed in an appropriate buffer with compounds or antibodies at varying concentrations for which the IC50 value is going to be determined.
  • the transporter/compound or antibody complex is incubated for a specific time until a steady state of binding and dissociation has formed.
  • the radiolabeled compound or peptide is added to the reaction mix.
  • the radiolabeled compound and the non-radiolabelled compounds/antibodies compete for the binding site of the transporter.
  • the unbound radiolabeled compound/peptide is separated from the receptor bound radiolabeled compound/peptide by means of filtration and subsequent washing with an appropriate buffer.
  • the transporter membrane/radiolabeled compound complex is bound to the filtration membrane, which is dried and an appropriate scintillator is added so the radioactive signal can be recorded by a suitable counter.
  • the bound and unbound separation is achieved by binding of the transporter membrane/compound complex to specific beads in a scintillation proximity assay (SPA). Only by binding of the receptor bound radiolabeled compound in a close proximity to the scintillation beads a scintillation signal can be recorded by a suitable counter. Radiolabeled compounds not in such a close proximity as the transporter membrane/compound complex don't give a signal.
  • SPA scintillation proximity assay
  • SLC22A5 is not a general drug transporter, but it efficiently translocates mildronate, Drug Metab Dispos, 37 (2009) 330-337.

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Abstract

Cette invention concerne l'identification de modulateurs du transporteur SLC22A13, la fonction de SLC22A13, et des utilisations thérapeutiques associées. Dans un mode de réalisation, cette invention concerne un procédé pour identifier et/ou obtenir un composé apte à moduler l'activité de transport de SLC22A13. Ledit procédé comprend la mise en contact d'un composé d'essai avec un système de mesure de ladite activité de transport, ledit système comprenant un polypeptide SLC22A13 ou un fragment fonctionnel de celui-ci, et un substrat pour mesurer l'activité de transport à l'aide dudit système; et la détection d'un niveau modifié de l'activité de transport du polypeptide SLC22A13 ou du fragment fonctionnel de celui-ci en présence du composé d'essai comparativement à l'activité de transport décrite en l'absence du composé d'essai et/ou en présence d'un témoin.
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