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WO2000070347A1 - Dosages pour agonistes, antagonistes et agonistes inverses d'hormone concentrant la melanine (mch) se liant au recepteur du type somatostatine (slc-1) - Google Patents

Dosages pour agonistes, antagonistes et agonistes inverses d'hormone concentrant la melanine (mch) se liant au recepteur du type somatostatine (slc-1) Download PDF

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Publication number
WO2000070347A1
WO2000070347A1 PCT/SE2000/001010 SE0001010W WO0070347A1 WO 2000070347 A1 WO2000070347 A1 WO 2000070347A1 SE 0001010 W SE0001010 W SE 0001010W WO 0070347 A1 WO0070347 A1 WO 0070347A1
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WO
WIPO (PCT)
Prior art keywords
mch
test compound
slc
receptor
agonists
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PCT/SE2000/001010
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English (en)
Inventor
Sultan Ahmad
Jack Cao
Eric Grazzini
Paola Lembo
Philippe Walker
Original Assignee
Astrazeneca Ab
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 Astrazeneca Ab filed Critical Astrazeneca Ab
Priority to CA002372980A priority Critical patent/CA2372980A1/fr
Priority to EP00937431A priority patent/EP1183539A1/fr
Priority to AU52605/00A priority patent/AU5260500A/en
Publication of WO2000070347A1 publication Critical patent/WO2000070347A1/fr

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Classifications

    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • G01N2333/575—Hormones
    • 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
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • 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
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • the present invention is directed to assay methods that can be used to determine whether a test compound can be used to modulate the binding of MCH to the SLC-1 receptor.
  • Compounds identified as being effective modulators have potential use as therapeutic agents in treating obesity and eating disorders.
  • MCH Melanin Concentrating Hormone
  • MCH Melanin concentrating hormone
  • MCH and its agonists have been proposed as a treatment for anorexia nervosa and weight loss due to AIDS, renal disease, or chemotherapy.
  • antagonists of MCH can be used as a treatment for obesity and other disorders characterized by compulsive eating and excessive body weight.
  • MCH has been known for over two decades, its specific receptor has not been structurally characterized and cloned. This has limited the ability to search for therapeutic agents that act by mimicking or inhibiting MCH.
  • G protein coupled receptors constitute a family of proteins sharing a common structural organization characterized by an extracellular N-terminal end, seven hydrophobic alpha helices putatively constituting transmembrane domains and an intracellular C-terminal domain. GPCRs bind a wide variety of ligands that trigger intracellular signals through the activation of transducing G proteins (Caron, et al, Rec. Prog. Horm. Res. 48:277- 290 (1993); Freedman, et al, Rec. Prog. Horm. Res. 57:319-353 (1996)).
  • GPCRs More than 300 GPCRs have been cloned thus far and it is generally assumed that there exist well over 1,000 such receptors. Roughly 50-60% of all clinically relevant drugs act by modulating the functions of various GPCRs (Gudermann, et al, J. Mol Med. 75:51-63 (1995)). Many of the clinically relevant receptors are located in the central nervous system.
  • the present invention is based upon the discovery that MCH serves as a ligand for the SLC-1 receptor.
  • Recombinant cells expressing either rat or human SLC-1 can be used in conjunction with MCH in screening assays designed to identify agonists and antagonists.
  • the invention is directed to a method of assaying a test compound for its ability to bind to the SLC-1 receptor. This is accomplished by incubating cells expressing the receptor gene with MCH and test compound. The extent to which the binding of MCH is displaced is then determined. Radioligand assays or enzyme-linked immunosorbent assays may be performed in which either MCH or the test compound is detectably labeled.
  • heterologous refers to any SLC-1 gene transfected into a cell, i.e., the term refers to any non-endogenous SLC-1.
  • the invention is also encompasses methods of determining if a test compound is an agonist, antagonist, or inverse agonist of MCH binding based upon a functional assay.
  • One way to carry out such assays is to incubate a cell expressing SLC-1 with the test compound and to then determine whether intracellular adenyl cyclase activity or intracellular calcium concentration changes. Results should typically be compared with those obtained when incubations are performed in a similar manner but in the absence of test compound. In general, functional assays of this type will be performed in conjunction with binding assays of the sort described above.
  • the preferred cell for use in the assays is a recombinant cell that has been transformed with a heterologous SLC-1 gene.
  • Test compounds that act as agonists should produce an increase or decrease in adenyl cyclase activity or increase in intracellular levels of calcium.
  • Inverse agonists may reduce adenyl cyclase activity or intracellular calcium levels, particularly if assays are performed in the presence of a fixed amount of MCH.
  • Antagonists should block the binding of MCH to receptor but not produce the opposite reponse in terms of adenyl cyclase activity or intracellular calcium that is the hallmark of an inverse agonist.
  • the present invention is directed to assays that can be used to screen compounds for their ability to modulate the binding of MCH to the SLC-1 receptor.
  • Any form of MCH that has been reported may be used, but the preferred peptide is 19 amino acids in length and has the sequence: Asp-Phe-Asp-Met-Leu-Arg-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp- Gln-Val (SEQ ID NO:l).
  • the peptide assumes a cyclic conformation as the result of a disulfide between the two cysteines.
  • This peptide may be obtained commercially (Sigma, St. Louis, MO) or can be synthesized using standard methodology well known in the art.
  • the peptide may be detectably labeled with radioisotopes such as 125 I or, alternatively, fluorescent or chemi luminescent labels can be incorporated. Also, the peptide can be joined to enzymes that are readily detectable such as horseradish peroxidase.
  • the SLC-1 receptor may be cloned from human cells using the procedure described by Kolakowski, et al. (FEBS Lett. 59 ⁇ ?:253-258 (1996)) or from rat cells using the procedure described by Lakaye, et al. (Biochim. Biophys. Acta. 1401:216-220 (1998)).
  • the Examples section provides a detailed description of a procedure that may be used in cloning SLC-1 which, is also referred to herein as clone 1-18.
  • the SLC-1 sequence should be incorporated into an expression vector with a promoter active in mammalian cells (Sambrook, et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989)).
  • promoters examples include that of the mouse metallothionein I gene (Hamer, et al, J. Mol Appl Gen. 7:273-288 (1982)); the immediate-early and TK promoter of herpes virus (Yao, et al, J. Virol. 59:6249-6258 (1995); McKnight, Cell 37:355-365 (1982)); the SV 40 early promoter (Benoist, et al, Nature 290:304-310 (1981)); and, the CMV promoter (Boshart, et al, Cell 41:521-530 (1985)). Vectors may also include enhancers and other regulatory elements.
  • expression vectors Once expression vectors have been constructed, they can be introduced into a mammalian cell line by methods such as calcium phosphate precipitation, microinjection, electroporation, liposomal transfer, viral transfer or particle mediated gene transfer.
  • methods such as calcium phosphate precipitation, microinjection, electroporation, liposomal transfer, viral transfer or particle mediated gene transfer.
  • HEK-293 cells have been found to give successful results and a procedure for expressing SLC-1 in these cells is described in the Examples section. Standard procedures for selecting cells and for assaying them for the expression of SLC-1 (e.g., by Northern analysis) may be performed.
  • assays may be performed to determine whether test compounds have any effect on binding.
  • assays can be performed using standard methods well known in the art. For example, in radioligand binding assays, cells expressing SLC-1 are incubated with MCH and with a compound being tested for binding activity.
  • the preferred source of SLC-1 is recombinantly transformed HEK-293 cells. Other cells may also be used provided they do not express other proteins that strongly bind MCH. This can easily be determined by performing binding assays on cells transformed with SLC-1 and comparing the results obtained with those obtained using their untransformed counterparts.
  • Assays may be performed using either intact cells or with membranes prepared from the cells (see e.g., Wang, et al, Proc. Natl Acad. Sci. U.S.A. 90:10230-10234 (1993)).
  • the membranes, or cells are incubated with MCH and with a preparation of the compound being tested.
  • receptor is separated from the solution containing ligand and test compound, e.g., by filtration, and the amount of binding that has occurred is determined.
  • the ligand used is detectably labeled with a radioisotope such as 125 I.
  • a radioisotope such as 125 I.
  • other types of labels can also be used.
  • fluorescent labeling compounds include fluorescein, isothiocynate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin o-phthaldehyde and fluorescamine.
  • useful chemiluminescent compounds include luminol, isoluminol, theromatic of acridinium ester, imidazole, acridinium salt, and oxalate ester.
  • Nonspecific binding may be determined by carrying out the binding reaction in the presence of a large excess of unlabeled ligand.
  • labeled MCH may be incubated with receptor and test compound in the presence of a thousandfold excess of unlabeled MCH.
  • Nonspecific binding should be subtracted from total binding, i.e., binding in the absence of unlabeled ligand, to arrive at the specific binding for each sample tested.
  • Other steps such as washing, stirring, shaking, filtering and the like may be included in the assays as necessary.
  • wash steps are included after the separation of membrane-bound ligand from ligand remaining in solution and prior to quantitation of the amount of ligand bound, e.g., by counting radioactive isotope.
  • the specific binding obtained in the presence of test compound is compared with that obtained in the presence of labeled ligand alone to determine the extent to which the test compound has displaced receptor binding.
  • test compound In performing binding assays, care must be taken to avoid artifacts which may make it appear that a test compound is interacting with receptor when, in fact, binding is being inhibited by some other mechanism.
  • the compound being tested should be in a buffer which does not itself substantially inhibit the binding of MCH and should, preferably, be tested at several different concentrations. Preparations of test compound should also be examined for proteolytic activity and it is desirable that antiproteases be included in assays.
  • compounds identified as displacing the binding of MCH be reexamined in a concentration range sufficient to perform a Scatchard analysis on the results.
  • agents that inhibit the binding of MCH to receptor may be either agonists or antagonists.
  • Activation of receptor may be monitored using a number of different methods.
  • adenyl cyclase assays may be performed by growing cells in wells of a microtiter plate and then incubating the wells in the presence or absence of test compound.
  • cAMP may then be extracted in ethanol, lyophilized and resuspended in assay buffer. Assay of cAMP thus recovered can be carried out using any method for determining cAMP concentration.
  • adenyl cyclase assays will be performed separately from binding assays, but it may also be possible to perform binding and adenyl cyclase assays on a single preparation of cells.
  • Activation of receptor may also be determined based upon a measurement of intracellular calcium concentration.
  • transformed HEK-293 cells may be grown on glass cover slides to confluence. After rinsing, they may be incubated in the presence of an agent such as Fluo-3 or FURA-2 AM (Molecular Probe F-1221). After rinsing and further incubation, calcium displacement may be measured using a photometer. Other types of assays for determining intracellular calcium concentrations are well known in the art and may also be employed.
  • Assays that measure the intrinsic activity of the receptor may be used in order to determine the activity of inverse agonists.
  • inverse agonists produce a biological response diametrically opposed to the response produced by an agonist. For example, if an agonist promoted an increase in intracellular calcium, an inverse agonist would decrease intracellular calium levels.
  • radioligand and cell activation assays discussed above merely provide examples of the types of assays that can be used for determining whether a particular test compound alters the binding of MCH to the SLC-1 receptor and acts as an agonist or antagonist. There are many variations on these assays that are compatible with the present invention. Such assays may involve the use of labeled antibodies as a means for detecting MCH that has bound to receptor or may take the form of the fluorescent imaging plate reader assays described in the Examples section herein.
  • PCR-based strategy was used to clone the rat 1-18 gene (SLC-1).
  • Rat spinal cord mRNA was isolated using the FastTrackO kit (InVitrogen, San Diego, Ca).
  • the templates for PCR amplification were synthesized using GeneAmp RNA PCR kits (N808-0017 Perkin Elmer) with 200 ng of the rat spinal dorsal horn polyA+ RNA and were amplified using the following primers:
  • TM3-5 5'-G(C or T)G(A or C)(C or G)(A or G)(C or G)(C or T)ITIGA(C or T) CGCTA-3' (SEQ ID NO:2)
  • TM7-5 5'-AAGC(C or T)(A or G)TA(G or T)AI(A or C or G)AI(A or C)GG(A or G)TT-3' (SEQ ID NO:3).
  • the reaction mixture contained 200 pmoles of each of the TM3-5 and TM7-5 primers and 2.5 units of Taq DNA polymerase in 50 mM KC1, 1.5 mM MgCl 2 , 10 mM Tris(HCl), 200 mM dNTPs, pH 9.0.
  • the reaction tubes were heated at 95 °C for one minute and subjected to 39 cycles of denaturation (95 °C / 1 min), annealing (42 °C / 1 min)and extension (72 °C / 1 min).
  • the amplified fragments were analyzed and size fractionated on a 1% agarose gel.
  • Fragments between 500 bp and 800 bp were excised from the gel, purified using the Sephaglas BandPrepO kit from Pharmacia (cat# 27-9285-01), and subcloned into the pGEM-T vector from Promega (cat# A3600). Recombinant pGEM-T clones were selected randomly and plasmid DNA was prepared using the alkaline lysis method starting with 2 ml of bacterial culture. The Sanger dideoxy nucleotide chain termination method was used to sequence the DNA from these clones, with the T7 sequencing kit from Pharmacia (cat# 27-1682-01).
  • the insert DNA fragment of the clone pGEMT-1-18 was excised from the vector using Pst I and Sac II, isolated from an agarose gel and labeled with 32 P by random primed synthesis using the Ready-To-GoO DNA labeling kit (cat#27-9251-01)from Pharmacia.
  • This probe was used to screen a rat brain stem-spinal cord cDNA library in 1 ZAP II (Stratagene, cat# 936521). The filters were incubated with the probe for 18 hours at 65°C in 2x SSC, 5x Denhardt's solution and 0.2% SDS. The filters were rinsed twice in O.lx SSC, 0.2% SDS at room temperature.
  • the filters were then washed twice for 45 min in O.lx SSC, 0.2% SDS at 65°C, once for 45 min at 65°C in 5 mM EDTA, 0.2% SDS, pH 8.0 and finally rinsed with O.lx SSC at room temperature.
  • Hybridization-positive phages were purified and their inserts rescued by helper phage mediated excision to yield plasmid DNA.
  • the insert of plasmid pBS/1-18 was sequenced progressively with the 1-18-specific primers.
  • Kb Sma I - Xho I fragment from pBS/1-18 was isolated and subcloned into the Eco RV and Xho I sites of pcDNA3 (InVitrogen, San Diego, Ca). This expression vector was called pcDNA3-l-18. Plasmid DNA was prepared using the Qiaprep system from Qiagen.
  • HEK-293 cells were transfected with a mammalian expression construct coding for the 1 - 18 clone (pcDNA 3.0 vector, Invitrogen) using the Superfect reagent (Qiagen).
  • a stable receptor pool of 1-18 was developed by applying a selection marker (G418, 0.6 mg/ml) and the cells were maintained in this selection medium.
  • the presence of mRNA specific for clone 1-18 was assessed by Northern blot analysis and by the reverse transcriptase polymerase chain reaction (RT-PCR).
  • a collection of peptide and non-peptide ligands was obtained from commercial sources (Sigma, CalBiochem, American Peptide Company, Bachem, RBI). The compounds were dissolved in water/DMSO at 30 iM and placed in 96 well microplates. A total of 846 compounds (peptides and non-peptides) were prepared and tested.
  • a functional assay was performed with FLIPR (Fluorescent Imaging Plate Reader, Molecular Devices) using the fluorescent calcium indicator Fluo-3 (Molecular Probes) on a 96 well platform.
  • HEK-293 cells either expressing the receptor or wild type cells, were loaded with Fluo-3 as follows.
  • Stable HEK-293 clones expressing 1-18 or parental cells were plated at a density of 70,000 cells/well in a 96 well plate.
  • the 1-18 cells were loaded with fluorescent solution (Dulbecco's modified medium with 10% fetal bovine serum containing 4 ified medium with 10% fetal bovine serum containing 4 Probes) on a 96 well platform.
  • HEK-293 cells either expressing the receptor or wild type cells, were loaded with Fluo-3 as follows. Stable HEK-2 BSA (pH 7.4). The cells were analyzed using the FLIPR system to measure the mobilization of intracellular calcium in response to different compounds.
  • HEK-293 cells endogenously express some GPCRs such as bradykinin receptors which can be used as an internal control for assays.
  • the background signal was established with all of the compounds in the parental HEK-293 cells (non-transfected) using the FLIPR assay.
  • HEK-293 cells expressing the clone 1-18 were stimulated with all compounds and calcium responses were compared with those in parental HEK-293 cells. Only one compound, melanin concentrating hormone (MCH), consistently elicited signals in the transformed cells but not the wild type cells. This indicates that MCH is interacting with the recombinantly expressed receptor.
  • MCH melanin concentrating hormone
  • Screening assays can be performed using the FLIPR assay described above. Alternatively,
  • MCH can be iodinated and used as a tracer in radioligand binding assays on whole cells or membranes.
  • Other assays that can be used include the GTPaS assay, adenylate cyclase assays, assays measuring inositol phosphates, and reporter gene assays (e.g., those utilizing luciferase, aqueorin, alkaline phosphatase, etc.).

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Abstract

La présente invention concerne des dosages pouvant être utilisés pour détecter les composés capables d'agir comme agonistes ou antagonistes de l'hormone concentrant la mélanine (MCH). Ces dosages reposent sur la liaison de MCH au récepteur SLC-1.
PCT/SE2000/001010 1999-05-19 2000-05-19 Dosages pour agonistes, antagonistes et agonistes inverses d'hormone concentrant la melanine (mch) se liant au recepteur du type somatostatine (slc-1) WO2000070347A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002372980A CA2372980A1 (fr) 1999-05-19 2000-05-19 Dosages pour agonistes, antagonistes et agonistes inverses d'hormone concentrant la melanine (mch) se liant au recepteur du type somatostatine (slc-1)
EP00937431A EP1183539A1 (fr) 1999-05-19 2000-05-19 Dosages pour agonistes, antagonistes et agonistes inverses d'hormone concentrant la melanine (mch) se liant au recepteur du type somatostatine (slc-1)
AU52605/00A AU5260500A (en) 1999-05-19 2000-05-19 Assays for agonists, agonists and inverse agonists of melanin concentrating hormone (mch) binding to the somatostatin-like receptor (slc-1)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US13484499P 1999-05-19 1999-05-19
US60/134,844 1999-05-19
US13867599P 1999-06-14 1999-06-14
US60/138,675 1999-06-14

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WO2000070347A1 true WO2000070347A1 (fr) 2000-11-23

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AU (1) AU5260500A (fr)
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7078187B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7078484B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7125885B2 (en) 2001-05-04 2006-10-24 Amgen Inc. Fused heterocyclic compounds
US7189856B2 (en) 2001-12-28 2007-03-13 Gideon Shapiro Non-peptide somatostatin receptor ligands
US7253179B2 (en) 2002-11-06 2007-08-07 Amgen Inc. Fused heterocyclic compounds

Citations (3)

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Publication number Priority date Publication date Assignee Title
WO1996018651A1 (fr) * 1994-12-16 1996-06-20 Smithkline Beecham Corporation Recepteur humain somatostatinoide
WO1996039162A1 (fr) * 1995-06-06 1996-12-12 Joslin Diabetes Center, Inc. Regulation du comportement alimentaire
WO2000040725A1 (fr) * 1998-12-28 2000-07-13 Takeda Chemical Industries, Ltd. Methode d'analyse

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996018651A1 (fr) * 1994-12-16 1996-06-20 Smithkline Beecham Corporation Recepteur humain somatostatinoide
WO1996039162A1 (fr) * 1995-06-06 1996-12-12 Joslin Diabetes Center, Inc. Regulation du comportement alimentaire
US5849708A (en) * 1995-06-06 1998-12-15 Joslin Diabetes Center, Inc. Promotion of eating behavior
WO2000040725A1 (fr) * 1998-12-28 2000-07-13 Takeda Chemical Industries, Ltd. Methode d'analyse

Non-Patent Citations (4)

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Title
BAECHNER D. ET AL.: "Identification of melanin concentratinghormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1)", FEBS LETT., vol. 457, no. 3, September 1999 (1999-09-01), pages 522 - 524, XP002942554 *
CHAMBERS J. ET AL.: "Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1", NATURE, vol. 400, no. 6741, July 1999 (1999-07-01), pages 261 - 265, XP002947035 *
KOLAKOWSKI L.F. JR. ET AL.: "Characterization of a human gene related to genes encoding somatostatin receptors", FEBS LETT., vol. 398, no. 2-3, December 1996 (1996-12-01), pages 253 - 258, XP002926051 *
SAITO Y. ET AL.: "Molecular characterization of the melanin-concentrating-hormone receptor", NATURE, vol. 400, no. 6471, July 1999 (1999-07-01), pages 265 - 269, XP002152581 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7078187B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7078484B2 (en) 2001-04-19 2006-07-18 Neurogen Corporation Melanin concentrating hormone receptors
US7125885B2 (en) 2001-05-04 2006-10-24 Amgen Inc. Fused heterocyclic compounds
US7189856B2 (en) 2001-12-28 2007-03-13 Gideon Shapiro Non-peptide somatostatin receptor ligands
US7253179B2 (en) 2002-11-06 2007-08-07 Amgen Inc. Fused heterocyclic compounds

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CA2372980A1 (fr) 2000-11-23
AU5260500A (en) 2000-12-05
EP1183539A1 (fr) 2002-03-06

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