WO2005023232A2 - Nouvelle utilisation et nouveaux procedes associes - Google Patents

Nouvelle utilisation et nouveaux procedes associes Download PDF

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WO2005023232A2
WO2005023232A2 PCT/SE2004/001277 SE2004001277W WO2005023232A2 WO 2005023232 A2 WO2005023232 A2 WO 2005023232A2 SE 2004001277 W SE2004001277 W SE 2004001277W WO 2005023232 A2 WO2005023232 A2 WO 2005023232A2
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cells
cbl
cbl receptor
compound
population
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WO2005023232A3 (fr
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Birgitta Sander
Birger Christensson
Edvard Smith
Eva Kimby
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Affibody Ab
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5383—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/164—Amides, e.g. hydroxamic acids of a carboxylic acid with an aminoalcohol, e.g. ceramides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/5052—Cells of the immune system involving B-cells
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia

Definitions

  • the present invention relates to the elucidation of a novel association between modulation of the CBl recep- tor and an effect on cells involved in lymphoproliferative disorders. More specifically, the present invention relates to methods and uses connected to this association, which enable the treatment and diagnosis of said disorders, the identification of new effective substances for their treatment, and other aspects apparent from the following disclosure.
  • CB cannabinoid receptors
  • lymphocytes express both CBl and CB2.
  • the cannabinoid recep- tor type 1 (CBl) was cloned in 1991 (Gerard CM et al (1991), Biochem J 279 (Pt 1): 129-34) and the peripheral cannabinoid receptor (CB2) in 1993 (Munro S et al (1993), Nature 365 (6441) : 61-5) .
  • the CB2 receptor is also autoactivated, and its activity inhibited by pertussis toxin or SR144528, which functions as an inverse agonist.
  • pertussis toxin or SR144528 which functions as an inverse agonist.
  • the activities and interactions of the CB receptors with SR141716A and SR144528 have been reviewed in Shire D et al (1999), Life Sci 65 (6-7) : 627-35. These two highly specific substances have been of tremendous value for studying various as- pects of cannabinoid interactions with human, urine and rat cells.
  • CBl and CB2 in lymphoid tissue Expression of CBl was initially described only in brain and testis, but was later investigated in lymphocytes. Expression of cannabinoid receptors in human leukocytes were investigated by PCR already in 1993 (Bouaboula M et al (1993), Eur J Biochem 214 (1) : 173-80) . According to this report, the highest expression was seen in human B cells. In 1995, the same research group repeated their experiments with RT-PCR specific for CBl and CB2, and could then detect CBl mRNA expression in human immune tissue, but to much lower levels than in the brain (Galiegue S et al (1995), Eur J Biochem 232 (1) : 54-61) .
  • THC induces apoptosis in both T cells and B cells (McKallip RJ et al (2002), J Phar Exp Ther 302 (2) :451-65) .
  • the apoptotic effect of THC could be inhibited by SR144528 but not by SR141716, and was thus attributed to CB2 specific effects.
  • THC induced reduced proliferation and apoptosis (McKallip RJ et al (2002), Blood 100(2) : 627-34) .
  • anandamide, THC or the CB1/CB2 agonist CP 55,940 reduced proliferation in response to mitogen stimulation of T or B lymphocytes.
  • the cannabinoid receptor agonist WIN 55,212-2 did not induce apoptosis in these cell lines.
  • anandamide and THC, but not CP55,940, in- prised apoptosis (Schwarz H et al (1994), supra) .
  • Production of both 2-AG and anandamide has been found in human lymphoma cells such as U937 (Maccarrone M et al (2001) , J Neurochem 76 (2) : 594-601) .
  • CB receptors protect against VR-receptor mediated cell death induced by anandamide. It has since been confirmed, in other celltypes, that the apoptosis inducing effects by anandamide is mediated via VR and not by the CB1/CB2 system (Contassot E et al (2004), Gynecol Oncol 93 (1) : 182-8 ) .
  • cannabinoids can be used in treatment of malignant cancer diseases, mainly in cells of epithelial origin (reviewed in Bifulco M and Di Marzo V (2002), Nature Medicine 8 ( 6) : 547-50) , but no connection between the CBl system and disorders related to lymphocytes has been demonstrated.
  • T lymphoblastic disorders targeting of CB2 receptors induced apoptosis (McKallip RJ et al (2002), Blood 100 (2) : 627-34 ) .
  • Mantle cell lymphoma is a rare type of lymphoma (5-10% of all malignant lymphomas) .
  • the normal cel- lular counterpart is presumed to be a B cell found in small numbers in the mantle zone of lymphoid follicles.
  • MCL was originally classified as a low-grade lymphoma, but recent reports of outcome indicate that most cases behave as an intermediate-grade lymphoma. It has neither the long survival of low-grade tumors nor the response to aggressive chemotherapy of high-grade tumors.
  • One exception is the infrequent "mantle zone" variant, which may have a more indolent course. These tumors present in older adults with a median age of 65.
  • lymphoproliferative disorders such as lymphomas .
  • pharmaceutical products for treatment of lymphoproliferative disorders which act to kill, or reduce viability, of affected cells only, and which do not significantly influence normal cells.
  • identify signaling pathways in which it is possible to intervene to obtain a biological effect which is useful for curative treatment of lymphoproliferative disorders.
  • Still another object of the invention is to provide methods for identifying new compounds which affect such signaling pathways.
  • the invention relates to use of a compound capable of modulating CBl receptor signaling for the preparation of a medicament for treatment of a lymphoproliferative disorder involving cells with abnormal CBl receptor function.
  • the invention provides a novel linkage between modulation of the CBl receptor and an effect on cells involved in a lymphoproliferative disorder characterized by abnormal function of this recep- tor.
  • the effect on these cells of modulating the CBl receptor is to reduce or eliminate the negative effects of the cells in the progress of the disorder.
  • the present inventors have shown, for the first time, that lymphoid cells affected by certain lymphoproliferative disorders display an increased amount of the CBl receptor on their surface, and that the amount of CBl receptor in fact exceeds the amount of the CB2 re- ceptor.
  • the compounds capable of modulating CBl receptor signaling is a compound characterized as a "CBl agonist".
  • the compound in another embodiment, it is a compound characterized as a "CBl inverse agonist". In another embodiment, it is a compound characterized as a "CBl antagonist”. In yet an embodiment, the compound has been identified as having activity on the CBl receptor through the use of a screening assay according to the second aspect of this invention, as described below. The number of possible, known and unknown, compounds with the requisite activity is great. A non- limiting listing of compounds that have been described in the literature as capable of modulating CBl receptor signaling is presented below. In one embodiment of the in- vention, the compound capable of modulating CBl receptor signaling is chosen from this listing.
  • the skilled person will be able to se- lect suitable CBl ligands without having to exercise any undue experimentation or inventive skill.
  • the compound could be one selected from the group consisting of WIN 55,212-2, anandamide and AM-251.
  • the compound capable of modulating CBl receptor signaling is a polypeptide, which has a biospecific affinity for the CBl receptor.
  • such a polypeptide could be an antibody, or functional fragment thereof, which recognizes one or more epitopes on the CBl receptor structure.
  • the polypeptide is based on another protein.
  • it could be a variant of a certain scaffold protein, which has been selected from a library of many different, randomly created such variants, wherein the selection has been performed using the CBl receptor as target.
  • selection procedures for example phage display, protein complementation assay, selection on bacterial surfaces etc are known to the skilled person, as are suitable starting scaffolds for creating affinity ligands against a target molecule.
  • Such engineered proteins for use as affinity ligands to CBl in the invention may be constructed using as scaffold a protein domain selected from the group consisting of domains of bacterial receptins, fibronectins, protease inhibitors, retinol binding proteins, bilin binding proteins, amylase inhibitors, CTLA- 4, cytochromes and cellulose binding proteins.
  • a protein domain selected from the group consisting of domains of bacterial receptins, fibronectins, protease inhibitors, retinol binding proteins, bilin binding proteins, amylase inhibitors, CTLA- 4, cytochromes and cellulose binding proteins.
  • domains from bacterial re- ceptins are mentioned.
  • domains derived from the group consisting of staphylococcal protein A, streptococcal protein G and Peptostreptococcus magnus protein L are especially preferred.
  • the present invention also provides use of a polypeptide with a biospecific affinity for the CBl receptor for the preparation of a medicament for treatment of a lymphoproliferative disorder involving cells with abnormal CBl receptor function.
  • the polypeptide with a biospecific affinity for the CBl receptor is suitably as described immediately above.
  • the non-limiting examples given herein of compounds capable of modulating CBl receptor signaling may be used singly or in any combination or mixture thereof.
  • they may be admixed with any conventional pharmaceutical excipients in accordance with known practices.
  • the lymphoproliferative disorder to be treated may be any disorder or disease which fulfils the definition given herein.
  • the invention is applicable for example to lymphomas, such as B cell lymphomas, for example mantle cell lymphoma.
  • lymphomas such as B cell lymphomas
  • Other examples of such disorders are posttransplantational lymphoproliferative disorders, Castleman's disease, atypical EBV infection, HTLV1 infection, infection with Herpes virus type 8, angioimmuno- blastic lymphadenopathy and lymphomatoid granulomatosis .
  • the cells with abnormal CBl receptor function involved in the lymphoproliferative disorder are B lymphocytes.
  • the treatment of lymphoproliferative disorder for which the medicament to be prepared using the compound capable of modulating CBl receptor signaling is intended is performed in com- bination with at least one other treatment of the lymphoproliferative disorder.
  • the treatment is carried out as maintenance therapy under a long period of time, such as from months to years .
  • a method for screening for, or identification of, compounds that have the desired effect on the CBl receptor is presented, which method makes use of the association de- scribed above between modulation of CBl receptor and effect on cells affected by a lymphoproliferative disorder.
  • the invention provides a method for identification of a compound capable of modulating CBl receptor signaling, which method comprises: - providing a compound suspected of being capable of modulating CBl receptor signaling; - applying said compound to a test population of cells with abnormal CBl receptor function; - applying said compound to a control population of cells with normal CBl receptor function; - analyzing the viability of cells in said test population and in said control population; whereby reduced viability in the test population relative to the control population identifies said com- pound as capable of modulating CBl receptor signaling.
  • Another embodiment of this aspect of the invention provides a method for identification of a compound capable of modulating CBl receptor signaling, which method comprises : - providing a compound suspected of being capable of modulating CBl receptor signaling; - providing a test population and a control population of cells with abnormal CBl receptor function; - applying said compound to said test population but not to said control population; and - analyzing the viability of cells in said test population and in said control population; whereby reduced viability in the test population relative to the control population identifies said compound as capable of modulating CBl receptor signaling.
  • the discovered association is ex- ploited when investigating the effects on CBl signaling of a newly synthesized or previously known compound.
  • the compound tested could be investigated further, as an interesting candidate sub- stance.
  • it could be tested as a compound capable of modulating CBl receptor signaling in a medicament for treating lymphoproliferative disorders according to the present invention.
  • Other alternatives as to the uses of such a compound are readily apparent to the per- son skilled in the biology of cannabinoid systems, since there are many areas of research where such a CBl ligand could be useful.
  • cells with abnormal CBl receptor function may be transformed B lymphocytes, in particular mantle cell lymphoma cells.
  • Another aspect of the present invention provides a method for the manufacture of a medicament for treatment of a lymphoproliferative disorder involving cells with abnormal CBl receptor function, comprising admixing a compound capable of modulating CBl receptor signaling with at least one pharmaceutically acceptable excipient .
  • Yet another aspect of the present invention provides a method for treatment of a lymphoproliferative disorder involving cells with abnormal CBl receptor function, which method comprises administering, to a subject in need thereof, a pharmaceutically active amount of a compound capable of modulating CBl receptor signaling.
  • a pharmaceutically active amount of a compound capable of modulating CBl receptor signaling may, again, be derived from the foregoing discussion of the first aspect of the invention.
  • Another aspect of the invention provides a method for the diagnosis of a lymphoproliferative disorder involving cells with abnormal CBl receptor function in a subject, which method comprises - taking cells from said subject; - culturing said cells to obtain a first cell population; - dividing said first cell population into a test population and a control population; - applying a compound capable of modulating CBl re- ceptor signaling to said test population but not to said control population; and - analyzing the viability of cells in said test and control populations; whereby reduced viability in the test population relative to the control population implies the presence of said disorder.
  • a furhter aspect of the present invention is a method for elimination of malignant lymphocytes from a cell population comprising malignant lymphocytes and non- malignant cells, comprising treating said cell population with a compound capable of modulating CBl receptor signaling.
  • a "purging" method serves to eliminate or reduce in number unwanted, malignant cells from cell preparations that are to be used in a context where it is of importance that no malignant cells are present. For example, such a context could be the preparation of cells for stem cell transplantation.
  • the following definitions are provided. Thereafter, a non-limiting listing of compounds capable of modulating CBl receptor signaling is provided.
  • a compound capable of modulating CBl receptor signaling is a compound, synthetic or biological or modified biological, which binds to the CBl receptor, or in other ways modifies the structure or localization of the CBl receptor or its association with other molecules, in such a way that the effect of the re- ceptor on signaling pathway (s) is modified so that the cell's fate or function is changed.
  • a compound capable of modulating CBl receptor signaling is sometimes also referred to as "a CBl receptor ligand” or "a CBl ligand”.
  • agonists compounds that are pre- sented in the literature as "agonists”, “inverse agonists” or “antagonists” to the CBl receptor are contemplated to fall within this definition.
  • compound capable of modulating CBl receptor signaling may be selective for the CBl receptor over other receptors, notably the CB2 receptor, but it is equally possible that the compound is capable also of modulating other receptors.
  • a lymphoproliferative disorder is used in this context to indicate a group of diseases, which includes lymphoid proliferations with proven and non-proven malignant potential.
  • Non-limiting examples of such disor- ders include lymphomas, such as B cell lymphomas, for example mantle cell lymphoma; posttransplantational lymphoproliferative disorders; Castleman' s disease; atypical EBV infections; HTLV1 infection; infection with Herpes virus type 8; angioimmunoblastic lymphadenopathy and lym- phomatoid granulomatosis .
  • lymphomas such as B cell lymphomas, for example mantle cell lymphoma; posttransplantational lymphoproliferative disorders; Castleman' s disease; atypical EBV infections; HTLV1 infection; infection with Herpes virus type 8; angioimmunoblastic lymphadenopathy and lym- phomatoid granulomatosis .
  • a lymphoproliferative disorder involving cells with abnormal CBl receptor function is a disease or disorder as defined above, in which individual lymphocytes or groups of lymphocytes have an increased amount of CBl (functional or non-functional) within, on or around the cell and/or in which altered CBl function is instrumental in, or associated with, the disease or disorder, in such a way that without the increase in CBl and/or altered CBl function no disease or disorder would be discernible.
  • a compound is said to be "selective for the CBl receptor over the CB2 receptor" if the compound binds to, or in other ways modifies the structure, localization or the association with other molecules of, the CBl receptor in such a way that the effect of the receptor on signaling pathway (s) is modified so that the cell's fate or function is changed, whereas, in the presence of the CB2 receptor only, this effect is not discernible .
  • a polypeptide has "a biospecific affinity for the CBl receptor" if the compound interacts with the CBl receptor through specific recognition between the polypeptide and the receptor, which recognition is based on the specific tertiary structure and/or charge resulting from the specific amino acid sequence of the polypeptide.
  • the interaction of polypeptide with receptor through bio- specific affinity leads to a biological result which is such that the effect of the receptor on signaling pathway (s) is modified so that the cell's fate or function is changed.
  • the interaction of polypeptide with receptor through biospecific affinity inhibits the binding of natural or modified CBl specific ligands so that the receptor's effect on the cell function is not discernible.
  • a functional fragment of an anti- body is a part of the antibody molecule that retains some or all of the antibody molecule's capacity to associate with the antigen.
  • the present invention contemplates use of a compound capable of modulating CBl receptor signaling.
  • a compound capable of modulating CBl receptor signaling can be found in the literature.
  • the present inven- tion provides screening methods by which it is possible to identify new such compounds.
  • compounds and general guidelines on the preparation and identification of compounds are presented.
  • a CBl binding ligand has to be highly flexible, and that acyl chains in the ligand should be able to assume a tightly folded conformation (U-shaped) .
  • the head can be either polar or non-polar but should not be bulky (Reggio PH and Traore H (2000), Chem Phys Lipids 108 (1-2) : 15-35) .
  • AM 281 (l-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4- methyl-iV-4-morpholinyl-li ⁇ -pyrazole-3-carboxamide) (Cosenza et al (2000), Synapse 38:477); AM 251 (_V-(piperidin-l-yl)-5- (4-iodophenyl) -1- (2, 4- dichlorophenyl) -4-methyl-li ⁇ -pyrazole-3-carboxamide) (Gat- ley et al (1996), Eur J Pharmacol 307:331); AM 630 (6-iodo-2-methyl-l-[2-(4-morpholinyl)ethyl]- li ⁇ -indol-3-yl] (4-methoxyphenyl) methanone) (Hosohata e
  • Virodhamine (0- (2-Aminoethyl) -5Z, 8Z, 11Z, 14Z- eicosatetraenoate) (Porter et al (2002) , J Pharmacol Exp Ther 301:1020) ; O-2050 ( (6a£, lOai?) -3- (1-methanesulfonylamino-4- hexyn-6-yl) -6a, 7,10, 10a-tetrahydro-6, 6, 9-trimethyl-6i ⁇ - dibenzo [b,d] pyran) (Martin et al (2002), "Symposium on the Cannabinoids", International Cannabinoid Research Society) ; CP 55,940 ( (-) - cis-3- [2-hydroxy-4- (1, 1-dimethyl- heptyl) phenyl] -trans-4- (3-hydroxypropyl) cyclohexanol) (Wiley et al
  • Control (bar 1) , as well as effects of VR antagonist alone (bar 4) or of 100 mM of the topoi- somerase I inhibitor camptothecin (bar 5) are also shown.
  • Caspase-3 fluorimetric assay showing caspase activity of Rec-1 cells 24 hours after treatment with 0 ⁇ M (bar 1) 2.5 ⁇ M (bar 2) or 10 ⁇ M (bar 3) of the CBl antagonist AM 251, or with 10 ⁇ M of camptothecin (bar 4) .
  • the t(ll;14) translocation was confirmed by FISH analysis using the Vysis LSI IGH/CCND1 dual fusion probe (Vysis, Richmond, UK) .
  • RNA isolation and oligonucleotide array hybridisa tion Total RNA was prepared using the TRIzol method as directed by the supplier (Invitrogen Life Technologies, Carlsbad, CA) followed by Trizol purification (Quiagen) and quality control on an Agilent Bioanalyzer (Agilent Technologies, Inc. Palo Alto, CA) .
  • the cRNA synthesis for micro array experiments and the hybridisations were per- formed using Affymetrix GeneChipTM high-density oligonucleotide human U133A arrays (Affymetrix, Inc, Santa Clara, CA) containing more than 22 000 genes, or the human U95Av.2 chip containing 12 500 genes, according to standard Affymetrix protocols at the core facility at the Department of Biosciences, Karolinska Institutet, Novum, Huddinge, Sweden.
  • Affymetrix da ta analysis The data was analysed using Affymetrix Microarray Suite version 5.0, MicroDB 3.0 and DMT 3.0 as well as a beta test version of Geneweaver, provided by Affi- body/Inforsense . Comparative analyses were performed on the expression data using Affymetrix Datamining Tool version 3.0. Cell lines and freshly isolated, viable MCL tumor cells Two MCL cell lines were used, the Granta 519 (Jadayel DM et al (1997), Leukemia 11(1): 64-72) and the Rec-1 (Rimokh R et al (1994), Blood 83 (12) : 3689-96) .
  • the Granta 519 an EBV transformed MCL cell line, was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen and cultured in Dulbecco' s MEM (DMEM) (Gibco, UK) (4.5% glucose) with 2 mM L-glutamine and 0.11 g/1 Na Pyr with pyridoxine and 50 ⁇ g/ml of gentamycin.
  • DMEM Dulbecco' s MEM
  • the Rec-1 cell line was kindly donated by Dr C Bastard, Centre Henri Becquerel, Rouen, France and cultured in RPMI-1640 with GlutaMax and 25 mM HEPES (RPMI) (Gibco) and 50 ⁇ g/ml of gentamycin.
  • both cell lines were cultured in medium with 10% fetal calf serum.
  • the translocation t(ll;14) was confirmed by FISH analysis and up-regulation of Cyclin Dl protein by immunocytochemistry.
  • the AtT20 cell line transfected with cDNA for the rat CBl receptor (AtT2 ⁇ tCBl) (Hsieh C et al (1999), J Neurochem 73 (2) : 493-501) or with low endogenous levels of CBl (AtT20) was kindly provided by Dr K Mackie (Dept of Anaesthesiology, University of Washington, Seattle, WA.
  • Immunof lucres cent staining for CBl Cells on cytospin preparations or imprints from lymph nodes were fixed in 3.7% formaldehyde (Sigma, St Louis, MA) during 4 minutes, rinsed three times in PBS and incubated for 30 minutes in PBS containing 0.1% Triton X-100 (Sigma) and 0.1% BSA (Sigma).
  • Rabbit polyclonal antiserum against CBl was kindly provided by Dr K Mackie and diluted 1:400 in PBS-Tween-BSA before being added to the cell preparation for 1 hour at room temperature in a moisture chamber.
  • Western blotting Cell extracts were prepared by lysing the cells in ice-cold sample buffer (50 mM HEPES, 500 mM NaCl, 0.05% Tween 20, 0.1% Triton x-100) to which 1000 x diluted protease inhibitor cocktail (p8340, Sigma) had been added. Protein concentration was measured with BCA protein assay reagent kit (Pierce Biotechnology) as described by the manufacturer. Proteins (40 ⁇ g/lane) were resolved by 10% SDS/PAGE and transferred onto PVDF membrane (Bio-Rad) .
  • Caspase-3 assay DEVD-dependent caspase activity was measured with the Caspase-3/CPP32 Fluorimetric Assay (Nordic Biosite) according to the manufacturer's instructions.
  • MTT assay Cell viability was determined using 3- (4, 5- dimethylthiazol-2-yl) -2, 5-diphenyl-tetrazolium bromide (MTT) assay (Roche Diagnostics) according to the manufac- turer's instructions. The assay is based on cleavage of the yellow tetrazolium salt MTT to purple formazan crystals by metabolically active cells. The solubilized crystals can be quantified with a spectrophotometer . Briefly, 200 ⁇ l of treated cells were seeded in triplicates in 96- well plates and incubated at 37 °C for 24 or 48 hours.
  • Rec-1 cells were cultured at 1 x lOVml in fresh RPMI and Granta cells at 1 x 10 6 /ml in DMEM. Serum was added as indicated in the figure legends.
  • Viable tumor cells from MCL tumor biopsies were cultured at 1 x 10 6 /ml in RPMI with 0.5% fetal calf serum. Cells were cultured in flat-bottomed 96 well plates (Costar) . After 24 hours, the cell viablity was determined by cell counting using trypan blue exclusion.

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Abstract

L'invention concerne l'utilisation d'un composé capable de moduler la signalisation du récepteur CB1 ou d'un polypeptide avec une affinité biospécifique pour le récepteur CB1, pour la préparation d'un médicament destiné au traitement d'un trouble lymphoprolifératif impliquant des cellules avec une fonction anormale du récepteur CB1. En outre, l'invention concerne des procédés d'identification d'un composé capable de moduler la signalisation du récepteur CB1, un procédé pour le diagnostic d'un trouble lymphoprolifératif impliquant des cellules avec une fonction anormale du récepteur CB1, ainsi qu'un procédé pour l'élimination de lymphocytes malins d'une population cellulaire.
PCT/SE2004/001277 2003-09-04 2004-09-06 Nouvelle utilisation et nouveaux procedes associes WO2005023232A2 (fr)

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EP1736775A1 (fr) * 2004-04-12 2006-12-27 Takeda Pharmaceutical Company Limited Nouveau ligand de protéine de récepteur couplé à une protéine g et utilisation de celui-ci
WO2014210205A1 (fr) * 2013-06-26 2014-12-31 Amgen Inc. Protéines de liaison à l'antigène se liant aux récepteurs cb1 et leurs utilisations
CN106232626A (zh) * 2014-03-27 2016-12-14 鸟石生物公司 结合人大麻素1(cb1)受体的抗体
CN109419789A (zh) * 2017-08-31 2019-03-05 清华大学 治疗和/或预防免疫紊乱疾病的化合物及其应用
US11421026B2 (en) 2015-09-30 2022-08-23 Bird Rock Bio, Inc. Antibodies that bind human cannabinoid 1 (CB1) receptor

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1736775A4 (fr) * 2004-04-12 2008-03-12 Takeda Pharmaceutical Nouveau ligand de protéine de récepteur couplé à une protéine g et utilisation de celui-ci
US7927821B2 (en) 2004-04-12 2011-04-19 Takeda Pharmaceutical Company Limited Methods of screening for compounds which bind G protein-coupled receptors
EP1736775A1 (fr) * 2004-04-12 2006-12-27 Takeda Pharmaceutical Company Limited Nouveau ligand de protéine de récepteur couplé à une protéine g et utilisation de celui-ci
US10227406B2 (en) 2013-06-26 2019-03-12 Amgen, Inc Cannabinoid receptor-1 (CB1) monoclonal antibodies
WO2014210205A1 (fr) * 2013-06-26 2014-12-31 Amgen Inc. Protéines de liaison à l'antigène se liant aux récepteurs cb1 et leurs utilisations
JP2016523910A (ja) * 2013-06-26 2016-08-12 アムジェン インコーポレイテッド Ｃｂ１受容体抗原結合タンパク質及びその使用
JP2020196763A (ja) * 2013-06-26 2020-12-10 アムジェン インコーポレイテッド Ｃｂ１受容体抗原結合タンパク質及びその使用
AU2014302410B2 (en) * 2013-06-26 2019-06-13 Amgen Inc. CB1 receptor antigen-binding proteins and uses thereof
US10308712B2 (en) 2014-03-27 2019-06-04 Bird Rock Bio, Inc. Antibodies that bind human cannabinoid 1 (CB1) receptor
CN106232626A (zh) * 2014-03-27 2016-12-14 鸟石生物公司 结合人大麻素1(cb1)受体的抗体
CN106232626B (zh) * 2014-03-27 2022-05-03 鸟石生物公司 结合人大麻素1(cb1)受体的抗体
US11566069B2 (en) 2014-03-27 2023-01-31 Bird Rock Bio, Inc. Treatment of disease responsive to modulation of cannabanoid 1(CB1) receptor signaling
US11421026B2 (en) 2015-09-30 2022-08-23 Bird Rock Bio, Inc. Antibodies that bind human cannabinoid 1 (CB1) receptor
WO2019041596A1 (fr) * 2017-08-31 2019-03-07 清华大学 Composé pour le traitement et/ou la prévention de maladie liée à un trouble immunitaire
CN109419789A (zh) * 2017-08-31 2019-03-05 清华大学 治疗和/或预防免疫紊乱疾病的化合物及其应用

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