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WO1998038203A1 - Co-transporteur de sodium-phosphate dans le cadre d'une therapie au lithium aux fins du traitement de maladies mentales - Google Patents

Co-transporteur de sodium-phosphate dans le cadre d'une therapie au lithium aux fins du traitement de maladies mentales Download PDF

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Publication number
WO1998038203A1
WO1998038203A1 PCT/US1998/002875 US9802875W WO9838203A1 WO 1998038203 A1 WO1998038203 A1 WO 1998038203A1 US 9802875 W US9802875 W US 9802875W WO 9838203 A1 WO9838203 A1 WO 9838203A1
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Prior art keywords
lithium
seq
sodium
leu
val
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PCT/US1998/002875
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English (en)
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Robert B. Gunn
Richard T. Timmer
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Emory University
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Priority to AU63263/98A priority Critical patent/AU6326398A/en
Publication of WO1998038203A1 publication Critical patent/WO1998038203A1/fr
Priority to US10/314,790 priority patent/US20040002079A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • Na-PO 4 cotransport is the primary mechanism for the regulation of total body phosphate balance. It mediates both the gastrointestinal uptake and renal reabsorption of PO . Whereas 70% (high phosphate diet) to 85% (low phosphate diet) of dietary phosphate is absorbed by the GI tract the major control is in proximal reabsorption by the kidney of about 70% of the filtered load and in distal discretionary reabsorption of some fraction of the remainder. Na-PO 4 cotransport has been found in the plasma membrane of every mammalian cell examined.
  • phosphate metabolism includes most important biological molecules: nucleotides, DNA, RNA, glycolytic intermediates, phospholipids, and most proteins through regulatory or structural phosphorylations.
  • Na-PO 4 cotransport is essential for ATP synthesis.
  • Na-PO 4 cotransporters exist in the kidney, as two isoforms, type I and type II.
  • a related cotransporter is also present in liver where the protein has been partially purified, reconstituted in liposomes, and expressed in oocytes from liver mRNA. Its function in rat hepatocytes in primary culture is stimulated by insulin.
  • Na-PO cotransport activity has been extensively characterized in the duodenum/upper jejunum of many higher vertebrates. Neither the liver nor intestinal forms of the cotransporter have been cloned from mammalian sources. In contrast to mammals, teleosts appear to have an isoform of the Type II transporter present in the intestine.
  • BNPI brain-specific cDNA
  • the brain transporter is specific to the brain and mRNA transcripts are found in the neurons of the cerebral cortex, hippocampus, and cerebellum.
  • the neuronal transporter is also found in peripheral nerves and transports arsenate and Li + can substitute for Na + .
  • the erythrocyte form represents a third type of Na-PO 4 cotransporter, which applicants have discovered is also a retroviral receptor.
  • Retroviruses require specific cell-surface receptors for cell recognition and infection.
  • Two widely expressed mammalian retrovirus receptors PiT-1 (Glvr-1; Genbank L20859, U.S.Patent 5,414,076) and PiT-2 (Ram-1; Genbank L19931, U.S. Patent 5,550,221) have been cloned and shown to share 30% homology with Pho-4 + ', a phosphate uptake gene in Neurospora crassa and when these two mammalian genes are expressed in oocytes they induce sodium dependent phosphate cotransporter activity.
  • a murine cationic amino acid transporter has been shown to be a retrovirus receptor, thus, indicating there are at least two classes of transporters that are retrovirus receptors.
  • the two sodium phosphate cotransporters/retrovirus receptors (PiT-1 and PiT-2) are widely expressed in tissues and cells (thymus, marrow, lung, liver, heart, kidney, muscle and brain) and appear to be the ubiquitous housekeeping sodium-phosphate cotransporters that every cell requires in order to maintain the intracellular concentration of phosphate above electrochemical equilibrium.
  • the cotransporter/receptor isoforms in different species (human and mouse; rat and hamster) together with the differences in retroviral envelope proteins define the species specificity for susceptibility to infection by each retrovirus.
  • the ability to transfect cell lines from one species with the transporter/receptor isoform from another species and/or alter the envelope protein provides novel model systems and the means to design vectors that allow increased gene transfer into human hematopoietic progenitor cells and other cells.
  • a third cotransporter BNPI There are different isoforms of these three genes in different people.
  • cDNA and cRNA probes to PiT-1 or to PiT-2 and their mRNA products and antibodies to these proteins distinguish between individuals who are responders or non-responders to lithium treatment.
  • the sodium-phosphate cotransporter is the same cell membrane protein as the lithium-sodium countertransporter.
  • the present invention provides a readily performed diagnostic test to evaluate patient status, by measuring a combination of sodium, phophate or lithium flux in an in vitro membrane-based translation system.
  • Applicants have identified the gene product of PiT-1 as the lithium-sodium countertransporter across cell membranes.
  • the PiT-1 gene product is the erythrocyte isoform. Probes for this gene distinguish between responders and non-responders to lithium treatment.
  • Applicants have also identified the lithium-sodium countertransporter as the physiological mechanism for the extrusion of lithium from cells. It regulates the cell concentration of lithium. The activity of this transporter determines the therapeutic effect of lithium.
  • This invention provides a simple molecular biological test for the ability of cells to extrude lithium.
  • the only test to determine the activity of a lithium transporter is a laboratory measurement of lithium flux into or out of cells using chemical assays for lithium. See, e.g., Sarkadi, B. et al., J. Gen.Physiol.72 : 249 (1978).
  • the lithium-sodium countertransporter has significance for determining the responsiveness of humans with mental disorders to treatment with lithium salts. At present about half of patients treated with lithium do not improve. There are no techniques at present to diagnose whether a patient will be helped by lithium treatment, except by a time-consuming therapeutic trial.
  • the diagnostic test of the present invention allows genetic screening to predict whether a patient will respond to lithium transport. The test is also a screen for susceptibility to and extent of manic depressive illness. Further, the test is suitable to screen newborns in families with depression for their potential to develop the illness and whether they can respond to lithium treatment.
  • the sodium-phosphate cotransporter is identified as the same protein as the lithium-sodium countertransporter, and is suitable for diagnostic assays for mental illnesses susceptible to lithium therapy, including manic depression.
  • Various methods for evaluating the flux of lithium and other cations in appropriate cells are also disclosed, including reticulocytes.
  • Figure 1 is a schematic diagram of a cell 1 showing that the sodium-phosphate cotransporter is the same cell membrane protein as the sodium-lithium countertransporter.
  • the present invention relates to a purified DNA molecule coding for a lithium-sodium countertransporter. It also relates to a purified DNA molecule coding for an amino acid sequence selected from the group consisting of hPiT-1, hPiT-2, and hBNPI, said molecule useful for measuring lithium-sodium countertransport in human cells. Specifically, the present invention relates to a novel utility for the sequences identified as SEQ.ID.NO.: 1, SEQ.LD.NO.: 2, SEQ.LD.NO.: 3, SEQ.LD.NO.: 4, SEQ.LD.NO.: 5, and SEQ.LD.NO.: 6.
  • the human amphotrophic retrovirus receptor is useful as a lithium-sodium countertransporter, including the sequences identified as SEQ.LD.NO.: 1, SEQ.LD.NO.: 2, SEQ.LD.NO.: 3, SEQ.LD.NO.: 4, SEQ.LD.NO.: 5, and SEQ.LD.NO.: 6.
  • a first method of evaluating sensitivity to lithium therapy in manic depressive patients comprising the steps of
  • a second method of evaluating sensitivity to lithium therapy in manic depressive patients comprising the steps of (a) providing a sample of patient blood;
  • the first and second methods are drawn to sequences to any lithium-sodium countertransporter selected from the group consisting of hPiT-1, hPiT- 2, and hBNPI, said sequences identified as SEQ.LD.NO.: 1, SEQ.LD.NO.: 2, SEQ.LD.NO.: 3, SEQ.LD.NO.: 4, SEQ.LD.NO.: 5, and SEQ.LD.NO.: 6.
  • a third method of evaluating sensitivity to lithium therapy in manic depressive patients comprising the steps of
  • a fourth method of evaluating lithium-sodium countertransport in patients with mental illness comprising the steps of
  • Phosphorus is a major dietary element essential to most important biological molecules. Its absorption from the gut and reabsorption from the glomerular filtrate is by secondary active transport on a family of Na-PO 4 cotransporters.
  • the gene product responsible for this function in erythrocytes is pharmacologically distinct from the previously characterized renal brush border Na-PO cotransporter. Both the brain and peripheral nerve forms (PiT-1, PiT-2, BNPI) and the red blood cell form of the Na- PO 4 cotransporter can use Li + as a congener for Na + . Also, arsenate is transported by nerve membranes and probably by red blood cells. Therefore, these two tissues most likely have the same cotransporter/receptor.
  • the renal Na-PO 4 cotransporter presumably the apical isoform, has been cloned from several species. Applicants have identified the erythrocyte isoform as PiT-1.
  • FIG. 1 schematically shows a simplified diagram, with cell 1.
  • Lithium cation, Li + enters the cell as the anion LiCO 3 " , by the action of the AEl (Anion Exchange Protein, Band 3) countertransporter.
  • the lithium cation leaks into the cells by a minor unknown leak pathway.
  • Lt is pumped out by the sodium-phosphate cotransporter, which applicants have identified to be also the lithium-sodium countertransporter.
  • the hPiT-1 gene or other coding sequences for the lithium-sodium countertransporter are prepared for the expression systems and diagnostic assays of the present invention.
  • These polynucleotide sequences are prepared by ligation of other sequences, restriction endonuclease digestion, cloning, mutagenesis, organic synthesis, or combination thereof, in accordance with the principles and practice of constructing DNA sequences.
  • sequencing DNA e.g., verification of a construct at the end of a series of steps, dideoxy DNA sequencing is the preferred method.
  • Other DNA sequencing methods are well known.
  • Phosphoramidite chemistry in solid phase is the preferred method for the organic synthesis of oligodeoxynucleotides and polydeoxynucleotides. Many other organic synthetic methods are available and are readily adapted to the particular sequences of this invention by a person skilled in the art.
  • Amplification of DNA or cDNA is a common step in the detection of specific sequences in the diagnostic tests of the present invention. It is typically performed by the polymerase chain reaction (PCR). See, e.g., Mullins, K. et al., U.S. Pat. No.
  • the basic principle of PCR is the exponential replication of a DNA sequence by successive cycles of primer extension.
  • the extension products of one primer when hybridized to another primer, becomes a template for the synthesis of another nucleic acid molecule.
  • the primer template complexes act as substrate for DNA polymerase which, in performing its replication function, extends the primers.
  • the conventional enzyme for PCR applications is the thermostable DNA polymerase isolated from Thermus aquaticus, or Taq DNA polymerase. Numerous variations in the PCR protocol exist, and a particular procedure of choice in any given step in the constructions of this invention is readily performed by a skilled artisan.
  • primers for hPiT-1 are organically synthesized, based on its known sequence, and are hybridized to a sample of patient DNA. PCR in combination with reverse transcriptase, so-called RT-PCR, is then carried out to amplify the patient hPiT-1 genes. Subsequent analysis, e.g., by restriction fragment length polymorphism (RFLP), provides information on patient status.
  • RFLP restriction fragment length polymorphism
  • the source for mRNA is derived from enzymological manipulations, rather than isolation of naturally transcribed mRNA from, e.g., cell lines such as the erythroleukemic cell line K562.
  • Synthetic capped mRNA is preferably prepared by in vitro transcription of the appropriate linearized cDNA constructs containing the appropriate promoter for an RNA polymerase, e.g., T7 RNA polymerase. See, e.g., Fletcher, L. et al., J.Biol. Chem.265 : 19582 (1990), herein incorporated by reference for these purposes. Under these conditions, high yields of capped mRNA coding for hPiT-1, hPiT-2 or BNPI sequences are obtained, which migrate as a discrete band in gel electrophoresis.
  • the capped mRNA is then subjected to in vitro membrane-based translation, e.g., in Xenopus oocytes, microsomes or cultured cells, in an expression system designed to permit flux analysis of Na, PO 4 , and Li.
  • Preferred expression systems include Xenopus oocytes, and transfected HEK 293 cells.
  • Other suitable transfection systems include Dictyostelium discoideum cells, baculovirus-infected ceected Sf9 cells, and CHO cells. Selection of the appropriate cell system, as well as adjusting the experimental parameters to enhance translation, is readily determined within the skill of the art.
  • the gene for the countertransporter proteins such as the hPiT-1 gene, is also suitable for expression in an expression vector in a recombinant expression system.
  • the constructed sequence need not be the same as the original, or its complimentary sequence, but instead may be any sequence determined by the degeneracy of the DNA code. Conservative amino acid substitutions may also be employed, or other modifications, such as an amino terminal methionine.
  • a ribosome binding site active in the host expression system is Hgated to the 5' end of the chimeric coding sequence, giving a synthetic gene.
  • the resulting synthetic gene can be inserted into any one of a large variety of vectors for expression, by ligating to an appropriately linearized plasmid.
  • Expression in E. coli is suitable for expression of active lithium-sodium countertransporter protein, e.g., E. coli BL21.
  • a regulatable promoter is also suitable for the expression of these coding sequences, e.g., under the control of the E. coli lac promoter.
  • Other suitable regulatable promoters include trp, tac, recA, T7, lambda promoters.
  • the flux of a molecule is a measure of the number of molecules that cross the cell membrane per unit time and per unit of membrane (expressed either as area or number of cells or amount of cell protein).
  • the flux is measured by determining the appearance or disappearance (or both) of the molecule on one side of the membrane.
  • the amount on one side of the membrane is measured at different known times either by a chemical determination or by a radioactive determination if a tracer of the atoms or molecules is used.
  • Lithium, atomic number 3, atomic weight 6.9 Daltons has no radioactive isotopes of use for biological measurements. Chemical detemination must be used instead of a radioisotope.
  • the amount of lithium is most often determined by atomic absorption spectroscopy or emission spectroscopy.
  • the assay of lithium-sodium countertransport flux rate is made by the following steps:
  • the cells are mixed in a standard buffered solution containing sodium and lithium chloride solution, and subjected to repeated suspension, centrifugation, removal of supernatant fluid, and resuspension;
  • the cells in the standard solution are incubated in the presence of inhibitors of the Na, K, ATPase (e.g., ouabain at 10 "5 M) and in the presence of inhbitors of Anion Exchange protein (e.g., denitrostilbenedisulfonate at 2.5 x 10 "4 M), in suspension at body temperature;
  • inhibitors of the Na, K, ATPase e.g., ouabain at 10 "5 M
  • inhbitors of Anion Exchange protein e.g., denitrostilbenedisulfonate at 2.5 x 10 "4 M
  • RFLP restriction fragment length polymorphism
  • ASO allele-specific ohgonucleotides
  • Tagging or labeling the desired polynucleotide fragments can take various forms.
  • the radioisotope 32 P and other radioactive labels are not preferred because of laboratory safety and waste disposal requirements.
  • Alternative methods of labeling include chemical analogs, such as biotinylated analogs of TTP and UTP, which incorporate into the resulting DNA and RNA, respectively.
  • the biotin-labeled probe can be coupled to avidin, or streptavidin, and the complex detected by chemiluminescence, immunofluorescence, immunoperoxidase, immune colloidal gold techniques, or the like.
  • the biotin-labeled probe can also be detected with avidin conjugated to poly AP (calf intestinal alkaline phosphatase), assayed with the appropriate AP substrates.
  • Digoxigenin is a useful substitute for avidin in many applications, and it is readily detected with antibodies specific for digoxigenin.
  • Various combinations of such labels are readily carried out, e.g., a biotin-labeled probe detected with streptavidin conjugated to poly AP, or a biotin labeled probe detected with anti- biotin antibodies linked to AP, or other secondary labeling systems.
  • a biotin-labeled probe detected with streptavidin conjugated to poly AP or a biotin labeled probe detected with anti- biotin antibodies linked to AP, or other secondary labeling systems.
  • Monoclonal antibodies are the reagent of choice in the present invention, and a specifcally used to analyze patient cells for specific characteristics of the lithium-sodium countertransporter.
  • Monospecific antibodies to the lithium-sodium countertransporter are purified from mammalian antisera containing antibodies reactive against the lithium- sodium countertransporter or are prepared as monoclonal antibodies reactive with the lithium-sodium countertransporter using the technique of Kohler and Milstein, Nature 256: 495-497 (1975).
  • Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for the lithium-sodium countertransporter.
  • Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the lithium-sodium countertransporter, as described above.
  • the lithium-sodium countertransporter specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats* horses and the like, with an appropriate concentration of the lithium-sodium countertransporter either with or without an immune adjuvant.
  • Preimmune serum is collected prior to the first immunization.
  • Each animal receives between about 0.1 mg and about 1000 mg of the lithium-sodium countertransporter associated with an acceptable immune adjuvant.
  • acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA.
  • the initial immunization consists of the lithium-sodium countertransporter in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally ( P) or both.
  • SC subcutaneously
  • P intraperitoneally
  • Each animal is bled at regular intervals, preferably weekly, to determine antibody titer.
  • the animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of the antigen in Freund's incomplete adjuvant by the same route.
  • Booster injections are given at about three week intervals until maximal titers are obtained.
  • the animals are bled, the serum collected, and aliquots are stored at about -20° C.
  • Monoclonal antibodies (mAb) reactive with the lithium-sodium countertransporter are prepared by immunizing inbred mice, preferably Balb/c, with the lithium-sodium countertransporter.
  • the mice are immunized by the LP or SC route with about 0.1 mg to about 10 mg, preferably about 1 mg, of the lithium-sodium countertransporter in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred.
  • the mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks.
  • Immunized mice are given one or more booster immunizations of about 0.1 to about 10 mg of the lithium-sodium countertransporter in a buffer solution such as phosphate buffered saline by the intravenous (IN) route.
  • Lymphocytes from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art.
  • Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas.
  • Fusion partners may include, but are not limited to: mouse myelomas P3/ ⁇ Sl/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred.
  • the antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%.
  • Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.
  • DMEM Dulbecco's Modified Eagles Medium
  • Supernatant fluids are collected from growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using the lithium-sodium countertransporter as the antigen.
  • SPIRA solid phase immunoradioassay
  • the culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb.
  • Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.
  • Monoclonal antibodies are produced in vivo by injection of pristane primed Balb/c mice, approximately 0.5 ml per mouse, with about 2x10 6 to about 6x10 6 hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
  • In vitro production in anti-lithium-sodium countertransporter mAb is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb.
  • the mAb are purified by techniques known in the art.
  • Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, buy are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of the lithium-sodium countertransporter in body fluids or tissue and cell extracts.
  • Schizophrenic disorders are mental diseases with a tendency toward chronicity and are characterized by psychotic symptoms involving disturbances of thinking, feeling, and behavior.
  • Affective disorders also known as mood disorders, are psychopathologic states in which a disturbance of mood is either a primary determinant or constitutes the core manifestation.
  • a clinically useful division of affective disorders is bipolar (with periods of depression and elevation) and unipolar (depressions only) mood disturbances. Such bipolar mood disturbances are commonly known as manic depression.
  • Lithium usually given as a carbonate salt, attenuates bipolar mood swings, without affecting normal mood. It also appears to be useful in the treatment of aggressive personality disorders, which are typically classified outside of affective disorders. About 50% of bipolar patients respond to lithium therapy. Various clinical attributes are useful in assessing response to lithium, including the presence of manic episodes as the primary mood disorder, an episode frequency of less than about 2 years, as well as past or family history of lithium response. Applicants now provide another attribute to evaluate response to lithium, that is, lithium-sodium countertransport.
  • Lithium is commonly used to treat affective disorders.
  • the site of action of lithium is believed to be in the brain.
  • the steady state ratio of intracellular red blood cell lithium concentration to plasma lithium concentration during therapy shows great interindividual variation, although the lithium ratio is relatively constant for any one individual. Individual fluctuations of the lithium ratio have also been reported. The relative constancy of the ratio in an individual may be genetically determined.
  • the steady state lithium ratio across the red cell membrane is the result of three lithium transport processes: the Na,K,ATPase which is inhibited by ouabain and other cardiac glycos des, the anion exchange protein (AEl, band 3) and the lithium-sodium countertransport system.
  • the Na, K, ATPase pumps Li into the cell by substituting Li + at a normal K + binding site, but at therapeutic levels of Li + (l-2mM) and normal plasma Na and normal plasma K, the Na,K ATPase carries Li poorly ( «0.025 mmol/lit cell • h; «75 ⁇ mol/kg Hgb • h).
  • the anion exchanger is the principle mediator of the inward leak of Li as the ion pair LiCO 3 " . This transport is inhibited by stilbene disulfonates (SITS, DNDS, DLDS), phloretin and dipyridamol.
  • the lithium-sodium countertransporter normally pumps Li out of the cell against its electrochemical gradient so that [Li] ce ⁇ is lower than [Li] p ⁇ and [Li] ce ⁇ /[Li] p ⁇ , which lithium ratio is 0.2 to 0.8 in different individuals.
  • a higher steady ratio is expected in alkalosis (higher plasma HCO 3 " , CO 3 2" , and LiCO 3 ' ) due to increased AEl mediated leak into the cell, or when the lithium-sodium countertransport extrusion of lithium is slowed, either because cell Na + gradient is decreased or because the countertransporter is less effective.
  • the lithium-sodium countertransporter as the product of PiT-1 gene previously identified as retrovirus receptor and a NaPO 4 cotransporter.
  • the red cell NaPO 4 cotransporter transports Li instead of Na (i.e., LiPO 4 cotransport) and that is performs Na/Na exchange and lithium-sodium countertransport.
  • the lithium ratio has been implicated in the responsiveness of polar disease to lithium treatment, the development of essential hypertension (hypertension of unknown etiology), the'susceptibility of individuals to affective (bipolar) disorders, and the toxic side effects of Li therapy.
  • Lithium influxes, 32 PO influxes, sodium effluxes were measured in human red blood cells incubated in an isotonic media containing (mM): 150 (Na + Li + K)C1, 0.3 K 2 HPO 4 , 20 HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), 0.25 DNDS (4,4'-dinitro stilbene-2,2'-disulfonate to inhibit the Anion Exchange Protein Band3 pathway for phosphate transport), titrated to pH 7.64 with KOH at 20°C to give a pH of 7.40 at 37°C where the fluxes were performed.
  • hPiT-1 Human PiT-1 (hPiT-1) was cloned as the human isoform of the gibbon ape retrovirus receptor [Van Zeijl, M., et al (Proc. Nat. Acad. Sci. 91 : 1168 (1994)].
  • PCR primers were designed to amplify either the hPiT-1 or hPiT-2 isoforms.
  • the 1700 bp product was amplified by RT-PCR from total RNA isolated from K562 cells, and restriction analysis with Sphl identifies the product as being derived from hPiT-1 and not hPiT-2. This evidence, considered together with the kinetic evidence of preceeding Example, indicates that hPiT-1 is the sodium-phosphate cotransporter isoform present in both K562 cells and erythrocytes.
  • RT-PCR was carried out using 1 ⁇ g of total RNA isolated from K562 cells using the method of Chomczynski et al., Analytical Biochem. 162: 156 (1987).
  • the RT-PCR reaction was carried out in a single tube using recombinant Tth DNA- polymerase which is capable of reverse transcriptase activity under appropriate reaction condition.
  • the primers used in these experiments (FI and Rl) are based upon highly conserved regions between hPiTl and hPiT2 located in putative transmembrane domains in the N-terminal and C-terminal regions of the proteins.
  • the results from these experiments show that the products of Sphl digestion are 1000, 487 and 138 bp, which agree with the predicted sizes.
  • the gel patterns under the PiT-1 control after Sphl digestion are the same as for K562, after Sphl digestion; and both are different from those predicted for PiT-2 after Sphl digestion.
  • K562 cells have the human PiT-1
  • the template for expression of the Na-PO 4 cotransporter in the cell-free system or in Xenopus oocytes is capped mRNA prepared polymerase, prepared by in vitro transcription of linearized cDNA constructs containing the promoter for T7 RNA polymerase.
  • Samples of mRNAs are prepared as prepared as previously described in a 1 ml reaction mixture [Fletcher, L. et al., J. Biol. Chem.265; 19582(1990)]. These reaction conditions permit high yields of mRNA (-1000 mg or about 100-200 copies of RNA transcript per copy of template DNA) that are >80% capped and migrate as a single discrete band of the correct molecular weight on denaturing PAGE.
  • T7 RNA polymerase is purified as described by Davanloo, et al. Proc. Natl.Acad.Sci.Sl;,2035(l9S4).
  • the Na-PO cotransporter mRNA is expressed in a wheat germ cell-free system containing in vitro transcribed mRNA, dog pancreatic microsomes and signal recognition particle (SRP).
  • Cell-free translation is carried out in the presence of 34 mM [ 14 C]leucine (50 cpm/pmol), 50 mM each of the other 19 amino acids, unfractionated wheat germ tRNA and other components a previously described [Erickson, AH et al., Methods Enzymol. 96;38 (1983)].
  • the expression of the sodium-phosphate cotransporter is carried out in several heterologous expression systems, e.g. Dictyostelium discoideum cells, Xeonpus oocytes, HEK 293 cells, baculovirus-infected Sf9 cells, and CHO cells. Transfection, growth and selection of transformants are performed by well known techniques.
  • the expression of the Na-PO 4 cotransporter is assessed by immunoprecipitation with Na- PO 4 - specific antibodies of the protein from cells (Dictyostelium, HEK 293, etc.) grown in medium supplemented with 35 S-methionine.
  • the functional expression of the Na-PO transporter, both native and mutant forms, in transfected cells is monitored by determining the Na-dependent 32 Na-PO 4 flux , as described in an Example below. Negative controls include determining background levels of Na-dependent Na-PO 4 transport from cells transfected with the construct in the anti-sense orientation.
  • the two principal expression systems for heterologous expression of the erythrocyte Na- PO 4 cotransporter are injected Xenopus oocytes and transfected Dictostelium.
  • the cotransporter is expressed in another expression system such as HEK 293 or baculovirus-infected Sf9 cells.
  • stage V and IN oocytes are removed using standard anesthetic (0.17% 3-aminobenzoic acid) and surgical procedures.
  • the oocytes are placed in OR-2 medium and collagenase treated (2mg/ml) for 2.5 h.
  • oocytes are washed and defolliculated if needed by trituration and co-injected with 2.5 ng capped SEAP cR ⁇ A and 5-50 ng of capped transporter of cR ⁇ A (prepared as described above).
  • Capped SEAP cR ⁇ A prepared by in vitro transcription o ⁇ HIndlll linearized pGEM-SEAP.
  • the pGEM-SEAP construct contains the human placental alkaline phosphatase with a site-specific mutation at codon 489 to create a termination codon[Tate, S.S. et al., FASEB J. 4; 228 (1990)]. This stop codon results in a secreted form of alkaline phosphatase rather than a membrane anchored form.
  • cR ⁇ As are injected in a total volume of 50 nl using a ⁇ arishige injector. Following incubation overnight in Barth's medium, oocytes are sorted and placed in single wells of
  • alkaline phosphatase activity is measured by chromogenic assay.
  • the secreted alkaline phosphatase catalyzes the dephosporylation of nicotinamide adenine dinucleotide phosphate (NADP + ); the NAD formed then catalytically activates an NAD + -specific oxidation-reduction cycle driven by the enzymes alcohol dehydrogenase and diaphorase.
  • the chromophore formed is a violet colored formazan product of LNT-violet. Only those oocytes that express SEAP
  • Dictyostelium A second expression system for heterologous expression of the cloned cotransporter is Dictyostelium.
  • a significant advantage of the Dictyostelium expression system is that these cells are grown in suspension culture and are handled like red cells for flux measurements.
  • the principle expression vector e.g. pBS18 and its derivatives
  • Tn5 gene neomycin phosphotransferase II
  • the insert of interest is driven by the actin 8 promoter and the 2H3 transcriptional terminator.
  • the Tn5 gene permits selection of permanent transfectants in media containing G-418, to which the native slime mold is highly sensitive.
  • HEK-239 cells C. HEK-239 cells. Heterologus expression is also readily carried out with HEK- 293 cells, ATCC Accession No. CRL 1573.
  • HEK-293 (human embryonic kidney) cells were obtained from the American Type Culture Collection (ATCC) at passage 31. These cells were used to prepare seed stocks at passage 32. Cells were used until passage 45, after which fresh cultures were started from frozen passage 32 cells. The cells were grown in Minimal Essential Media (MEM) with Hank's salts and supplemented with L-glutamine and 5% fetal calf serum at 37°C in 5% CO 2 / 95% air. Transfection was carried out using standard calcium phosphate precipitation methods.
  • MEM Minimal Essential Media
  • FLUX MEASUREMENTS A. Flux measurements in cell-free expression system. The flux is measured by an adaptation of a rapid filtration method according to Macintyre, J.D. et al. Biochim. Biophys. Acta 644; 351 (1981). Briefly, microsomes are suspended, equilibrated and mixed in media containing either sodium or choline or N-methyl-alpha- glucamine as the dominant cation in a thermostatically controlled chamber. The flux is initiated by additional of 32 PO to the flux medium. Aliquots are removed at different times and filtered under vacuum using prewashed mixed cellulose-ester filters.
  • microsomes retained by the filter are rapidly washed with stopping solution containing 323 mM MgSO 4 (isotonic for microsomes).
  • a sample of flux suspension is used to measure total protein and specific activity.
  • the flux (pmol PO- ⁇ ig protein-min or ions/cotransporter molecule-min) is calculated from the slope of cpm/aliquot versus time.
  • Preloaded microsomes are used to verify the quantitative recovery of microsomes, the replication of sample counts, and the effectiveness of the wash in removing a extracellular marker, usually 14 C-PEG at
  • Oocytes Flux measurements in oocytes.
  • Oocytes are prepared and injected as described in Example above. Briefly, eight to ten oocytes are placed in individual wells of a 96- well culture plate in medium containing either Na or choline as the dominant cation. The flux is initiated by addition of 32 PO 4 to each well. As known times, oocytes are removed and washed three times in ice cold choline medium. The oocyte is then dissolved in 0.2 ml 10% SDS and the counted in a liquid scintillation counter. A sample of the 32 PO 4 incubation fluid is counted to calculate the extracellular specific activity. The influx (pmol/oocyte/hr) is calculated from the specific activity and the uptake. The difference between the flux in Na and choline media is the calculated
  • HL-5 medium contains 20 + 3 mM K. This is not a defined medium so the composition must be determined for each flux.
  • " -Cells are grown to a density of 1-3 x 10 6 /ml at 20°C in a shaking incubator. Approximately 10 7 cells are required for each data point on the influx curve. The cells are resuspended to 4 x 10 7 /ml in HL-5 in a thermostatted stirred chamber at a known pH. At time zero tracer (-0.6 ⁇ Ci/ml) is added and at known times thereafter samples (0.4 ml) are removed.
  • the samples are transferred to 7 ml of ice-cold stop solution (58 mM MgSO 4 ), immediately centrifuged for 30 seconds at 3000 x g in a rotor, the supernatant aspirated and discarded as radioactive waste.
  • the pellet of cells is resuspended thrice in 6 ml of stop solution, pelleted, and the supernatant aspirated.
  • To the drained pellets 1 ml of 0.1% DOC in 1 N NaOH is added for solubilization. Aliquots are counted for radioactivity or are assayed for protein.
  • the data are calculated as pmoles/mg protein and the data vs. time are fitted to a single exponential by nonlinear regression analysis and the inital slope (flux) and asymptote value constant (pmoles/mg protein) calculated.
  • HEK-293 Human embryonic kidney cells (HEK-293) are purchased from the American Type Culture Collection (ATCC, accession number CRL 1573) at passage 32 and grown in MEM and 5% fetal calf serum in a 5% CO /95% air 37°C incubator. They are maintained in T75 flasks and split weekly. At passage 45 decreased expression is observed, so frozen stocks at passage 34 are brought up.
  • the cells are transfected with cDNA harvested from bacteria and purified on an anion exchange column. At 24 hours the cells are plated onto 24-well plates at 3-5 x 10 5 cells/cm 2 and transport measured at 48-72 hr. The expression is low by 96 hr and absent at 5 and 10 days.
  • the medium is aspirated and washed once with 0.5 ml of a Na-free (143 mM N-methyl-D- glucamine Cl) HCO 3 -free MEM-like HEPES buffered medium for 3-5 min. Then it is preincubated at 37°C in room air for 30 minutes. The flux is initiated by adding 32 PO 4 or 22 Na containing media. Cells transfected with the vector only (e.g., pRBG4) are always treated and fluxed in parallel. 32 PO 4 influx in the absence of Na is always " measured. All fluxes are performed in duplicate.
  • a Na-free (143 mM N-methyl-D- glucamine Cl) HCO 3 -free MEM-like HEPES buffered medium for 3-5 min. Then it is preincubated at 37°C in room air for 30 minutes. The flux is initiated by adding 32 PO 4 or 22 Na containing media. Cells transfected with the vector only (e.g., pRBG4) are always
  • the plates are placed on a water thermostatted table for 5 minutes and the flux initiated by aspirating the preincubation medium from the last column of wells and adding the tracer solution at known times ( ⁇ 0.2 sec). This is done to successive columns of cells at approximately 30 minutes, 15 minutes, 10 minutes, 5 minutes, 2 minutes and 1 minute prior to terminating the influx simultaneously for all wells on the plate by 3 rapid ice cold washes (over 15 seconds total elapsed time) with (mM): 150 NaCl, 1.5 CaCl 2 , 1 MgCl 2 solution. Residual wash solution is aspirated and the cells in the dry wells are solubilized in 0.5 ml of 25 mM NaOH with 0.5% deoxycholate.
  • a 50 ⁇ l sample from each well is used to measure protein and 400 ⁇ l sample is counted in a liquid scintillation counter.
  • Quadruplicate 10 ⁇ l samples of the influx solution are counted contemporaneously with the flux samples for specific activity determination.
  • the pmol/ ⁇ g protein in each well is calculated and the slope of the linear least squares best fit to these values against sample times is the computed flux, according to Sarkadi, B. et al., J.Gen.Physiol.72 249 (1978).
  • 5 or 6 of the data points are used to calculate each flux.
  • Each condition is always measured in duplicate in both vector-only transfected and vector+insert transfected cells.
  • the hPiT-1 DNA was isolated, cloned and sequenced according to 5,414,076, herein incorporated by reference for this purpose. It is set forth as SEQUENCE LD NO.:l and SEQUENCE ID NO.:2.
  • the hPiT-2 DNA was isolated, cloned and sequenced according to 5,550,221, herein incorporated by reference for this purpose. It is set forth as SEQUENCE LD NO.:3 and SEQUENCE LD NO.:4.
  • BNPI DNA was isolated, cloned and sequenced according to Ni, B et al., J.Neurochem.66, 2227 (1996), herein incorporated by reference for this purpose. It is set forth as SEQUENCE LD NO.:5 and SEQUENCE ID NO.:6.
  • Polyclonal antibodies are prepared according to England, B.J. et al.,
  • hPiT-1 RESTRCITION LENGTH FRAGMENT POLYMORPHISM ANALYSIS A.
  • primers for hPiT-1 for example, CAGTTCAGTC AAGCCGTCAG ( SEQ ID NO : 7 ) and CCAGCCAACA GACACAACAG ( SEQ ID NO : 8 )
  • the hPiT-1 sequence is amplified by PCR and ASO, by the methods of Connor, B.J.et al., Proc.Natl.Acad.Sci. 80: 278 (1983), and Saiki, R.K. et al., Nature 324: 163
  • hPiT-2 sequence is amplified by PCR and ASO, by the methods of Connor, B.J.et al., Proc.Natl.Acad. Sci. 80: 278 (1983), and Saiki, R.K. et al., Nature 324: 163
  • BNPI sequence is amplified by PCR and ASO, by the methods of Connor, B.J.et al., Proc.Natl.Acad.Sci. 80: 278 (1983), and Saiki, R.K. et al, Nature 324: 163
  • CTCCGTGGGA GCCAATGATG TAGCAAATTC TTTTGGTACA GCTGTGGGCT CAGGTGTAGT 540
  • AGCACTTTCC AGAGCAAACT CACTGTTTAC AAGAACTCTT GGCCTTACGA AGTTTATAAC 180
  • CTCCGGGCAC TCCCAGTATT CTATGCTGCT ACCATAGCAA TCAATGTCTT TTCCATCATG 840
  • CTCCGCCGAA ACAACAGTTA CACCTGCTAC ACCGCAGCCA TTTGTGGGCT GCCAGTGCAC 1440
  • GGCTCCATCC CACACTGTTC ACCCAGGCTG CGGAGACTCA CCTTCCCGAG CTAACTTAAC 2400 TACTGTACAT AATAATATGT ATTAAACTGG TATCGTGGTG ATATAATGTG GTGCAGTTAC 2460
  • CCGCCGCATC ATGTCCACCA CCAACGTGCG CAAGTTGATG AACTGCGGAG GCTTCGGCAT 1620
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FEATURE
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

Le co-transporteur de sodium-phosphate présent dans presque toutes les cellules humaines, qui est identifié comme étant la même protéine que celle du contre-transporteur de lithium-sodium, se prête fort bien à des épreuves diagnostiques de maladies mentales sensibles à une thérapie au lithium, au nombre desquelles la psychose maniaco-dépressive.
PCT/US1998/002875 1997-02-27 1998-02-11 Co-transporteur de sodium-phosphate dans le cadre d'une therapie au lithium aux fins du traitement de maladies mentales WO1998038203A1 (fr)

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AU63263/98A AU6326398A (en) 1997-02-27 1998-02-11 Sodium-phosphate cotransporter in lithium therapy for the treatment of mental illness
US10/314,790 US20040002079A1 (en) 1998-02-11 2002-12-09 Sodium-phosphate cotransporter in lithium therapy for the treatment of mental illness

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10147006A1 (de) * 2001-09-24 2003-04-17 Gruenenthal Gmbh Screeningverfahren für verschiedene Indikationen mit BNPI und/oder DNPI

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5288678A (en) * 1990-10-19 1994-02-22 Beckman Instruments, Inc. Indirect potentiometric method and diluent for analysis of lithium
US5414076A (en) * 1989-08-24 1995-05-09 American Cyanamid Company DNA encoding gibbon ape leukemia virus receptor
US5550221A (en) * 1993-04-16 1996-08-27 American Cyanamid Company Nucleic acid molecule encoding the amphotropic virus receptor
US5618918A (en) * 1995-04-27 1997-04-08 Eli Lilly And Company Human brain sodium dependent inorganic phosphate cotransporter

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5414076A (en) * 1989-08-24 1995-05-09 American Cyanamid Company DNA encoding gibbon ape leukemia virus receptor
US5288678A (en) * 1990-10-19 1994-02-22 Beckman Instruments, Inc. Indirect potentiometric method and diluent for analysis of lithium
US5550221A (en) * 1993-04-16 1996-08-27 American Cyanamid Company Nucleic acid molecule encoding the amphotropic virus receptor
US5633348A (en) * 1993-04-16 1997-05-27 American Cyanamid Company Amphotropic virus receptor
US5618918A (en) * 1995-04-27 1997-04-08 Eli Lilly And Company Human brain sodium dependent inorganic phosphate cotransporter
US5618677A (en) * 1995-04-27 1997-04-08 Eli Lilly And Company Human brain sodium dependent inorganic phosphate cotransporter assay
US5686266A (en) * 1995-04-27 1997-11-11 Eli Lilly And Company Human brain sodium dependent inorganic phosphate cotransproter and related nucleic acid compounds

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JOHANN S. V., ET AL.: "A HUMAN AMPHOTROPIC RETROVIRUS RECEPTOR IS A SECOND MEMBER OF THE GIBBON APE LEUKEMIA VIRUS RECEPTOR FAMILY.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 91., 1 February 1994 (1994-02-01), US, pages 1168 - 1172., XP002910069, ISSN: 0027-8424, DOI: 10.1073/pnas.91.3.1168 *
KLINGER H. P., ET AL.: "CHARACTERIZATION OF A HUMAN GENE CONFERRING SENSITIVITY TO INFECTION BY GIBBON APE LEUKEMIA VIRUS.", CELL GROWTH AND DIFFERENTIATION., THE ASSOCIATION, PHILADELPHIA, PA, US, vol. 01., 1 January 1990 (1990-01-01), US, pages 119 - 127., XP002910068, ISSN: 1044-9523 *
MILLER D. G., ET AL.: "CLONING OF THE CELLULAR RECEPTOR FOR AMPHOTROPIC MURINE RETROVIRUSES REVEALS HOMOLOGY TO THAT FOR GIBBON APE LEUKEMIA VIRUS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 91., 1 January 1994 (1994-01-01), US, pages 78 - 82., XP002910070, ISSN: 0027-8424, DOI: 10.1073/pnas.91.1.78 *
NADI N. S., ET AL.: "CLONING AND EXPRESSION OF A CDNA ENCODING A BRAIN-SPECIFIC NA+-DEPENDENT INORGANIC PHOSPHATE COTRANSPORTER.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 91., 1 June 1994 (1994-06-01), US, pages 5607 - 5611., XP002910071, ISSN: 0027-8424, DOI: 10.1073/pnas.91.12.5607 *
WILSON C. A., ET AL.: "PROPERTIES OF A UNIQUE FORM OF THE MURINE AMPHOTROPIC LEUKEMIA VIRUS RECEPTOR EXPRESSED ON HAMSTER CELLS.", JOURNAL OF VIROLOGY., THE AMERICAN SOCIETY FOR MICROBIOLOGY., US, vol. 68., no. 12., 1 December 1994 (1994-12-01), US, pages 7697 - 7703., XP002910072, ISSN: 0022-538X *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10147006A1 (de) * 2001-09-24 2003-04-17 Gruenenthal Gmbh Screeningverfahren für verschiedene Indikationen mit BNPI und/oder DNPI

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