Use of Norgestimate As a Selective Inhibitor of Trpc3

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(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: (2006.01) of the grant of the patent: G01N 33/68 31.07.2013 Bulletin 2013/31 (86) International application number: PCT/EP2008/008671 (21) Application number: 08842179.7 (87) International publication number: (22) Date of filing: 14.10.2008 WO 2009/052972 (30.04.2009 Gazette 2009/18)

(54) USE OF NORGESTIMATE AS A SELECTIVE INHIBITOR OF TRPC3, TRPC6 AND TRPC7 ION CHANNELS VERWENDUNG VON NORGESTIMAT ALS SELEKTIVER HEMMER DER IONENKANÄLE TRPC3, TRPC6 UND TRPC7 UTILISATION DE NORGESTIMATE EN TANT QU’INHIBITEUR SÉLECTIF DE CANAL D’ION TRPC3, TRPC6 ET TRPC6 ET TRPC7

(84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR WO-A-2004/041289 WO-A-2006/074802 HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT WO-A-2006/102596 US-A1- 2001 044 584 RO SE SI SK TR US-A1- 2003 119 796 Designated Extension States: AL BA MK RS • LUCKY A W ET AL: "Effectiveness of norgestimate and ethinyl estradiol in treating (30) Priority: 26.10.2007 EP 07291301 moderate acne vulgaris." JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY NOV (43) Date of publication of application: 1997, vol. 37, no. 5 Pt 1, November 1997 (1997-11), 14.07.2010 Bulletin 2010/28 pages 746-754, XP005119497 ISSN: 0190-9622 • YLIKORKALA O ET AL: "Effects on serum lipid (60) Divisional application: profiles of continuous 17beta-estradiol, 10185309.1 / 2 266 575 intermittent norgestimate regimens versus continuous combined 17beta-estradiol/ (73) Proprietor: SANOFI norethisterone acetate hormone replacement 75008 Paris (FR) therapy." CLINICAL THERAPEUTICS MAY 2000, vol. 22, no. 5, May 2000 (2000-05), pages 622-636, (72) Inventors: XP002495105 ISSN: 0149-2918 • MIEHE, Susanne • OLSON W H ET AL: "The duration of response to 65926 Frankfurt am Main (DE) norgestimate and ethinyl estradiol in the • KLEEMANN, Heinz-Werner treatment of acne vulgaris." INTERNATIONAL 65926 Frankfurt am Main (DE) JOURNAL OF FERTILITY AND WOMEN’S • STRUEBING, Carsten MEDICINE 1998 NOV-DEC, vol. 43, no. 6, 65926 Frankfurt am Main (DE) November 1998 (1998-11), pages 286-290, XP008090635 ISSN: 1534-892X (74) Representative: Körner, Kathrin et al Sanofi-Aventis Deutschland GmbH Patente Deutschland Industriepark Höchst Gebäude K801 65926 Frankfurt am Main (DE)

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• DATABASE BIOSIS [Online] BIOSCIENCES • DIETRICH ALEXANDER ET AL: "In vivo TRPC INFORMATION SERVICE, PHILADELPHIA, PA, functions in the cardiopulmonary vasculature" US; 16 October 2007 (2007-10-16), WARD CARY CELL CALCIUM, vol. 42, no. 2, August 2007 C ET AL: "Tid1 interacts with TRPC6 and (2007-08), pages 233-244, XP002528898 ISSN: negatively regulates calcium entry in 0143-4160 cardiomyocytes" XP002495106 Database • M.J. O’NEIL, ET AL.: "The Merck Index -An accession no. PREV200800198117 & Encyclopedia of Chemicals, Drugs and CIRCULATION, vol. 116, no. 16, Suppl. S, October Biologicals, 13th Edition" 2001, MERCK 2007 (2007-10), page 249, 80TH ANNUAL RESEARCH LABORATORIES , NJ, USA , SCIENTIFIC SESSION OF THE AMERICAN- XP002528899 page 1201, column 1, paragraph HEART-ASSOCIATION; ORLANDO, FL, USA; 6735 - column 2 NOVEMBER 04 -07, 2007 ISSN: 0009-7322 • DATABASE MEDLINE [Online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; August 2001 (2001-08), MATTOX J H ET AL: "Combined oral hormone replacement therapy formulations."XP002495107 Database accession no. NLM11521121 & AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY AUG 2001, vol. 185, no. 2 Suppl, August 2001 (2001-08), pages S38-S46, ISSN: 0002-9378

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Description [0007] Studies in transgenic mice already have proven to be valuable in unravelling possible physiological func- FIELD OF THE INVENTION tions of certain TRPCs (Freichel et al., 2001). But these model systems do not exist for each of the seven mam- [0001] The present invention relates to the non-medi- 5 malian TRPC channels so far, their generation and anal- cal use of norgestimate as a selective inhibitor of mem- ysis is time- and cost- consuming and they also have lim- bers of the TRPC3/6/7 ion channel subfamily, which al- itations as they are vulnerable to compensatory effects lows to pharmacologically distinguishing between said by closely related channels (Dietrich et al., 2005c). subfamily members and members of other TRPC sub- [0008] Lucky et al., Journal of the American Academy families. It also relates to the use of norgestimate to iden- 10 of Dermatology, vol. 37, no. 5, 1997: 746-754 teaches the tify, characterize and modulate TRPC ion channels in effectiveness of norgestimate and ethinyl estradiol in cellular models, tissues or animal models. treating moderate acne vulgaris. Ylikorkala et al., Clinical Therapeutics, vol. 22, no. 5, 2000: 622-636 suggests that BACKGROUND OF THE INVENTION continuous administration of 1 mg E2 and 90 mg norges- 15 timate is beneficially effective on lipids and lipoproteins [0002] TRPC channels are nonspecific cation chan- in healthy postmenopausal women. Olson et al., Inter- nels widely expressed in human tissues (Montell 2005). national Journal of Fertility and Women’s Medicine, vol. They were the first found mammalian homologues of Dro- 43, no. 6, 1998:286-290 describes that a triphasic com- sophila TRP (Wes et al., 1995, Zhu et al., 1995). Seven bination of norgestimate and ethinyl estradiol improves proteins, referred to as TRPC1 - 7, constitute the canon- 20 the interval of recurrence of acne lesions. US ical (or classical) TRP subfamily that is the closest related 2003/119796 A1 teaches the treatment of cardiovascular to the archetypal Drosophila TRP (30 - 40 % identity, disease-related disease conditions and obesity in post- Okuhara et al., 2007). menopausal women by administering compositions com- [0003] Based on amino acid sequence homology, tis- prising norgestimate. WO 2004/041289 A also teaches sue distribution and functional similarities, TRPCs can 25 norgestimate for preventing and treating cardiovascular be subclassified into four groups (Clapham et al., 2001; disease-associated disorders in women. US Montell, 2001). Being quite unique within the TRPC fam- 2001/044584 A1 relates to the use of norgestimate to ily, TRPC1 and TRPC2 each constitute a subfamily by reduce blood viscosity for treating atherosclerosis. WO themselves while TRPC4 and -5 are merged just as 2006/102596 A discloses treatment of traumatic central TRPC3, -6, and -7. The closely related TRPC3, TRPC6 30 nervous system injury by administering norgestimate. and TRPC7 channels share a common activation mech- Mattox et al., DATABASE MEDLINE , [Online] anism and diacylglycerol (DAG) has been identified as XP002495107 suggests that continuous 17beta- estradi- their endogenous ligand (Hofmann et al., 1999; Okada ol/intermittent norgestimate hormone replacement ther- et al., 1999). apy may hold benefits for treating cardiovascular dis- [0004] According to the WHO, 30% of all deaths world- 35 ease, colon cancer and Alzheimer’s disease. WO wide were caused by various cardiovascular diseases in 2006/074802 A discloses a method of screening a mod- 2005. Thus, there is an immense medical need for new ulator of the TRPC channel comprising the steps of con- medicines that prevent and treat cardiovascular diseas- tacting a TRPC-expressing cell or an inactivating mutant es. TRPC channels are considered important pharma- thereof, stimulating calcium influx by a channel activator cological targets for the development of novel medicines 40 before, simultaneously or after contacting the cell with a for several cardiovascular pathologies including cardio- test compound, and determining a change in the TRPC myopathy, vascular remodelling, hypertension and high channel activity. endothelial permeability (Dietrich et al., 2007). [0009] Thus, the aim of the present invention was to [0005] But so far, there are many open questions re- develop a new pharmacological tool that allows to dis- garding their native composition and activation mecha- 45 criminate between and within the TRPC subfamilies, nisms, physiological functions, and roles in pathophysi- thereby allowing to elucidate the roles of the different ology and disease. In situ identification of native TRPC channels under physiological as well as patho-physio- channels is complicated by their wide and partially over- logical conditions. lapping distribution, potential heteromultimerization, sim- [0010] According to the present invention, this is ilar electrophysiological properties and a paucity of tool 50 achieved with the use of norgestimate to selectively in- compounds to unequivocally trace these channels (Mo- hibit TRPC3, TRPC6 and TRPC7. Thus, norgestimate ran et al., 2004). allows to pharmacologically distinguishe channels be- [0006] Known TRPC organic inhibitors such as 2- ami- longing to different TRPC subfamilies. In addition, norg- noethoxydiphenyl borate (2-APB) (Xu et al., 2005), SKF estimate does not interfere with the common G protein- 96365 (Boulay et al., 1997; Zhu et al., 1998) and BTP2 55 coupled receptor, Gq, phospholipase Cβ pathway that (He et al., 2005), and inorganic blockers such as Gd3+ mediates TRPC channel activation in many cells. These and La3+ are not potent and specific enough (Liet al., properties make norgestimate a preferable tool for the 2004). identification and modulation of TRPC3, TRPC6 and

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TRPC7. The inventive use is claimed with the proviso above methods are fluorescent cells. that it is not a use in a method of treatment of the animal [0021] Preferred cells according to the present inven- or human body. tion are MDCK, HEK 293, HEK 293 T, BHK, COS, NIH3T3, Swiss3T3 or CHO cells, in particular a HEK293 SUMMARY OF THE INVENTION 5 cell. [0022] TRPC channel activity can be measured or de- [0011] One subject matter of the present invention is tected by measuring or detecting a change in ion fluxes, directed to the non- medical use of norgestimate as a se- in particular Ca 2+ fluxes, by e.g. patch clamp techniques, lective inhibitor of a TRPC ion channel. A TRPC ion chan- whole cell currents, radiolabeled ion fluxes, or in partic- nel selectively inhibited by norgestimate is preferably 10 ular fluorescence e.g. using voltage-sensitive dyes or TRPC3, TRPC6 or TRPC7. Norgestimate may be used ion-sensitive dyes (Vestergarrd-Bogind et al. (1988), J. to selectively inhibit a TRPC ion channel in vitro and in Membrane Biol., 88:67-75; Daniel et al. (1991) J. Phar- vivo. macol. Meth. 25:185-193; Hoevinsky et al. (1994) J. [0012] Inparticular, norgestimate is at least4- foldmore MembraneBiol., 137: 59-70; Ackerman et al. (1997), New potent on TRPC3/6/7 subfamily members than 15 on Engl. J. Med., 336:1575-1595; Hamil et al. (1981), TRPC4/5 subfamily members (Figures 1 and 2). The 4- Pflugers. Archiv., 391:185). fold more potency of norgestimate on TRPC3/6/7 sub- [0023] An example for the measurement of the TRPC family members than on TRPC4/5 subfamily members ion channel activity is an assay comprising the steps of: was evaluated with fluorometric measurements and based on the calculated inhibition by 10m M norgesti- 20 (a) contacting a fluorescent cell expressing a TRPC mate. ion channel, [0013] These fluorometric measurements were exem- (b) stimulating Ca 2+ influx by a channel activator be- plary validated by means of patch clamp recording in the fore, simultaneously or after contacting the fluores- whole-cell configuration (Figure 3). cent cell with the modulator or test compound, and [0014] As a selective inhibitor of TRPC3, TRPC6 and 25 (c) measuring or detecting a change in fluorescence. TRPC7, norgestimate maybe used as a pharmacological tool which allows characterising channels belonging to [0024] Another subject of the present invention is di- different TRPC subfamilies, that is distinguishing be- rected to a method for profiling selectivity of norgestimate tween TRPC3/6/7 subfamily members and members of for a TRPC channel, which comprises assessing the other TRPC ion channel subfamilies (Figures 1, 2 and 3). 30 norgestimate’s ability to inhibit the TRPC channel activity [0015] As such an inhibitor, norgestimate may further and comparing the inhibition potency of norgestimate be- be used as a tool compound to develop and verify assays tween and within the different TRPC subfamilies. that measure the activity of the respective ion channel. An example of such an assay is shown in Figure 1. DETAILED DESCRIPTION OF THE INVENTION [0016] Another subject matter of the present invention 35 is directed to the use of norgestimate to make a differ- [0025] The term "TRPC ion channel" or "TRPC" in con- ential analysis of the channel functions of, respectively, text of the present invention refers to Ca2+ permeable the TRPC3/6/7 subfamily members and the TRPC4/5 non-selective cation channels. It shall mean any one of subfamily members under physiological and patho- phys- the list of the following transient receptor potential ca- iological conditions. This can be done e.g. as described 40 nonical ion channels: TRPC 1, TRPC2, TRPC3, TRPC4, in the Exemplification. The analysis can be made in a TRPC5, TRPC6 and TRPC7. Especially preferred are cell, a tissue or an animal. The animal may be a rodent, TRPC3, TRPC6 and/or TRPC7. preferably a mouse or a rat. [0026] Such TRPC ion channels could be derived from [0017] According to a preferred embodiment, the mod- any vertebrate and in particular mammalian species (e.g. ulation of native TRPCs by norgestimate can be investi- 45 dog, horse, bovine, mouse, rat, canine, rabbit, chicken, gated using the A7r5 cell line, which is a validated model anthropoid, human or others). The TRPC could be iso- system for the study of endogenously expressed TRPC lated from tissue probes of such vertebrate organisms or ion channels. Further details of such preferred assay sys- could be manufactured by means of recombinant biolog- tems are given in the Exemplification and in Figure 4. ical material that is able to express the TRPC protein. [0018] Another subject matter of the present invention 50 [0027] The term may refer to native polypeptides, pol- is directed to a method of determining the effect of norg- ymorphic variants, mutants, and interspecies homo- estimate on the TRPC channel activity. Preferred TRPC logues. ion channels are TRPC3, TRPC6 and TRPC7. [0028] "Norgestimate"is also known under itschemical [0019] In general, a cell expressing a TRPC ion chan- name D-17.beta.-acetoxy-B-ethyl-17.alpha. ethinyl-gon- nel is brought into contact with norgestimate and the in- 55 4-en-3-one oxime. It is a gestagen which is a synthetic fluence of norgestimate on the TRPC ion channel activity form of the naturally occurring female sex hormone pro- is measured or detected. gesterone. Norgestimate is a molecule used in the prior [0020] In preferred embodiments the cells used in the art in the composition of hormonal contraceptives.

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[0029] In the present invention, norgestimate is used and the like. as a selective inhibitor of a TRPC3, TRPC6 and TRPC7. [0038] Examples of TRPC activators are diacylglycer- [0030] The term "selective inhibition" in context of the ols, in particular 1-Oleyl-2acetyl-sn-glycerol (OAG), Gq- present invention refers to the potency of an inhibitor, coupled receptor agonists, such as phenylephrine and e.g. norgestimate, to inhibit one or more members of the 5 in particular trypsin, an agonist that stimulates receptor TRPC family with a higher efficiency than one or more tyrosine kinases such as epidermal growth factor (EGF) other members. In a preferred embodiment, norgesti- or diacylglycerol generating enzymes such as phosphol- mate is more effective on the TRPC3/6/7 subfamily than ipases or activators thereof. on the TRPC4/5 subfamily. In a more preferred embod- [0039] An example for the measurement of the TRPC iment, norgestimate is at least 4-fold more effective on 10 ion channel activity modulation in presence of a test com- the TRPC3/6/7 subfamily than on the TRPC4/5 subfami- pound is as following: ly. [0040] In general, cells are provided which express a [0031] The term "pharmacological tool" refers to a TRPC channel. Such cells can be produced using genetic compound which functional properties allow to study how methods known to a person skilled in the art. After having drugs interact with living organisms to produce a change 15 induced the expression of the TRPC channel, cells are in functions of interest, thereby allowing to study new usually plated into e.g. microtiter plates and grown. Usu- drug composition and properties, interactions, toxicolo- ally the cells grow at the bottom of multiwell plates and gy, therapy, medical applications and antipathogenic ca- are fixed. Thereafter, the cells are generally washed and pabilities. Furthermore, the term refers to a compound loaded with a dye in a suitable loading buffer, preferably which may be used to characterize potential targets for 20 with a fluorescent dye such as fluo4am. After having re- the development of novel medicines, e.g. characterize moved the loading buffer, the cells are incubated with their native composition, activation mechanisms, physi- the test compound or modulator, in particular with a bio- ological functions, and roles in pathophysiology and dis- chemical orchemical test compound as described above, ease. e.g. in the form of a chemical compound library. Ca2+ [0032] The term "differential analysis" refers to an anal- 25 measurements can be carried out using e.g. a Fluores- ysis which allows to characterize channel function differ- cence Imaging Plate Reader (FLIPR). To stimulate Ca 2+ ences between TRPC3/6/7 subfamily members and influxthrough theTRPC channela channel activator such TRPC4/5 subfamily members under the same condi- as OAG should generally be applied. tions. [0041] The expected effects of inhibitors would be a [0033] According to a preferred embodiment, cells ex- 30 reduction of e.g. the fluorescence increase. Activators pressing respectively TRPC6 ion channels and TRPC5 would lead to a further increase of e.g. an activator- ion channels are brought into contact with norgestimate evoked fluorescence or induce e.g. an activator-inde- in two parallel experiments, and the influence of norges- pendent fluorescence increase. timate on the respective TRPC ion channel activities is [0042] Thereafter, suitable modulators, in particular in- measured or detected, allowing to compare functions of 35 hibitors can be analyzed and/or isolated. For the screen- the respective channels under the same conditions. ing of chemical compound libraries, the use of high- [0034] The term "TRPC ion channel modulator" means throughput assays are preferred which are known to the a modulating molecule of the TRPC channel, in particular skilled person or which are commercially available. an inhibitory or activating molecule ("inhibitor" or "activa- [0043] The term "cell expressing TRPC" refers to a cell tor"), especially an inhibitor of the TRPC channel identi- 40 expressing the ion channel of interest endogenously or fiable according to the method of the present invention. a recombinant cell. [0035] An inhibitor is generally a compound which, e.g. [0044] The cell will usually be a mammalian cell, such bindsto, partially or totally blocks activity, decreases, pre- as a human cell, a mouse cell, a rat cell, a Chinese ham- vents, delays activation, inactivates, desensitizes, or ster cell, etc. Cells that are found to be convenient include down-regulates the activity or expression of at least one 45 MDCK, HEK 293, HEK 293 T, BHK, COS, NIH3T3, of the TRPC channels as preferably described above in Swiss3T3 and CHO cells, preferably HEK293 cells. details. [0045] The term "tissue" as used herein, can refer to [0036] An activator is generally a compound which, any type of tissue preparation or taken part of tissue or e.g. increases, opens, activates, facilitates, enhances organ (e.g. brain, liver, spleen, kidney, heart, blood ves- activation, sensitizes, agonizes, or up-regulates the ac- 50 sels, muscle, skin, etc. and refers also to any kind of body tivity or expression of at least one of the TRPC. channels fluid such as blood, saliva, lymphatic fluid, synovial fluid as preferably described above in details. etc.), preferably if derived from a vertebrate, and more [0037] Such modulators include genetically modified preferably from a mammal such as a human. Tissue sam- versions of the TRPC channels, preferably an inactivat- ples can be gained by well-known techniques, such as ing mutant of the TRPC channels, as well as naturally 55 taking of blood, tissue punction or surgical techniques. occurring or synthetic ligands, antagonists, agonists, [0046] Examples of dyes used in such measurements peptides, cyclic peptides, nucleic acids, antibodies, an- are Di-4-ANEPPS (pyridinium 4-(2(6-(dibutylamino)-2- tisense molecules, ribozymes, small organic molecules naphthalenyl)ethenyl)-1-(3-sulfopropyl) hydroxide), CC-

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2-DMPE (12- ,ditetradecanoyl- sn-glycero-3-phos- HEK293 FITR cells preincubated with 30 mM norgesti- phoethanolamine triethylammonium), DiSBAC2 (bis-(1 , mate or buffer only (10 min). Kinetics and quantity of cal- 2-dibarbituric acid)-trimethine oxanol), DisBAC3cium store release in cells treated with 30 mM norgesti- ((bis-(1 ,3-dibarbituric acid)-trimethine oxanol), SBFI- mate were not changed compared to untreated cells. AM (13- , benzenedicarboxylic acid,4,4’-[1,4,10-trioxa- 5 Therefore, norgestimate is not a PAR antagonist and 7,13-diazacyclopentadecane- 7,13-diylbis(5-methoxy-6, TRPC4 and TRPC5 activation via PAR stimulation is suit- 12-benzofurandiyl)]bis-(tetrakis-[(acetyloxy)methyl]es- able to measure the channels inhibition by norgestimate. ter)), fluo3am (1-[2-Amino-5-(2,7-dichloro-6-hydroxy-3- [0050] 30 mM norgestimate had only small inhibitory oxy-9-xanthenyl)phenoxy]-2-(2’- amino-5<1>-methyl- effects on TRPC4 and TRPC5 (fig. 2 A, C). An IC 50 > 30 phenoxy)ethane- N,N,N’,N’-tetraacetic), fluo4am (1-[2- 10 mM was determined on both TRPC4 (n = 2, Figure 2 B) Amino-5-(2,7- dichloro-6-hydroxy-4-oxy-9-xanthenyl) and TRPC5 (n = 4, Figure 2 D). phenoxy]-2-(2’-amino-5’- methylphenoxy)ethane-N,N, [0051] In summary these FLIPR measurements have N’,N’-tetraacetic) or fura2am (1-t2-(5’-carboxyoxazol-2’- shown that norgestimate inhibits channels of the yl)- 6-aminobenzofuran-5-oxy]-2-(2’-amino-<1>-methyl- TRPC3/6/7 subfamily as well as of the TRPC4/5 sub- phenoxy)-ethane-N,N,N’,N’- tetraacetic). 15 family when applied in micromolar concentrations. Norg- [0047] The term "profiling" refers to the characterisa- estimate was more effective on the TRPC3/6/7 than on tion of a compound, e.g. norgestimate, through functional the TRPC4/5 subfamily. Therefore, norgestimate was and structural categories, potency, specificity, selectivity chosen for further experiments to test its discriminating and mechanism-of-action. In the present invention, it re- capability. fers in particular to the selectivity study of the inhibition 20 effect of norgestimate on a TRPC ion channel. 2. Patch Clamp recordings

EXEMPLIFICATION 2.1. Differential effect of Norgestimate on recombinant homomeric TRPC5 and TRPC6 channels 1. FLIPR measurements 25 [0052] Inhibition of TRPC6- and TRPC5-mediated TRPC3/6 Ca2+-influx by norgestimate monitored in fluorometric experiments with cell populations was validated and com- [0048] TRPC3 and TRPC6 were directly activated by pared with whole-cell patch clamp recordings of single application of oleoyl- 2-acetyl-sn-glycerol (OAG), a mem- 30 cells. Channels were indirectly excited with the same brane permeable diacylglycerol analogue. TRPC3- and stimulus for better comparability of the norgestimate ef- 2+ - TRPC6-mediated Ca influx following application of 30 fect. Aluminium tetrafluoride (AlF 4 ) applied intracellular- mM OAG was strongly reduced in cells preincubated with ly via the patch pipette activates G- proteins by mimicking 30 mM norgestimate (Fig. 1 A, C).The IC50 of norgesti- GTP (Sternweis & Gilman, 1982; Bigay et al., 1985). Sub- mate on TRPC3 was 2.8 6 0.4 mM (n = 2, Fig. 1B). Norg- 35 sequently TRPC channels are stimulated and mediate estimate was similarly active on TRPC6 with an IC50 of non-specific cationic currents (fig. 3 A, B) 5.2 6 0.4 mM (n = 4, Fig. 1 D). [0053] When 10 mM norgestimate were applied to activated TRPC6 channels, 89.9 6 3.1% (n = 15) of the TRPC4/5 current measured at resting membrane potential were 40 blocked. This inhibition was reversible as current ampli- [0049] When these experiments were performed, no tude increased after norgestimate wash out (fig. 3 A). direct physiological stimuli of TRPC4 and TRPC5 were The adjacent block by 10 mM lanthanum (La 3+) was com- known. Therefore, both channels had to be stimulated plete and reversible and thus used for background (leak) indirectly, e.g. by application of trypsin, a protease-acti- calculation. vated receptor (PAR) stimulating protease. Trypsin is 45 [0054] By contrast, application of 10 mM norgestimate able to activate all four known PAR subtypes and the to stimulated TRPC5 channels had a minor effect on the mRNA of three of them (PAR1, PAR2 and PAR3) was current, 25.9 6 5.7% (n = 20) were blocked. Since these shown to be endogenously present in HEK293 cells (Ka- channels are potentiated by micromolar La 3+ concentra- wabata et al., 1999). PAR are G protein- coupled and their tions (Jung et al., 2003), 2-aminoethoxydiphenyl borate activation leads to calcium store depletion from endo- 50 (2-APB), a characterized TRPC5 blocker (Xu et al., 2005) plasmic reticulum (ER). This so called phosphatidylinosi- was used instead. Background was calculated by appli- tol (PI) response is independent of the channel’s pres- cation of 10 mM 2-APB that completely and reversibly ence but consequently leads to activation of store- oper- blocked the channel. ated channels like TRPC4 and TRPC5. Basic prerequi- [0055] So far all norgestimate effects were investigat- site to measure the effect of norgestimate on both chan- 55 ed on homomeric channels heterologously expressed in nels under these conditions is to exclude PAR antago- HEK293 cells. It is well known that TRPC channels heter- nism of this potential channel inhibitor. Hence, calcium omultimerize under physiological conditions (reviewed release from ER was compared in non-induced TRPC5 by Schaefer, 2005). Taking the numerous possible stoi-

6 9 EP 2 205 247 B1 10 chiometrically compositions of these heteromultimers in- norgestimate (NG). to account, it is common use to start pharmacological Measurements were performed in 96er well examinations of TRPC channels in artificial model sys- plates using FLIPR (Molecular Devices, Sunny- tems. But it is even more interesting to test whether norg- vale, CA, USA) estimate inhibits native TRPC channels too. 5

(B, D) Determination of norgestimates IC50 val- 2.2 Effect of norgestimate on native heteromeric ue on TRPC4 (B) and TRPC5 (D). Data are rep- TRPC6/7 channels resented as means of 2 wells (B) and 4 wells (D) with 47,000 cells per well. [0056] The A7r5 cell line (derived from rat thoracic aor- 10 ta smooth muscle cells) is a model system to investigate Figure 3: endogenous TRPC channels (Jung et al., 2002;Soboloff et al., 2005). The channels were indirectly stimulated by Effect of 10 mM norgestimate (NG) on whole- 8 - Arg -vasopressin (AVP), a vasoconstricting peptide that cell currents evoked by AlF4 infusion into in- 15 activates the endogenous vasopressin V1A receptor in duced TRPC6 - (A) and TRPC5 HEK 293 (B) these cells (Thibonnier et al., 1991). Before whole-cell cells. Whole cell currents recorded at - 70 mV patch clamp recordings were performed, it was shown in (left panels) and the corresponding current- volt- calcium imaging experiments that norgestimate does not age (I-V) relationships are shown (right panels). generally suppress V1 receptors (fig. 4) For background correction channels were com- [0057] Perfusion of A7r5 cells with 100 nM AVP stim- 20 pletely blocked with 10 mM La3+ (A) or 10 mM 2- ulated non-selective cationic currents that bore the bio- APB (B). The curves were obtained during volt- physical hallmarks of TRPC channels (fig. 5). The dou- age ramps from -100 to +80 mV. ble-rectifying I-V relationship was similar to those exhib- (C) Statistical analysis of the norgestimate effect ited by the heterologously expressed TRPC6 homomers on TRPC5- and TRPC6-mediated currents. 10 (Fig. 3 A ). When 10 mM norgestimate were applied to 25 mM norgestimate inhibit 25.9 6 5.7% (n = 20) of AVP-stimulated A7r5 cells, 86.5 6 6.0% (n = 8) of the TRPC5-mediated currents and 89.9 6 3.1% (n current measured at resting membrane potential were = 15) of TRPC6-mediated currents (P < 0.001, blocked and this effect was reversible (fig. 5). Wilcoxon test, two-sided).

DESCRIPTION OF THE FIGURES 30 Figure 4:

2+ [0058] Time-dependent changes of [Ca ]i of fura-2- loaded A7r5 cells. Cells were preincubated with Figure 1: or without (control) 10 mM norgestimate (NG) in 35 calcium-free standard extracellular solution (1 (A, C) Time-dependent changes of intracellular mM EGTA) for 5 min before stimulating the V1 2+ 2+ Ca concentration ([Ca ]i) of fluo-4-loaded in- receptor by application of 100 nM AVP. Data are duced TRPC3 HEK293 cells (A) and TRPC6 represented as means of 33 cells (control) and HEK293 cells (C). TRPC-mediated Ca2+ influx 36 cells (10 mM norgestimate). following application of 30 mMOAG was strongly 40 Measurements were performed using a stand- reduced in cells preincubated with 30 mM norg- ard imaging setup (TILL Photonics, Gräfelfing, estimate (NG). Germany). Measurements were performed in 96er well plates using FLIPR (Molecular Devices, Sunny- Figure 5: vale, CA, USA) 45 (B, D) Determination of norgestimates IC 50 val- Effect of 10 mM norgestimate (NG) on whole- ue on TRPC3 (B) and TRPC6 (D). Data are rep- cell currents evoked by 100 nM AVP on A7r5 resented as means of 2 wells (B) and 4 wells cells in standard extracellular solution (200 mM 2+ (D) with 45,000 cells per well. [Ca ]o). Whole cell currents recorded at -60 mV 50 (left panels) and the corresponding I- V relation- Figure 2: ships are shown (right panels). The curves were obtained during voltage ramps from -100 to +80 (A, C) Time-dependent changes of intracellular mV. L-type voltage-gated Ca2+ channels were 2+ 2+ Ca concentration ([Ca ]i) of fluo-4-loaded in- blocked by 5 mM nimodipine (ND) during the duced TRPC4 HEK293 cells (A) and TRPC5 55 whole experiment. HEK293 cells (C). TRPC-mediated Ca2+ influx following application of 200 nM trypsin was slightly reduced in cells preincubated with 30 mM

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Claims a fluorescent cell.

1. Use of norgestimate for inhibition of a TRPC ion 11. The method according to claim 9 or 10, wherein the channel, wherein the TRPC ion channel is selected cell is selected from a MDCK, HEK 293, HEK 293 T, from the list consisting of: TRPC3, TRPC6 and5 BHK, COS, NIH3T3, Swiss3T3 or CHO cell. TRPC7, and wherein norgestimate is used as a pharmacological tool which allows characterising chan- 12. The method according to any of the claims 9-11, nels belonging to different TRPC subfamilies, wherein the TRPC ion channel activity is measured with the proviso that said use is not a use in a method or detected by measuring or detecting a change in of treatment of the human or animal body. 10 Ca2+ fluxes, by patch clamp techniques, whole cell currents, radiolabeled ion fluxes, or fluorescence us- 2. Use of norgestimates according to claim 1, which is ing voltage-sensitive dyes or ion-sensitive dyes. carried out in vitro. 13. A method for profiling selectivity of norgestimate for 3. Use of norgestimate according to claim 1, which is 15 a TRPC ion channel, which comprises assessing the carried out in vivo in a non-human animal. norgestimate’s ability to inhibit the TRPC ion channel activity and comparing the inhibition potency of norg- 4. Use of norgestimate according to claim 1, wherein estimate between and within the different TRPC sub- norgestimate is used to make a differential analysis families. of the channel functions of, respectively, 20 the TRPC3/6/7 subfamily members and the TRPC4/5 subfamily members under physiological and patho- Patentansprüche physiological conditions. 1. Verwendung von Norgestimat zum Inhibieren eines 5. Use of norgestimate according to claim 4, wherein 25 TRPC-Ionenkanals, wobei der TRPC-Ionenkanal the analysis is made in a cell. aus der folgenden Liste ausgewählt ist, bestehend aus: 6. Use of norgestimate according to claim 4, wherein the analysis is made in a tissue. TRPC3, TRPC6 und TRPC7, und wobei Norge- 30 stimat als pharmakologisches Werkzeug ver- 7. Use of norgestimate according to claim 4, wherein wendetwird, mitdem es möglich ist,zu verschie- the analysis is made in a non-human animal. denen TRPC-Unterfamilien gehörende Kanäle zu charakterisieren, 8. A method for selectively inhibiting a TRPC ion chan- mit der Maßgabe, dass es sich bei der Verwen- nel, which comprises contacting a cell expressing a 35 dung nicht um eine Verwendung in einem Ver- TRPC ion channel with norgestimate, wherein the fahren zur Behandlung des menschlichen oder TRPC ion channel is selected from the list consisting tierischen Körpers handelt. of: 2. Verwendung von Norgestimat nach Anspruch 1, wel- TRPC3, TRPC6 and TRPC7, and which is car- 40 che in vitro durchgeführt wird. ried out in vitro or which is carried out in vivo in a non-human animal, wherein the animal is a 3. Verwendung von Norgestimat nach Anspruch 1, wel- mouse or a rat, che in vivo in einem nichtmenschlichen Tier durch- with the proviso that said method is not a method geführt wird. of treatment of the human or animal body. 45 4. Verwendung von Norgestimat nach Anspruch 1, wo- 9. A method for determining the effect of norgestimate bei Norgestimat für eine differentiale Analyse der Ka- on the TRPC ion channel activity, which comprises: nalfunktionen der Mitglieder der TRPC3/6/7-Unter- familie beziehungsweise der Mitglieder der a) contacting a cell expressing a TRPC ion chan- 50 TRPC4/5-Unterfamilie unter physiologischen und nel, pathophysiologischen Bedingungen verwendet b) stimulating Ca2+ influx by a channel activator wird. before, simultaneously or after contacting the cell with norgestimate, and 5. Verwendung von Norgestimat nach Anspruch 4, wo- c) measuring or detecting a change of the TRPC 55 bei die Analyse in einer Zelle erfolgt. ion channel activity. 6. Verwendung von Norgestimat nach Anspruch 4, wo- 10. The method according to claim 9, wherein the cell is bei die Analyse in einem Gewebe erfolgt.

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7. Verwendung von Norgestimat nach Anspruch 4, wo- nal ionique TRPC, le canal ionique TRPC étant choi- bei die Analyse in einem nichtmenschlichen Tier er- si dans la liste constituée de : TRPC3, TRPC6 et folgt. TRPC7, et le norgestimate étant utilisé en tant qu’outil pharmacologique qui permet la caractérisa- 8. Verfahren zur selektiven Inhibierung eines TRPC- 5 tion de canaux appartenant à différentes sous-fa- Ionenkanals, bei dem man eine einen TRCP- Ionen- milles de TRPC, kanal exprimierende Zelle mit Norgestimat in Kon- à condition que ladite utilisation ne soit pas une uti- takt bringt, wobei der TRPC- Ionenkanal aus der aus: lisation dans un procédé de traitement du corps hu- TRPC3, TRPC6 und TRPC7 bestehenden Liste aus- main ou animal. gewählt ist, und welches in vitro durchgeführt wird 10 oder in vivo in einem nichtmenschlichen Tier durch- 2. Utilisation de norgestimate selon la revendication 1, geführt wird, wobei es sich bei dem Tier um eine qui est conduite in vitro. Maus oder eine Ratte handelt, mit der Maßgabe, dass es sich bei dem Verfahren 3. Utilisation de norgestimate selon la revendication 1, nicht um ein Verfahren zur Behandlung des mensch- 15 qui est conduite in vivo chez un animal non humain. lichen oder tierischen Körpers handelt. 4. Utilisation de norgestimate selon la revendication 1, 9. Verfahren zur Bestimmung der Wirkung von Norge- le norgestimate étant utilisé pour effectuer une ana- stimat auf die TRPC-Ionenkanalaktivität, bei dem lyse différentielle des fonctions du canal, respecti- man: 20 vement, des membres de la sous- famille TRPC3/6/7 et des membres de la sous-famille TRPC4/5 dans a) eine einen TRPC-Ionenkanal exprimierende des conditions physiologiques et physiopathologi- Zelle mit Norgestimat in Kontakt bringt, ques. b) den Ca2+-Einfluss vor, gleichzeitig mit oder nach dem Inkontaktbringen der Zelle mit Norge- 25 5. Utilisation de norgestimate selon la revendication 4, stimat mit einem Kanalaktivator stimuliert und l’analyse étant effectuée dans une cellule. c) eine Veränderung der TRPC- Ionenkanalakti- vität misst oder nachweist. 6. Utilisation de norgestimate selon la revendication 4, l’analyse étant effectuée dans un tissu. 10. Verfahren nach Anspruch 9, wobei es sich bei der 30 Zelle um eine fluoreszente Zelle handelt. 7. Utilisation de norgestimate selon la revendication 4, l’analyse étant effectuée chez un animal non hu- 11. Verfahren nach Anspruch 9 oder 10, wobei die Zelle main. aus einer MDCK-, HEK 293-, HEK 293 T-, BHK-, COS-, NIH3T3-, Swiss3T3- oder CHO- Zelle ausge- 35 8. Procédé pour inhiber sélectivement un canal ionique wählt ist. TRPC, qui comprend la mise en contact d’une cellule exprimant un canal ionique TRPC avec du norges- 12. Verfahren nach einem der Ansprüche 9-11, wobei timate, le canal ionique TRPC étant choisi dans la die TRPC-Ionenkanalaktivität durch Messen oder liste constituée de : TRPC3, TRPC6 et TRPC7, et Nachweis einer Veränderung der 2+ Ca-Ströme 40 qui est conduit in vitro ou qui est conduit in vivo chez durch Patch-Clamp-Methoden, Ganzzellströme, un animal non humain, l’animal étant une souris ou Ströme radioaktiv markierter Ionen oder Fluores- un rat, à condition que ledit procédé ne soit pas un zenz mit spannungssensiblen Farbstoffen oder io- procédé de traitement du corps humain ou animal. nensensiblen Farbstoffen gemessen beziehungsweise nachgewiesen wird. 45 9. Procédé pour déterminer l’effet de norgestimate sur l’activité du canal ionique TRPC, qui comprend : 13. Verfahren zum Erstellen eines Selektivitätsprofils von Norgestimat für einen TRPC-Ionenkanal, bei a) la mise en contact d’une cellule exprimant un dem man die Fähigkeit von Norgestimat zum Inhi- canal ionique TRPC avec du norgestimate, bieren der TRPC- Ionenkanalaktivität untersucht und 50 b) la stimulation de l’influx de Ca 2+ par un acti- das Inhibierungsvermögen von Norgestimat zwi- vateur de canal avant, simultanément ou après schen und innerhalb der verschiedenen TRPC-Un- la mise en contact de la cellule avec du norges- terfamilien vergleicht. timate, et c) la mesure ou la détection d’un changement 55 de l’activité du canal ionique TRPC. Revendications 10. Procédé selon la revendication 9, la cellule étant une 1. Utilisation de norgestimate pour l’inhibition d’un ca- cellule fluorescente.

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11. Procédé selon la revendication 9 ou 10, la cellule étant choisie parmi une cellule MDCK, HEK 293, HEK 293 T, BHK, COS, NIH3T3, Swiss3T3 ou CHO.

12. Procédé selon l’une quelconque des revendications 5 9 à 11, l’activité du canal ionique TRPC étant mesu- rée ou détectée en mesurant ou en détectant un changement des flux de Ca2+, par des techniques « patch clamp », les courants de cellules totales, les flux d’ion radiomarqué, ou la fluorescence en utili- 10 sant des colorants sensibles au voltage ou des colorants sensibles aux ions.

13. Procédé de profilage de la sélectivité du norgestimate pour un canal ionique TRPC, qui comprend 15 l’évaluation de la capacité du norgestimate à inhiber l’activité du canal ionique TRPC et la comparaison du pouvoir inhibiteur du norgestimate entre et dans les différentes sous-familles de TRPC. 20

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

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