+

WO2002052003A2 - Regulation du canal calcium humain - Google Patents

Regulation du canal calcium humain Download PDF

Info

Publication number
WO2002052003A2
WO2002052003A2 PCT/EP2001/015088 EP0115088W WO02052003A2 WO 2002052003 A2 WO2002052003 A2 WO 2002052003A2 EP 0115088 W EP0115088 W EP 0115088W WO 02052003 A2 WO02052003 A2 WO 02052003A2
Authority
WO
WIPO (PCT)
Prior art keywords
calcium channel
polypeptide
polynucleotide
seq
channel protein
Prior art date
Application number
PCT/EP2001/015088
Other languages
English (en)
Other versions
WO2002052003A3 (fr
Inventor
Alex Smolyar
Original Assignee
Bayer Aktiengesellschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Aktiengesellschaft filed Critical Bayer Aktiengesellschaft
Publication of WO2002052003A2 publication Critical patent/WO2002052003A2/fr
Publication of WO2002052003A3 publication Critical patent/WO2002052003A3/fr

Links

Classifications

    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • 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

Definitions

  • Ion channels are integral membrane proteins, typically comprising multiple subunits, which form selective and highly regulated pores in cellular membranes. Each of these pores controls the influx and efflux of a given ion (e.g., sodium, potassium, calcium, or chloride) across the plasma membrane or the membranes of intracellular compartments.
  • a given ion e.g., sodium, potassium, calcium, or chloride
  • Many important physiological processes depend on the control of ion gradients by ion channels. Such processes include synaptic transmission, secretion, fertilization, muscle contraction, and regulation of intracellular and extracellular ion concentrations and pH.
  • Ion channels open in response to various stimuli. For example, there are ligand-gated channels, second messenger-gated channels, voltage- gated channels, and shear- or stress-gated channels.
  • Fig. 12 shows the BLASTP - alignment of 405 (SEQ ID NO:2) against swiss
  • Human calcium channel is 26% identical over 315 amino acids to swiss
  • Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions.
  • Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • cDNA Complementary DNA
  • species homologs, and variants of calcium channel polynucleotides that encode biologically active calcium channel polypeptides also are calcium channel polynucleotides.
  • Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO:l or its complement also are calcium channel polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.
  • Nucleotide sequences which hybridize to calcium channel-like polynucleotides or their complements following stringent hybridization and/or wash conditions also are calcium channel polynucleotides.
  • Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR
  • pIN vectors Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989
  • pGEX vectors Promega, Madison, Wis.
  • 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.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical).
  • Sequences encoding a calcium channel-like polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980).
  • a calcium channel-like polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation.
  • a Hposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Patent 5,705,151).
  • a reagent such as an antisense oligonucleotide or ribozyme
  • from about 0.1 ⁇ g to about 10 ⁇ g of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 ⁇ g to about 5 ⁇ g of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 ⁇ g of polynucleotides is combined with about 8 nmol liposomes.
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
  • cloned DNA segments can be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
  • the antisense oligonucleotides are administered to a patient with hypertension.
  • the severity of the patient' s hypertension is decreased.
  • test oligonucleotide for seven days results in significantly reduced expression of human calcium channel as determined by Western blotting. This effect is not observed with the control oligonucleotide.
  • the number of cells in the cultures is counted using an automatic cell counter. The number of cells in cultures treated with the test oligonucleotide (expressed as 100%) is compared with the number of cells in cultures treated with the control oligonucleotide. The number of cells in cultures treated with the test oligonucleotide is not more than 30% of control, indicating that the inhibition of human calcium channel has an anti-proliferative effect on cancer cells.
  • Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal
  • Inflammatory pain is induced mainly in rats by injection of 0.75 mg carrageenan or complete Freund's adjuvant into one hind paw.
  • the animals develop an edema with mechanical allodynia as well as thermal hyperalgesia.
  • Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer,
  • Compounds are tested against uninflamed as well as vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c, intradermal, transdermal
  • Forelimb akinesia is assessed three weeks following lesion placement using a modified stepping test protocol.
  • the animals are held by the experimenter with one hand fixing the hindlimbs and slightly raising the hind part above the surface.
  • One paw is touching the table, and is then moved slowly sideways (5 s for 1 m), first in the forehand and then in the backhand direction.
  • the number of adjusting steps is counted for both paws in the backhand and forehand direction of movement.
  • the sequence of testing is right paw forehand and backhand adjusting stepping, followed by left paw forehand and backhand directions.
  • the test is repeated three times on three consecutive days, after an initial training period of three days prior to the first testing.
  • Forehand adjusted stepping reveals no consistent differences between lesioned and healthy control animals. Analysis is therefore restricted to backhand adjusted stepping.
  • Balance Test
  • a modified version of the staircase test is used for evaluation of paw reaching behavior three weeks following primary and secondary lesion placement.
  • Plexiglass test boxes with a central platform and a removable staircase on each side are used.
  • mice are perfused transcardially with 0.01 M PBS (pH 7.4) for 2 min, followed by 4% paraformaldehyde (Merck) in PBS for 15 min.
  • the brains are removed and placed in 4% paraformaldehyde for 24 h at 4 °C. For dehydration they are then transferred to a 20% sucrose (Merck) solution in 0.1 M PBS at 4 °C until they sink.
  • the brains are frozen in methylbutan at -20 °C for
  • mice with specific brain lesions which impair cognitive functions, or animals treated with compounds such as scopolamine or MK-801, which interfere with normal learning, or aged animals which suffer from cognitive deficits are used.
  • the T-maze spontaneous alternation task is used.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Toxicology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des réactifs régulant les canaux calcium humains et des réactifs se liant à des produits géniques de type canal calcium humain, qui peuvent jouer un rôle dans la prévention, l'amélioration ou l'atténuation de dysfonctionnements ou de maladies comprenant notamment, mais pas exclusivement, le cancer, des troubles du SNC, des troubles cardio-vasculaires et des affections hématologiques.
PCT/EP2001/015088 2000-12-26 2001-12-19 Regulation du canal calcium humain WO2002052003A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US25729700P 2000-12-26 2000-12-26
US60/257,297 2000-12-26
US28011301P 2001-04-02 2001-04-02
US60/280,113 2001-04-02

Publications (2)

Publication Number Publication Date
WO2002052003A2 true WO2002052003A2 (fr) 2002-07-04
WO2002052003A3 WO2002052003A3 (fr) 2002-12-27

Family

ID=26945881

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2001/015088 WO2002052003A2 (fr) 2000-12-26 2001-12-19 Regulation du canal calcium humain

Country Status (1)

Country Link
WO (1) WO2002052003A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999046383A1 (fr) * 1998-03-13 1999-09-16 Brown University Research Foundation Isoforme de canal calcique humain de type n et utilisations
EP1328633A2 (fr) * 2000-07-28 2003-07-23 Lexicon Genetics Incorporated Nouvelles proteines du canal ionique humain et polynucleotides codant ces dernieres

Also Published As

Publication number Publication date
WO2002052003A3 (fr) 2002-12-27

Similar Documents

Publication Publication Date Title
US20040038365A1 (en) Regulation of human lysosomal acid lipase
WO2002066627A1 (fr) Regulation de la dipeptidyle peptidase 8 humaine
WO2002072824A2 (fr) Regulation du canal potentiel humain du recepteur transitoire humain
US20040115675A1 (en) Regulation of human transient receptor potential channel
US20040152092A1 (en) Regulation of human phosphatidic acid phosphatase type 2c-like protein
WO2002088363A2 (fr) Regulation de nouvelles prolyl 4-hydroxylases humaines
US20040241796A1 (en) Regulation of human nek-like serine/threonine protein kinase
WO2002062974A2 (fr) Regulation de la proteine humaine elongase hselo1
WO2002034920A2 (fr) Regulation de la proteine de type ceruloplasmine humaine
WO2002052003A2 (fr) Regulation du canal calcium humain
WO2002055556A2 (fr) Regulation de la proteine a canal de potassium a porte de tension humaine kv2.2
WO2002055710A2 (fr) Regulation de l'acide phosphatase pourpre humain
WO2002048324A1 (fr) Regulation de l'enzyme e2 humaine conjuguant l'ubiquitine
WO2003018815A2 (fr) Regulation de la grk humaine
WO2002064761A2 (fr) Accelerateurs promoteurs d'adhesion de cable d'acier
WO2003033709A2 (fr) Regulation de la proteine kinase serine/threonine humaine
WO2002038776A1 (fr) Régulation de la ligase coa d'acides gras humains
WO2003025174A2 (fr) Regulation de la proteine humaine de type mrp1
WO2003000874A2 (fr) Regulation de la proteine kinase serine/threonine humaine nek3
WO2002022791A2 (fr) Regulation de la pyridoxine 5'-phosphate oxydase humaine
US20040171539A1 (en) Regulation of human protein kinase-like protein
WO2002090543A2 (fr) Régulation de la protéine semblable à celle de type 2c de la phosphatase de l'acide phosphatidique humain
WO2002036753A2 (fr) Régulation de la lipase triacylglycérol humaine
WO2003000873A2 (fr) Régulation de la sérine/thréonine protéine kinase humaine nek1
EP1360281A2 (fr) Regulation de la serine/threonine proteine kinase humaine de type wee1

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载