TREATMENT OF TINNITUS THROUGH MODULATION OF CHLORIDE CO-TRANSPORTER NKCC1 IN THE AUDITORY SYSTEM

Abstract

The present invention relates to the treatment or prevention of tinnitus. More precisely, the present invention relates to a compound modulating chloride co-transporter NKCC1 (chloride co-transporter modulator) for use in the treatment of tinntitus. In addition, the present invention concerns pharmaceutical compositions comprising such an NKCC1 chloride co-transporter modulator as an active agent, a method for the treatment or prevention of tinnitus by administering such a chloride co-transporter modulator, and a screening method for the identification and characterization of compounds capable of modulating chloride co-transporter NKCC1.

Claims

1. A compound modulating chloride co-transporter sodium potassium chloride co-transporter 1 (NKCC1) for use in the treatment or prevention of tinnitus.

2. Compound for use according to claim 1, wherein the compound antagonizes NKCC1.

3. Compound for use according to claim 1 or 2, wherein the compound antagonizes the function of NKCC1 by decreasing or inhibiting its activity and/or expression.

4. Compound for use according to any of claims 1 to 3, wherein the activity and/or expression of KCC2 is increased by NKCC1 antagonism.

5. Compound for use according to claim 3, wherein the compound decreases activity and/or expression of NKCC1 and increases activity and/or expression of KCC2.

6. Compound for use according to any of claims 1 to 5, wherein the compound is selected from the group consisting of thiazides and sulfonamides.

7. Compound for use according to any of claims 1 to 6, wherein the compound is a sulfonamide selected from the group consisting of acetazolamide, azosemide, bumetanide, chlorthalidone, clopamide, furosemide, hydrochlorothiazide (HCT, HCTZ, HZT), indapamide, mefruside, metolazone, piretanide, tripamide xipamide, dichlorphenamide (DCP), dorzolamide, ethoxzolamide, sultiame, or zonisamide or analogs thereof.

8. Compound for use according to any of claims 1 to 7, wherein the sulfonamide compound is a diuretic compound, preferably selected from the group consisting of bumetanide, furosemide, piretanide, azosemide, and torsemide or analogs thereof.

9. Compound for use according to any of claims 1 to 8, wherein diuretic compound is selected from the group consisting of (i) bumetanide, bumetanide aldehyde, bumetanide methyl ester, bumetanide cyanomethyl ester, bumetanide ethyl ester, bumetanide isoamyl ester, bumetanide octyl ester, bumetanide benzyl ester, bumetanide dibenzylamide, bumetanide diethylamide, bumetanide morpholinoethyl ester, bumetanide 3-(dimethylaminopropyl) ester, bumetanide N,N-diethylglycolamido ester, bumetanide N,N-dimethylglycolamido ester, bumetanide pivaxetil ester, bumetanide propaxetil ester, bumetanide methoxy(polyethyleneoxy).sub.n-1-ethyl ester, bumetanide benzyltrimethylammonium salt and bumetanide cetyltrimethylammonium salt, bumetanide [—(C═O)—SH] thioacid, bumetanide S-methyl thioester, bumetanide S-cyanotnethyl thioester, bumetanide S-ethyl thioester, bumetanide S-isoamyl thioester, bumetanide S-octyl thioester, bumetanide S-benzyl thioester, bumetanide S-(morpholinoethyl) thioester, bumetanide S—[3-(dimethylaminopropyl)] thioester, bumetanide S—(N,N-diethylglycolamido) thioester, bumetanide S—(N,N-dimethylglycolamido) thioester, bumetanide S-pivaxetil thioester, bumetanide S-propaxetil thioester, bumetanide S—[methoxy(polyethyleneoxy).sub.n-1-ethyl] thioester, bumetanide [(C═O)—S.sup.−] benzyltrimethyl-ammonium thioacid salt and bumetanide [—(C═O)—S.sup.−] cetyltrimethylammonium thioacid salt, metastable bumetanide [—(C═S)—OH] thioacid, bumetanide O-methyl thioester, bumetanide O-cyanomethyl thioester, bumetanide O-ethyl thioester, bumetanide O-isoamyl thioester, bumetanide O-octyl thioester, bumetanide O-benzyl thioester, bumetanide O-(morpholinoethyl) thioester, bumetanide O—[3-(dimethylaminopropyl)] thioester, bumetanide O—(N,N-diethylglycolamido) thioester, bumetanide, O—(N,N-dimethylglycolamido) thioester, bumetanide O-pivaxetil thioester, bumetanide O-propaxetil thioester, bumetanide O—[methoxy(poryethyleneoxy).sub.n-1-ethyl] thioester, bumetanide [—(C═S)—O.sup.−] benzyltrimemyl-ammonium thioacid salt and bumetanide [—(C═S)—O.sup.−] cetyltrimethylammonium thioacid salt, bumetanide thioaldehyde, bumetanide [—(C═S)—SH] dithioacid, bumetanide methyl dithioester, bumetanide cyanomethyl dithioester, bumetanide ethyl dithioester, bumetanide isoamyl dithioester, bumetanide octyl dithioester, bumetanide benzyl dithioester, bumetanide dibenzylthioamide, bumetanide diethylthioamide, bumetanide morpholinoethyl dithioester, bumetanide 3-(dimethylaminopropyl) dithioester, bumetanide N,N-diethylglycolamido dithioester, bumetanide N,N-dimethylglycolamido dithioester, bumetanide pivaxetil dithioester, bumetanide propaxetil dithioester, bumetanide methoxy(polyethyleneoxy).sub.n-1-ethyl dithioester, bumetanide benzyltrimethylammonium dithioacid salt and bumetanide cetyltrimethyl-ammonium dithioacid salt and (ii) furosemide, furosemide aldehyde, furosemide methyl ester, furosemide cyanomethyl ester, furosemide ethyl ester, furosemide isoamyl ester, furosemide octyl ester, furosemide benzyl ester, furosemide morpholinoethyl ester, furosemide 3-(dimethylaminopropyl) ester, furosemide N,N-diethylglycolamido ester, furosemide N,N-dimethylglycolamido ester, furosemide pivaxetil ester, furosemide propaxetil ester, furosemide methoxy(polyethyleneoxy).sub.n-1-ethyl ester, furosemide benzyltrimethylammonium acid salt and furosemide cetyltrimethylammonium acid salt. In particular embodiments, the compound is not furosemide. In further embodiments of the present invention, the compound of formula IV can be furosemide [—(C═O)—SH] thioacid, furosemide S-methyl thioester, furosemide S-cyanomethyl thioester, furosemide S-ethyl thioester, furosemide S-isoamyl thioester, furosemide S-octyl thioester, furosemide S-benzyl thioester, furosemide S-(morpholinoethyl) thioester, furosemide S—[3-(dimethylaminopropyl)] thioester, furosemide S—(N,N-diethylglycolamido) thioester, furosemide S—(N,N-dimethylglycolamido) thioester, furosemide S-pivaxetil thioester, furosemide S-propaxetil thioester, furosemide S—[methoxy(poryethyleneoxy).sub.n-1-ethyl] thioester, furosemide [—(C═O)—S.sup.−] benzyltrimethylammonium thioacid salt and furosemide [—(C═O)—S.sup.−]cetyltrimethylammonium thioacid salt, metastable furosemide [—(C═S)—OH] thioacid, furosemide O-methyl thioester, furosemide O-cyanomethyl thioester, furosemide O-ethyl thioester, furosemide O-isoamyl thioester, furosemide O-octyl thioester, furosemide O-benzyl thioester, furosemide O-(morpholinoethyl) thioester, furosemide O—[3-(dimethylaminopropyl)] thioester, furosemide O—(N,N-diethylglycolamido) thioester, furosemide O—(N,N-dimethylglycolamido) thioester, furosemide O-pivaxetil thioester, furosemide O-propaxetil thioester, furosemide O—[methoxy(polyethyleneoxy).sub.n-1-ethyl] thioester, furosemide [—(C═S)—O.sup.−] benzyltrimethyl-ammonium thioacid salt and furosemide cetyltrimethylammonium thioacid salt, furosemide thioaldehyde, furosemide [—(C═S)—SH] dithioacid, furosemide methyl dithioester, furosemide cyanomethyl dithioester, furosemide ethyl dithioester, furosemide isoamyl dithioester, furosemide octyl dithioester, furosemide benzyl dithioester, furosemide dibenzylthioamide, furosemide diethylthioamide, furosemide morpholinoethyl dithioester, furosemide 3-(dimethylamino[rho]ropyl) dithioester, furosemide N,N-diethylglycolamido dithioester, furosemide N,N-dimethylglycolamido dithioester, furosemide pivaxetil dithioester, furosemide propaxetil dithioester, furosemide methoxy(polyethyleneoxy).sub.n-1-ethyl dithioester, furosemide benzyltrimethylammonium dithioacid salt and furosemide cetyltrimethylammonium dithioacid salt and (iii) piretanide, piretanide aldehyde piretanide methyl ester, piretanide cyanomethyl ester, piretanide ethyl ester, piretanide isoarmyl ester, piretanide octyl ester, piretanide benzyl ester, piretanide dibenzylamide, piretanide diethylamide, piretanide morpholinoethyl ester, piretanide 3-(dimethylaminopropyl) ester, piretanide N,N-diethylglycolamide ester, piretanide dimethylglycolamide ester, piretanide pivaxetil ester, piretanide propaxetil ester, piretanide methoxy(polyethyleneoxy).sub.n-1-ethyl ester, piretanide benzyltrimethylammonium salt and piretanide cetyltrimethylammonium salt. In particular embodiments, the compound is not piretinide, piretanide [—(C═O)—SH] thioacid, piretanide S-methyl thioester, piretanide S-cyanomethyl thioester, piretanide S-ethyl thioester, piretanide S-isoamyl thioester, piretanide S-octyl thioester, piretanide S-benzyl thioester, piretanide S-(morpholinoethyl) thioester, piretanide S—[3-(dimethylaminopropyl)] thioester, piretanide S—(N,N-diethylglycolamido) thioester, piretanide S—(N,N-dimethylglycolamido) thioester, piretanide S-pivaxetil thioester, piretanide S-propaxetil thioester, piretanide S—[methoxy(polyethyleneoxy).sub.n-1-ethyl] thioester, piretanide [-(C═O)—S.sup.−] benzyltrimethylammonium thioacid salt and piretanide [—(C═O)—S.sup.−] cetyltrimethylammonium thioacid salt, metastable piretanide [—(C═S)—OH] thioacid, piretanide O-methyl thioester, piretanide O-cyanomethyl thioester, piretanide O-ethyl thioester, piretanide O-isoamyl thioester, piretanide O-octyl thioester, piretanide O-benzyl thioester, piretanide O-(morpholinoethyl) thioester, piretanide O—[3-(dimethylaminopropyl)] thioester, piretanide O—(N,N-diethylglycolamido) thioester, piretanide, O—(N,N-dimethylglycolamido) thioester, piretanide O-pivaxetil thioester, piretanide O-propaxetil thioester, piretanide O—[methoxy(polyethyleneoxy).sub.n-1-ethyl] thioester, piretanide [—(C═S)—O.sup.−] benzyltrimethylammonium thioacid salt and piretanide [—(C═S)—O.sup.−] cetyltrimethylammonium thioacid salt, piretanide thioaldehyde, piretanide [—(C═S)—SH] dithioacid, piretanide methyl dithioester, piretanide cyanomethyl dithioester, piretanide ethyl dithioester, piretanide isoamyl dithioester, piretanide octyl dithioester, piretanide benzyl dithioester, piretanide dibenzylthioamide, piretanide diethylthioamide, piretanide morpholinoethyl dithioester, piretanide 3-(dimethylaminopropyl) dithioester, piretanide N,N-diethylglycolamido dithioester, piretanide N,N-dimethylglycolamido dithioester, piretanide pivaxetil dithioester, piretanide propaxetil dithioester, piretanide methoxy(polyethyleneoxy).sub.n-1-ethyl dithioester, piretanide benzyltrimethylammoniurn dithioacid salt and piretanide cetyltrimethylarnmoniurn dithioacid salt and (iv) tetrazolyl-substituted azosemides, in particular methoxymethyl tetra-zolyl-substituted azosemides, methylthiornethyl tetrazolyl-substituted azosemides and N-mPEG350-tetrazolyl-substituted azosemides), azosemide benzyltrimethylammonium salt and/or azosemide cetyltrimethylammonium salt and (v) pyridine-substituted torsemide quaternary ammonium salts or the corresponding inner salts (zwitterions), in particular methoxymethyl pyridinium torsemide salts, methylthiomethyl pyridinium torsemide salts and N-mPEG350-pyridinium torsemide salts.

10. Compound for use according to any of claims 1 to 6, wherein the thiazide compound is selected from the group consisting of bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methylclothiazide, polythiazide, trichlor-methiazide, chlorthalidone, indapamide, metolazone or quinethazone or analogs thereof.

11. Compound for use according to any of claims 1 to 10, wherein the compound is used in combination with one or more carbonic anhydrase inhibitors, in particular acetazomalide, dichlorphenamide, dorzolamide, brinzolamide and/or methazolamide.

12. Compound for use according to any of claims 1 to 5, wherein the compound is an antibody, an antibody fragment or a humanized antibody antagonizing NKCC1 activity or an RNA selected from the group consisting of an siRNA, shRNA and anti-sense RNA, reducing NKCC1 expression.

13. Compound for use according to any of claims 1 to 5, wherein the compound is selected from CCC interacting protein (CIP1) or a functional peptide derived therefrom, N-ethylmaleimide (NEM) or staurosporin.

14. Compound for use according to any of claims 1 to 13, wherein the compound is used in combination with one or more GABAergic agonists and/or one or more glycine agonists, in a co-treatment protocol either simultaneously or in a staggered regimen protocol.

15. A pharmaceutical composition comprising a compound modulating chloride co-transporter NKCC1 as defined in any claims 1 to 14.

16. Pharmaceutical composition according to claim 15, further comprising one or more carbonic anhydrase inhibitor and/or GABAergic agonists and/or glycine agonists.

17. Pharmaceutical composition according to claim 15 or 16, wherein the pharmaceutical composition is provided in a liquid, semi-liquid or viscous form, preferably in a gel-like form.

18. Pharmaceutical composition according to any of claims 15 to 17, wherein the composition contains a, preferably biodegradable polymer, preferably selected from the group consisting of hyaluronic acid resp. hyaluronates, lecithin gels, (poly)alanine derivatives, pluronics, poly(ethyleneglycol), poloxamers, chitosans, xyloglucans, collagens, fibrins, polyesters, poly(lactides), poly(glycolide) or their co-polymers PLGA, sucrose acetate isobutyrate, and glycerol monooleate.

19. Pharmaceutical composition according to any of claims 15 to 18 for use in the treatment or prevention of tinnitus.

20. Pharmaceutical composition according to claim 19 for use in the treatment or prevention of tinnitus, whereby the pharmaceutical composition is administered locally.

21. Pharmaceutical composition according to claim 20 for use in the treatment or prevention of tinnitus, whereby the pharmaceutical composition is administered to the middle/inner ear interface, preferably to the round and/or oval window membrane.

22. A method for treatment tinnitus comprising administering a therapeutically effective amount of a compound modulating one or more chloride co-transporters as defined in claims 1 to 14 or a pharmaceutical composition according to claims 15 and 21.

23. Method for treatment tinnitus according to claim 22, further comprising co-administering of a carbonic anhydrase inhibitor and/or a GABAergic agonist and/or a glycine agonist.

24. Method for treatment according to claim 22 or 23, wherein administration is performed systemically or locally, preferably locally on or in the ear.

25. A screening method for the identification and characterization of compounds capable of modulating one or more chloride transporters, by (a) providing cells stably expressing the one or more chloride transporters, (b) adding a test compound to the cells, (c) adding a transporter cation and (d) measuring the cation transport across the cell membrane.

26. A screening method according to claim 25, wherein one or more of the following steps are carried out: (i) provision of cells with heterologous expression of one or more of the transporters, (ii) additionally adding a fluorescence dye binding to the transporter cation, and (iii) measuring the initial cation transport rate and (iv) blocking interfering transporter activity of another transporter.

27. A screening method according to claim 25 or 26, wherein the one or more chloride transporters are selected from NKCC1 and KCC2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0145] FIG. 1 shows that tinnitus inducing acoustic trauma (120 dB at 10 kHz for 2 hours) leads to a reduced KCC2 expression in inner hair cells and spiral ganglion neurons of the rat. [0146] A) In situ hybridization of spiral ganglion neurons at the midbasal turn of cochleae. 14 days after exposure a clear down-regulation of KCC2 mRNA in the noise exposed animal (AT) is observed (b). Comparison with the sham exposed control animal is provided by (a). [0147] B) Western Blot of KCC2. The first lane shows hippocampus tissue as a positive control with a clear band at 140 kDa, which is KCC2. The second lane corresponds to the cochlear tissue of a non-exposed control animal, the third lane to cochlear tissue of a noise exposed animal 7 days after exposure. A clear down-regulation of KCC2 protein can be seen in the noise exposed animal (right lane) compared to the control animal.

[0148] FIG. 2 shows that down-regulation of KCC2 in rat inner hair cells and spiral ganglion neurons at the cochlear midbasal turn 6 or 30 days following noise trauma (120 dB at 10 kHz for 1 hour respectively 1.5 hours) is inherent to the pathophysiology of tinnitus and not related to hearing loss. [0149] A) In situ hybridization of spiral ganglion neurons at the midbasal turn of cochleae. 6 days after noise trauma a clear down-regulation of KCC2 mRNA in the animal showing tinnitus behaviour is observed (a) compared with the animal that shows no tinnitus behaviour (b). The same observation is made 30 days post noise trauma (c) and (d). [0150] B) Count of inner hair cell ribbon synapses (as % of control) 2 weeks following noise or sham exposure of rat cochleae. Among those ears treated with AP, a significant decrease of ribbons was observed in noise exposed ears in the midbasal (middle columns) and basal (right columns) cochlear turns in comparison to sham exposed animals. In contrast, the decline in noise exposed ears that received NKCC1 antagonist bumetanide was not significant in comparison to sham exposed animals. But there was a significant difference in ribbon numbers between noise exposed ears treated with AP or bumetanide. Apical turns did not show as much change as inner hair cells. They are hardly affected by high-frequency trauma.

[0151] FIG. 3 shows that local administration of the NKCC1 inhibitor bumetanide to rat cochleae exposed to noise trauma protects from down-regulation of KCC2 and attenuates the morphological correlate of tinnitus. [0152] A) Real-time PCR for KCC2 expression in cochlear tissue 2 weeks following noise (black bar) or sham exposure (light grey bar) and treatment of rat cochleae by AP or bumetanide. Sham exposed cochleae treated with artificial perilymph (AP) served as control (white bar). 18S rRNA and (3-actin were used as housekeeping genes. A statistically significant down-regulation of KCC2 was observed in noise exposed animals treated with AP. No such down-regulation of KCC2 was seen in sham or noise exposed animals treated with bumetanide. There was also a statistically significant difference in KCC2 expression between noise exposed animals treated with AP or bumetanide. [0153] B) Count of inner hair cell ribbon synapses 2 weeks following noise or sham exposure of rat cochlea. The number of ribbons per Inner hair cell (IHC) is shown. Among those ears treated with AP, a significant decrease of ribbons was observed in noise exposed ears in the midbasal (middle columns) and basal (right columns) cochlear turns in comparison to sham exposed animals. In contrast, the decline in noise exposed ears that received bumetanide (black bar) was not significant in comparison to sham exposed animals (light grey bar). But there was a significant difference in ribbon numbers between noise exposed ears treated with AP (dark grey bar) or bumetanide (black bar). Apical turns (left) did not show much change as inner hair cells there are hardly affected by high-frequency trauma.

EXAMPLES

Example 1

[0154] Objective

[0155] The loop diuretic furosemide has been proposed as a (reversible) treatment of tinnitus. It has been hypothesized that it attenuates the firing of the auditory nerve by reducing the endocochlear potential (Risey et al., 1995). Yet, concurrently tinnitus is also known as a side effect of furosemide. The inventors sought to elucidate whether and, if yes, how the expression of chloride co-transporters in the cochlea changed following an insult to the cochlea susceptible of inducing tinnitus in order to identify starting points for the development of —a novel pharmacotherapy for tinnitus.

[0156] Materials and Methods

[0157] 13 anaesthetized adult female Wistar rats were exposed for 2 hours intra-aurally to a continuous 10 kHz tone at an intensity of 120 dB SPL in a sound attenuation booth. This exposure is susceptible of inducing tinnitus in rats (e.g. Tan et al., 2007). The acoustic stimulus was calibrated at the head level of the animal. Four anaesthetized control animals were placed in the sound attenuation booth for the same duration, but the speaker remained turned off. 14 days after the real or sham sound exposure animals were sacrificed. The cochleae were harvested and fixed by immersion in 2% paraformaldehyde (PFA), 125 mM sucrose in 100 mM phosphate buffered saline (PBS), pH 7.4, for 2 hours. They were decalcified in Rapid Bone Decalcifier (Eurobio, Les Ulis Cedex, France) followed by an overnight incubation in Hanks buffered saline with 25% sucrose. The next day cochleae were embedded in O.C.T. compound (Miles Laboratories, Elkhart, Ind., USA). Before use, tissue samples were cryosectioned at 10 μm thickness for in situ hybridization as well as immunohistochemistry, mounted on SuperFroste/plus microscope slides and stored at −20° C.

[0158] For immunohistochemistry slides were dried at room temperature for 30 minutes. Afterwards, they were permeabilized for 3 minutes with PBS+0.1% Triton, washed with PBS and blocked with 1% BSA/PBS. Slides were incubated over night with primary antibody diluted in 0.5% BSA/PBS (KCC2 1: 150, Upstate Biotechnology, Hamburg, Germany). On the next day slides were washed with PBS and incubated with secondary antibody (Cy3-anti-rabbit, Jackson Immuno Research, Suffolk, UK) for 1 hour. Slides were washed again and mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, Calif., USA). Slides were viewed using an Olympus AX70 microscope.

[0159] For in situ hybridization slides were incubated with anti-digoxigenin antibody conjugated to alkaline phosphatase (1:750, Roche, Mannheim, Germany). The sections were then allowed to develop in the substrate solution containing nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate (Sigma, Munich, Germany). The sections were observed at different time periods to monitor the development of the substrate into a colored product. Sections from controls and exposed animals were stopped at the same time, mounted and viewed using an Olympus AX70 microscope.

[0160] For Western Blot analysis proteins from rat cochleae and brain tissue were separated by SDS polyacrylamide gel electrophoresis using the XCell Sure Lock Mini Cell and NuPage Novex 4-12% Bis-Tris Gels (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. For immunoblotting, proteins (40 μg/lane) were transferred onto polyvinylidine difluoride transfer membranes using the Xcell II Blot Module (Invitrogen). The KCC2 anti-body (Blaesse et al., 2006; generously provided by H G Nothwang and E Friauf) was pre-incubated overnight at 4° C., in a reasonable dilution. Round antibody was visualized with the Enhanced Chemiluminescence Plus Western Blotting Detection Reagent (Amersham Biosciences, Freiburg, Germany). For quantification, densitometric analysis was performed using the CellAF software (Olympus, Hamburg, Germany). The intensity of the control band and the band from the exposed tissue were measured and compared against each other.

[0161] Results

[0162] Animals exposed to excessive noise that is susceptible of inducing tinnitus showed a clear down-regulation of KCC2 14 days after noise exposure (FIG. 1a). Immunohistochemistry showed a markedly reduced expression of the KCC2 protein in inner hair cells compared to non-exposed control animals. In situ hybridization revealed also a clear down-regulation of KCC2 mRNA in spiral ganglion neurons of noise exposed cochleae compared to non-exposed cochleae. Western Blots showed a clear down-regulation of KCC2 protein in cochlear tissue of noise exposed animals compared to control animals (7.8 times lower) as shown by FIG. 1b. The results from this experiment showed for the first time that KCC2 is down-regulated following a cochlear insult such as acoustic trauma. Whether this down-regulation is related to hearing loss, to tinnitus or both, remained to be elucidated as a result of that experiment. Accordingly Example 2 was set up for further elucidation.

Example 2

[0163] Objective

[0164] The aim of the 2nd experiment was to assess whether down-regulation of KCC2 observed in the first experiment was related to the pathophysiology of hearing loss or to that of tinnitus, or to both. This down-regulation could suggest that tinnitus induction by furosemide may be related to its known effect on KCC2, rather than through its attenuation of the endocochlear potential, as commonly suspected (e.g. Riscy et al., 1995). For this purpose, a behavioural model as described by Rüttiger et al., 2003 was used to discriminate between animals with and without tinnitus.

[0165] Materials and Methods

[0166] 36 adult female Wistar rats were trained in a conditioning chamber to actively access a liquid feeder whenever a constant sound was present. During silence, no reward was given. The conditioning was completed when animals performed more accesses to the reward feeder during periods of sound than during periods of silence.

[0167] On Day 0 conditioned rats were anaesthetized and exposed intra-aurally either for 1 hour (Group A; n=18) or for 1.5 hours (Group B; n=18) to a continuous 10 kHz tone at an intensity of 120 dB SPL in a sound attenuation box. The acoustic stimulus was calibrated at the head level of the animal. All animals were divided into groups corresponding to their tinnitus behaviour: in Group A, 5 animals exhibited tinnitus, 10 no tinnitus, and 3 died, while in Group B 5 animals developed tinnitus, 12 no tinnitus, and 1 died. Group A animals were sacrificed 6 days and Group B animals 30 days after the real or sham sound exposure.

[0168] Tissue preparation, immunohistochemistry and in situ hybridization were carried out as described for Example 1.

[0169] Ribbon synapses of inner hair cells were counted. Sections were viewed using an Olympus AX70 microscope equipped with epifluorescence illumination (100× objective, NA=1.35) and a motorized z-axis. Images were acquired using a CCD camera arid the imaging software Cell{circumflex over ( )}F (OSIS GmbH, Münster, Germany). For Otoferlin and CtBP2/RIBEYE immunopositive spot counting on cryo-sectioned cochleae were performed through imaging over a distance of 8 μm with the complete coverage of the IHC nucleus and beyond in an image-stack along the z-axis (z-stack). Typically z-stacks consisted of 30 layers with a z-increment of 0.276 μm, for each layer one image per fluorochrome was acquired. Z-stacks were 3-dimensionally deconvoluted using Cell{circumflex over ( )}F's RIDE module with the Nearest Neighbour algorithm (OSIS GmbH, Münster, Germany).

[0170] Results

[0171] In both Group A and Group B KCC2 was markedly down-regulated 6 days or 30 days after noise exposure in those animals showing the behavioural correlate of tinnitus compared with animals displaying no tinnitus behaviour (as shown in FIG. 2a). Immunohistochemistry shows a clearly reduced expression of the KCC2 protein in inner hair cells of tinnitus animals compared to non-tinnitus animals. In situ hybridization reveals also a clear down-regulation of KCC2 mRNA in spiral ganglia neurons of tinnitus animals compared to non-tinnitus cochleae. Further, IHC ribbons of hearing impaired tinnitus animals were significantly reduced in the midbasal and basal turns in comparison to no tinnitus animals (n=4 animals, p<0.001 for the two-way ANOVA, and p<0.02 for the post-test two-sided Student's t-test, both with α=0.05) (see FIG. 2b).

[0172] In conclusion, the results from Example 2 showed for the first time ever that down-regulation of KCC2 in inner hair cells and spiral ganglion neurons following noise trauma is not related to hearing loss, but rather inherent to the pathophysiology of tinnitus. This finding suggests that tinnitus induction by diuretics, such as furosemide acting on NKCC1 and KCC2, is likely the result of KCC2 inhibition, whereas their contrary effect is typically due to its NKCC1 antagonism at different concentrations. As a consequence, a strategy for tinnitus treatment consists of—down-regulating NKCC1 in order to lower intracellular Cl.sup.− levels.

Example 3

[0173] Objective

[0174] The aim of the third experiment was to assess whether pharmacological modulation of intracellular chloride levels in the cochlea following tinnitus-inducing noise trauma can suppress tinnitus. Since bumetanide has a much higher affinity for NKCC1 than for KCC2 (Payne et al., 2003), its administration should—in view of the previous findings of the present invention—allow for reducing intracellular Cl.sup.− levels as the inhibitory effect on NKCC1 would dominate any—undesired—inhibitory effect on KCC2.

[0175] Materials and Methods

[0176] 20 adult female Wistar rats were anaesthetized as described above for the previous experiments. First, auditory brainstem response (ABR) measurements were performed. As in Experiment 2, they were then exposed in a sound attenuation booth for 1.5 hours to a continuous 10 kHz tone at an intensity of 120 dB SPL (Group A; n=10) or sham exposed in the same setting (Group B; n=10).

[0177] Animals were treated bilaterally right after noise trauma with either artificial perilymph (AP) or bumetanide (Sigma B3023, Lot 027Ko988). AP was prepared freshly in accordance with Guitton et al., 2003 (140 mM NaCl, 4 mM KCl, 2 mM CaCl.sub.2, 2m M MgCl.sub.2, 10 mM glucose, 10 mM HEPES). Bumetanide (300 NM) was prepared as follows: 50 mg of bumetanide powder was diluted in 6.87 ml AP. This 20 mM stock solution was diluted 1:66 in AP (15.15 μL stock solution and 984.85 μL AP). 5 animals of Group A and 5 animals of Group B received AP (total 10 animals) and 5 animals of Group A and 5 animals of Group B received bumetanide (total 10 animals).

[0178] For the local treatment administration, the fur was removed behind the ears and the bulla exposed in a retro-auricular approach. A small hole was carefully drilled into the bony bulla just above the round window niche (0.6-1 mm in diameter). The mucosa was opened and the region around the round window carefully dried of fluid. Through the hole, a small gel-foam pellet (Gelita Tampon; Braun, Melsungen, Germany) was inserted into the round window dow niche. 5-8 μl of bumetanide solution or AP were applied on the gel foam by means of a precision pipette with gel loader tips, thus avoiding air bubbles under the gel. Visual inspection showed that the niche was completely filled and covered with the gel. The hole in the bulla was then covered from the outside with muscular tissue and the wound was sutured with surgical thread (Vicryl, Johnson & Johnson, Norderstedt, Germany). Postoperatively the animals were kept warm with the body temperature being controlled until wake-up.

[0179] For MR measurements only Dormitor was given as anesthetic. 15 minutes prior to surgery, Fentanyl was given additionally subcutaneously (s.c.). After the surgery Rimadyl s.c. was given as analgesic. After letting the animals sleep for several hours, the effect of Dormitor was antagonized by Antisedan s.c.

[0180] Total RNA was isolated as previously described (Tan et al., 2007) using the Qiagen Rneasy Mini Kit (Qiagen, Hilden, Germany). In brief, tissue was lysed using lysate buffer mixed with β-mercaptoethanol. After three freeze-thawing steps samples were centrifuged and the supernatant kept for further cleaning steps with washing buffer. RNA was eluted in 40 μl RNase free water. Total RNA was transcribed into cDNA as previously described (Tan et al., 2007) using the Sensiscript RT Kit (Qiagen). In brief, 50 ng of RNA were incubated 10 min at 65° C. with RNase free water and Oligo dT15 Primers. After adding RNAsin and the Sensiscript enzyme samples were incubated for 1 hour at 37° C. For the real-time PCR reaction 12.5 μl of SYBRGreen (QuantiFast Sybr Green, Qiagen), 1 μl Primer Mix, 7.5 μl ddH2O and 4 μl cDNA were used per well. All samples were run in triplicate for every primer used as well as for the negative control (no cDNA added). 18S rRNA and β-actin were used as housekeeping genes.

[0181] Immunhistochemistry was carried out as described above. Ribbon synapses of inner hair cells were counted as in Experiment 2.

[0182] ABRs were recorded in anesthetized animals as previously described (Knipper et al., 2000, Schimmang et al., 2003). In short, electrical brainstem responses to free field click (100 μs) and pure tone (3 ms, 1 ms ramp) acoustic stimuli were recorded with sub-dermal silver wire electrodes at the ear, the vertex and the back of the animal. After amplification (×100,000) the signals were averaged for 64-256 repetitions at each sound pressure presented (0-100 dB SPL in steps of 5 dB). The threshold was determined by the lowest sound pressure that produced potentials visually distinct from noise level. ABR measure points were just before noise respectively sham trauma, right after treatment, 1, 2, 7 and 15 days post treatment.

[0183] Results

[0184] Real-time PCR for KCC2 from cochlear tissue sampled 15 days following treatment shows no difference in sham exposed ears (Group B), regardless of whether they were treated with AP or bumetanide. However, in noise exposed ears (Group A), a statistically significant down-regulation of KCC2 was observed in AP treated ears compared with those that were only sham exposed (2 sided Student t-test p<0.05), but not in bumetanide treated ears. KCC2 expression in noise exposed, bumetanide treated ears was similar to the level in sham exposed ears, but statistically significantly different from noise exposed, AP treated ears (p<0.01) (see FIG. 3a).

[0185] A surprisingly similar outcome was observed when counting ribbon synapses of inner hair cells (see FIG. 3b). Among those ears treated with AP, a significant decrease of ribbons was observed in noise exposed ears in the midbasal and basal cochlear turns in comparison to sham exposed animals (p <0.01). In contrast, the decline in noise exposed ears that received bumetanide was not significant in comparison to sham exposed animals. But there was a significant difference in ribbon numbers between noise exposed ears treated with AP or bumetanide (p <0.02). Apical turns did not show much change as inner hair cells there are hardly affected by high-frequency trauma.

[0186] In summary, these results show for the first time ever that pharmacological modulation of the intracellular Cl.sup.− concentration in inner hair cells is feasible and that up-regulation of KCC2 expression in inner hair cells can be achieved by inhibiting NKCC1. Such inhibition is exerted by NKCC1 antagonists which however, do not exert any direct effect on the increase of the KCC2 activity or expression. Rather, the effect is mediated by NKCC1 inhibition, which—as such—exerts an influence on the KCC2 activity or expression, namely an increase. Application of the NKCC1 inhibitor bumetanide results in a reduced loss of inner hair cell ribbons. As a biomarker for the presence of tinnitus this shows that this therapeutic strategy allows for attenuation, respectively suppression of tinnitus.

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