Pharmaceutical compositions comprising small interfering RNA inhibitors of NOX3

Abstract

This invention relates to a method of identifying a modulator of an NADPH oxidase, whereby said modulator is suitable as a lead compound and/or as a medicament for the treatment and/or prevention of hearing loss and/or phantom hearing, the method comprising the steps of (a) contacting a test compound with a protein, wherein said protein (i) comprises or consists of the amino acid sequence of any one of SEQ ID NO: 1, 3 or 5, or (ii) is encoded by a nucleic acid comprising or consisting of the sequence of any one of SEQ ID NO: 2, 4, 6, 23 or 24, or (iii) is a fragment of the protein according to (i) or (ii) and exhibits NADPH oxidase activity, or (iv) has a sequence at least 75% identical with the protein according to (i) or (ii) or with the fragment according to (iii) and exhibits NADPH oxidase activity, and optionally with one or more NADPH oxidase subunits, under conditions allowing binding of said test compound to said protein or, if present, said subunit(s); (b) optionally determining whether said test compound binds to said protein or, if present, said subunit(s); and (c) determining whether (ca) said test compound, upon contacting in step (a); or (cb) said test compound, upon binding in step (b) modulates the expression and/or activity of said protein or, if present, said subunit(s). Also provided are pharmaceutical compositions, medical uses and diagnostic uses of compounds of the invention.

Claims

1. A pharmaceutical composition comprising an siRNA inhibitor of NOX3 and a pharmaceutically acceptable carrier wherein NOX3 protein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1, 3, or 5, and wherein the composition is suitable for administration to the inner ear of a human subject.

2. The pharmaceutical composition of claim 1, further comprising an ototoxic agent.

3. The pharmaceutical composition of claim 2, wherein said ototoxic agent is an antibiotic.

4. The pharmaceutical composition of claim 3, wherein said antibiotic is an aminoglycoside antibiotic.

5. The pharmaceutical composition of claim 1, wherein the siRNA, represses the expression of NOX3 protein.

6. The pharmaceutical composition of claim 2, wherein said ototoxic agent is a cytostatic.

7. The pharmaceutical composition of claim 6, wherein said cytostatic is bleomycine, bromocriptine, carboplatinum, cisplatin, methotrexate, nitrogen mustard, vinblastine, or vincristine.

8. The pharmaceutical composition of claim 4, wherein said aminoglycoside antibiotic is amikacin, gentamycin, kanamycin, neomycin, netilmycin, streptomycin or tobramycin.

9. The pharmaceutical composition of claim 3, wherein said antibiotic is erythromycin, vancomycin, minocycline, polymixin B, amphotericin B, or capreomycin.

10. The pharmaceutical composition of claim 2, wherein said ototoxic agent is a salicylate.

11. The pharmaceutical composition of claim 10, wherein said salicylate is aspirin or methyl salicylate.

12. The pharmaceutical composition of claim 2, wherein said ototoxic agent is a non-steroidal anti-inflammatory agent.

13. The pharmaceutical composition of claim 12, wherein said nonsteroidal anti-inflammatory agent is selected form the group consisting of diclofenac, etodolac, fenprofen, ibuprofen, indomethacin, naproxen, piroxicam, and sulindac.

14. The pharmaceutical composition of claim 2, wherein said ototoxic agent is a quinine derivative.

15. The pharmaceutical composition of claim 14, wherein said quinine derivative is selected from the group consisting of chloroquine phosphate, quinacrine hydrochloride, and quinine sulphate.

Description

(1) The Figures show:

(2) FIG. 1: Tissue distribution of NOX3 mRNA. A) NOX3 mRNA expression was evaluated in 12 rat tissues by RT-PCR (upper panel); GAPDH mRNA was used as a reference transcript (lower panel). No cDNA represents negative control PCR devoid of added cDNA. The first lane of both panels shows DNA size markers. B) Quantification of NOX3 RNA in 14 mouse tissues using real time PCR. NOX3 mRNA expression is shown relative to 18S rRNA expression. The amounts of NOX3 and 18S PCR products were measured using SYBR Green.

(3) FIG. 2: PCR detection of cDNAs encoding NOX activator and regulator subunits in the inner ear. A, RT-PCR amplification of NOXA1, NOXO1, and the reference GAPDH cDNA from the indicated rat tissues. B, RT-PCR amplification of p67.sup.phox and p47.sup.phox cDNA from the indicated rat tissues. The first lane of each panel shows DNA size markers.

(4) FIG. 3: Expression of NOX3 mRNA in specific regions of cochlea. The indicated regions of the rat inner ear were obtained by microdissection and NOX3 (upper panel) and GAPDH (lower panel) expression were assessed by RT-PCR. + symbols represent reverse transcribed (RT positive) samples; symbols represent not reverse transcribed (RT negative) samples. P0, P3, and P4 indicate the postnatal days when samples were taken. Positive control inner ear sample was isolated from adult rat.

(5) FIG. 4: Localization of NOX3 mRNA in inner ear by in situ hybridization. Mouse inner ear sections hybridized with digoxigenin-labeled antisense (A, C, and E) and sense (B, D, and F) probes of NOX3, shown at 20 (A, B) and 40 (C-F) magnifications. A, The antisense probe hybridized with the RNA of spiral ganglion neurons. B, The sense probe yielded only a weak, uniform signal and no labeling of spiral ganglion neurons. C, Hybridization of antisense NOX3 probe with the organ of Corti labeled the sensory epithelium. D, Hybridization of sense NOX3 probe with organ of Corti did not yield specific signals. E, Antisense NOX3 probe hybridized with the sensory epithelial cell layer of the saccule. F, Only a week uniform signal was observed with the sense NOX3 probe.

(6) FIG. 5: NOX3-dependent superoxide production in the absence of other NOX subunits. HEK293 cells were transfected with either pcDNA3.1 vector or NOX3, and superoxide generation was measured as cytochrome C reduction (upper panel) or as luminol-amplified chemiluminescence (lower panel) in the presence or absence of 100 nM PMA, as indicated. Upper panel shows the result of a single experiment representative of three independent studies. Lower panel shows statistical analysis of peak superoxide production. Chemiluminescence signals were measured with relative light units (RLU) and normalized to 1 second and 150,000 cells.

(7) FIG. 6: Subunit regulation of NOX3 activity. A, B, and C, HEK293 cells were transfected with different combinations of NOX3, NOXO1, NOXA1, p47.sup.phox, and p67.sup.phox, as indicated. Superoxide generation was measured as SOD sensitive cytochrome C reduction (lines and symbols) or as luminol-amplified chemiluminescence (bar graphs) in the presence or absence of PMA (100 nM), as indicated. Lines and symbols show typical experiments, representative of at least three independent studies. Bar graphs show statistical analysis of peak superoxide production. Chemiluminescence signals were measured with relative light units (RLU) and normalized to 1 second and 150,000 cells.

(8) FIG. 7: Cisplatin enhances NOX3-dependent superoxide production. Superoxide production of transfected HEK293 cells were measured either as luminol-amplified chemiluminescence (B, D, E and F) or with a luminol-based superoxide detection kit, Diogenes (A and C). Cells were pre-incubated in the presence or absence of 20 M cisplatin for 12 hours (A-E). A, HEK293 cells were transfected with NOX3 or control vector (pcDNA3.1) and incubated with or without cisplatin before superoxide measurement. 100 nM PMA and 5 M DPI were added as indicated. Traces represent a typical experiment, representative of three independent studies. B, HEK293 cells stably expressing NOX3/NOXA1/NOXO1 were pre-incubated with or without cisplatin before superoxide measurement. 5 M DPI was added as indicated. Traces show a typical experiment, representative of eight independent studies. C, Statistical analysis of peak superoxide production of NOX3 transfected HEK293 cells, after cisplatin- or control treatment, in the presence or absence of 100 nM PMA. D, Statistical analysis of peak superoxide production of HEK293 cells transfected with the indicated constructs and pre-incubated with or without cisplatin. The measurements were carried out in the absence or presence of 100 nM PMA, as indicated. E, Superoxide production of a HEK293 cell clone stably transfected with NOX3/NOXO1/NOXA1 after incubation with various concentrations of cisplatin for 12 hours. F, Superoxide production of a HEK293 cell clone stably transfected with NOX3/NOXO1/NOXA1 after incubation in the presence or absence of 20 M cisplatin for the indicated periods of time.

(9) The following examples illustrate the invention but should not be construed as being limiting.

EXAMPLE 1

Cloning of Mouse and Rat NOX3 cDNA

Experimental Procedures

(10) The first and the last exons of mouse and rat NOX3 genes were identified based on their homology with the human NOX3 gene using the Ensembl Genome Browser (www.ensembl.org). Inner ear samples of mouse (strain C57Bl6) and rat (strain Sprague-Dawley) were isolated and total RNA was purified as described [28]. Primers were designed and used to amplify the full length of coding sequences (mouse NOX3 forward 5-atg ccg gtg tgc tgg att ctg aac-3 and reverse 5-cta gaa gtt ttc ctt gtt gta ata gaa-3, rat NOX3 forward 5-gtg ttg gta gta aga gaa gtg tca tg-3 and reverse 5-c tag aag ttt tcc ttg ttg taa tag-3) with Taq DNA polymerase (Qiagen) under standard conditions. PCR products were subcloned into pcDNA3.1 vector (Invitrogen) and verified by sequencing.

(11) Results.

(12) So far, NOX3 mRNA has only been detected in human embryonic kidney, but expression levels were very low [22, 30] and hence the physiological relevance questionable. We reasoned that the physiologically relevant localization of NOX3 might have been missed because previous studies had restricted their analysis to commercially available human RNA sources. To overcome these limitations, we decided to work in mouse and rat and to prepare RNA from tissues that had not been investigated so far. As hitherto only the human NOX3 sequence was known, we identified mouse and rat NOX3 genes by searching genomic DNA databases and designedbased on these resultsmouse and rat NOX3 PCR primers.

(13) We then prepared RNA from a variety of mouse and rat tissues, including bone (femur, skull, shoulder blade), cartilage (joints of ribs, outer ear), and inner ear and analyzed them for NOX3 expression by RT-PCR. As shown on FIG. 1A, high levels of NOX3 transcript were detected only in the rat inner ear sample (despite its relatively low mRNA content demonstrated by the low amount of GAPDH PCR product). Using primer pairs designed from the first and the last exons of the mouse and rat NOX3 gene, respectively, we amplified whole length mouse and rat NOX3 coding sequences from inner ear samples. The predicted amino acid sequences of both mouse and rat NOX3 showed 81% sequence identity with the human sequence and 93.5% identity with each other.

EXAMPLE 2

Tissue Distribution of NOX3

Experimental Procedures

(14) Total RNA was isolated from different organs of rat and mouse and from specific regions of the rat inner ear using the TRIzol reagent. With the exception of RNA purified from parts of the inner ear, samples were DNase treated, then further purified with RNeasy kit (Qiagen). 2 g total RNA from each tissue was reverse transcribed using Superscript reverse transcriptase (Life Technologies, Inc.). PCR was carried out with Taq DNA polymerase using the following primers: mouse NOX3 forward 5-gtg ata aca ggc tta aag cag aag gc-3, reverse 5-cca ctt tcc cct act tga ctt tag-3; rat NOX3 forward 5-gcg tgt gct gta gag gac cgt gga g-3, reverse 5-gag cct gtc cct ctg ctc caa atg c-3; mouse GAPDH forward 5-ggg tgt gaa cca cga gaa at-3, reverse 5-gtc atg agc cct tcc aca at-3; rat GAPDH forward 5-cgg tgt caa cgg att tgg ccg tat t-3, reverse 5-act gtg gtc atg agc cct tcc acg a-3; rat NOXO1 forward 5-acc caa acc tct gga tct gga gcc c-3, reverse 5-gga tgg cac tca tac agg ggc gag t-3; rat NOXA1 forward 5-tac tgg ccg tag cac gcg aag act g-3, reverse 5-gga cct ccc agg ctt gca gtt tga a-3; rat p47.sup.phox forward 5-gca gga cct gtc gga gaa ggt ggt c-3, reverse 5-tct gtc gct ggg cct ggg tta tct c-3; rat p67.sup.phox forward 5-aag cag aag agc agt tag cat tgg c-3, reverse 5-gga gtg cct tcc aaa ttc ttg gct g-3. Standard PCR conditions were used, and the number of PCR cycles was 30 (FIGS. 1 and 2) or 28 (FIG. 3) for the amplification of GAPDH and 35 for all other amplifications.

(15) Quantitative PCR was carried out using ABI Prism 7900HT Sequence Detection System with standard temperature protocol and 2SYBR Green PCR Master Mix reagent (Applied Biosystems, Worrington, UK) in 25 l volume, in triplicates. 300 nM of the following primer pairs were used for the reactions: mouse 18S forward 5-aca tcc aag gaa ggc agc ag-3 and reverse 5-ttt tcg tca cta cct ccc cg-3; mouse NOX3 forward 5-cga cga att caa gca gat tgc-3, and reverse 5-aag agt ctt tga cat ggc ttt gg-3. All amplifications were carried out in a MicroAmp optical 96-well reaction plate with optical adhesive covers (PE Applied Biosystems). The accumulation of PCR products was detected by monitoring the increase in fluorescence of the reporter dye.

(16) Results.

(17) NOX3 is Predominantly Expressed in the Inner Ear

(18) Based on the cDNA sequence of mouse NOX3, we designed primers for real time PCR to study quantitative expression of NOX3 RNA in different mouse tissues. 18S RNA was used as a reference gene. The results of real-time PCR demonstrated that NOX3 was predominantly expressed in the inner ear (FIG. 1B). Low amounts of NOX3 RNA could also be detected in skull, brain, and embryonic kidney. However, inner ear contained 50-fold of the NOX3 content of skull and 870-fold of the one of embryonic kidney (FIG. 1B).

(19) Expression of Cytoplasmic NOX Subunits in the Inner Ear

(20) NOX1 and gp91.sup.phox/NOX2 require cytoplasmic organizer subunits (NOXO1, p47.sup.phox) and activator subunits (NOXA1, p67.sup.phox) to form a functional enzyme. As NOX3 shows a high degree of homology with NOX1 and gp91.sup.phox/NOX2 [31], we considered that it might also be a subunit-dependent enzyme and therefore investigated expression of cytoplasmic NOX subunits in the inner ear. RT-PCR analysis (using 35 PCR cycles) showed that mRNA of the activator subunit NOXA1, as well as mRNA of the organizer subunit p47.sup.phox was expressed in the inner ear (FIG. 2). mRNA of the activator subunit, p67.sup.phox, and the organizer subunit, NOXO1, could be detected only at very high cycle numbers (40 PCR cycles; data not shown). Since p47.sup.phox mRNA is expressed in phagocytic cells, its detection might be due to blood cell contamination. In contrast, NOXA1 is not expressed in blood cells [24] nor in tissues neighboring the inner ear (FIG. 2A); thus, it is most likely expressed within cells of the inner ear.

(21) Expression of NOX3 in Different Parts of the Cochlea

(22) In order to identify regions of the inner ear that express NOX3, we isolated distinct parts of rat cochlea such as organ of Corti, stria vascularis, and spiral ganglia from newborn rats (postnatal day 1 to 4) as described previously [32]. As a control tissue, we used dorsal root ganglia. Total RNA was extracted from these tissues and tested for NOX3 and GAPDH housekeeping gene expression by RT-PCR. Results showed that NOX3 is expressed in spiral ganglia and in the organ of Corti, while stria vascularis and dorsal root ganglia were devoid of NOX3 mRNA (FIG. 3). Our experiments demonstrated that i) NOX3 is expressed only in selected structures of the cochlea (i.e. organ of Corti and spiral ganglia), and ii) its expression is not a general property of the peripheral nervous system (i.e. it was absent from dorsal root ganglia).

EXAMPLE 3

In Situ Hybridization

Experimental Procedures

(23) For in situ hybridization experiments digoxigenin-labelled antisense and sense (negative control) cRNA probes (nucleotides 560-849 of mNOX3) were generated and used as described previously [19] on decalcified, 7 m thick inner ear sections.

(24) Results.

(25) To further define the site of NOX3 expression, we performed in situ hybridization of adult mouse inner ear sections. The antisense NOX3 probe labeled spiral ganglion neurons (FIG. 4A) and cells of the organ of Corti (FIG. 4C). The cellular structures within the organ of Corti were not sufficiently well preserved to identify NOX3-expressing cells more precisely. The sense probe gave only a weak, uniform background signal demonstrating the specificity of the antisense hybridization (FIGS. 4 B and D). Specific labeling for NOX3 was also observed in the vestibular system, namely in the sensory epithelial cell layer of the saccule (FIG. 4 E, F).

EXAMPLE 4

Measurement of Reactive Oxygen Species

Experimental Procedures

(26) Cell Culture and Transfection

(27) HEK293 were maintained in Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12 that was supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 g/ml), and 4 mmol/liter L-glutamine. NOX3-, NOXO1-, NOXA1-, p47.sup.phox, and p67.sup.phox cDNAs were subcloned into pcDNA3.1 (Invitrogen, Groningen, Netherlands) and transfected into HEK293 cells with the Effectene transfection system (Qiagen). To obtain stable clones, NOX3, NOXO1, NOXA1-transfected HEK293 cells were selected with 400 g/ml G418 starting on the 2nd day after the transfection. After 14 days of selection, 24 surviving clones were tested for superoxide production. The positive clones were verified to express NOX3-, NOXO1-, and NOXA1 RNA by RT-RCR.

(28) ROS generation was measured by the peroxidase-dependent luminol-amplified chemiluminescence technique (referred to as luminol-amplified chemiluminescence) in 96 well microplates using Luminometer Wallac 1420 Multilabel Counter (PerkinElmer Life Sciences). Measurements were performed in Hanks' balanced salt solution supplemented with 1 mg/ml D-glucose, 1 unit/ml horseradish peroxidase, and 250 M luminol. In some experiments, phorbol ester (PMA) was added during the measurements to 100 nM final concentration. When the effect of cisplatin or 5-Fluorouracil (5-FU) was investigated, these compounds were pre-incubated with the cells for the indicated time and concentration in cell culture medium. Before ROS measurements, the cell culture medium was exchanged with the assay solution and chemiluminescence or absorption (see below) was measured at 37 C. After measurements cells were counted, and the results were normalized to 150,000 cells. Extracellular superoxide production was measured in 96-well microplates at 550 nm as the SOD-sensitive reduction of 100 M ferricytochrome C (referred to as cytochrome C reduction technique). The O.sup..sub.2 production was calculated using an absorption coefficient of 21.1 mM.sup.1 cm.sup.1 and normalized to 10.sup.7 cells [29].

(29) Results.

(30) NOX3-Dependent Superoxide Generation in the Absence of Subunits

(31) To investigate its molecular function, we transiently expressed NOX3 in HEK293 cells, which do not show endogenous expression of the enzyme. Superoxide production was measured with cytochrome C reduction technique and with luminol-amplified chemiluminescence. Using either technique, NOX3-transfected cells generated low amounts of superoxide, but only in the presence of a protein kinase C activator (phorbol ester, PMA) (FIG. 5). Since both NOX1 and gp91.sup.phox/NOX2 have an obligatory subunit requirement, the stimulus-dependent and subunit-independent activity of NOX3 is a unique and distinguishing feature of this NOX isoform.

(32) Regulation of NOX3 by the Organizer and Activator Subunits of NOX1 and gp91.sup.phox/NOX2

(33) Since expression of NOX regulator and activator subunits was detected in the inner ear (see above, FIG. 2), we reasoned that they might influence NOX3 activity. Thus, we investigated superoxide generation by NOX3 upon co-transfection with cytoplasmic subunits. In the first series of experiments, NOX3 was co-transfected with the cytosolic subunits of the phagocyte NADPH oxidase, p67.sup.phox and p47.sup.phox. In these transfectants, the NOX3-dependent superoxide generation was markedly increased, even without an added stimulus (FIG. 6A). The addition of PMA, however, led to a strong enhancement of NOX3 activity (FIG. 6A). HEK293 cells, transfected with p47.sup.phox and p67.sup.phox but devoid of NOX3, did not produce any superoxide (not shown). Interestingly p67.sup.phox alone, in the absence of p47.sup.phox, was sufficient to double the PMA-induced superoxide generation of NOX3, while p47.sup.phox in the absence of p67.sup.phox, did not modify NOX3 activity (compare FIG. 5 with FIG. 6A). Next it was investigated whether NOX3 could be regulated by the NOXO1 and NOXA1 subunits, which are associated with NOX1 in the colon. Co-transfection of NOX3 with NOXO1 and NOXA1 resulted in a massive increase of superoxide production (FIG. 6B). The NOXO1/NOXA1-enhanced superoxide generation was insensitive to PMA (FIG. 6B). The co-expression of NOXA1 with NOX3, in the absence of NOXO1, had an enhancing effect on PMA-stimulated NOX3 activity. NOXO1 alone, however, did not influence NOX3-dependent superoxide production (FIG. 6B, lower panel).

(34) At a least on a biochemical level, there is promiscuity among the organizer and regulator subunits: NOXO1 is able to function with p67.sup.phox, and NOXA1 with p47.sup.phox [24-26]. Therefore, we investigated which combinations of organizer and activator subunits are capable to regulate NOX3, and what kind of properties those complexes may have. Expression of NOXO1, p67.sup.phox, and NOX3 in HEK293 cells, led to spontaneous superoxide generation that could not be further enhanced by PMA (FIG. 6C). However, when p47.sup.phox, NOXA1, and NOX3 were expressed, superoxide production by HEK293 cells was largely PMA-dependent (FIG. 6C). Thus, the organizer subunit (p47.sup.phox versus NOXO1) determines whether NOX3 activity is PKC-dependent or independent.

(35) Cisplatin Enhances NOX3 Activity

(36) Cisplatin is an ototoxic drug that exerts its toxic effect, at least in part, through induction of ROS generation in the inner ear [2]. We therefore investigated the effect of this drug on NOX3 activity. HEK293 cells were transfected with NOX3 or with a control vector (pcDNA3.1) and incubated for 12 hours in the presence or absence of 20 M cisplatin. Cisplatin alone elicited superoxide production in NOX3-transfected, but not in control-transfected cells (FIG. 7A, see traces before PMA addition and FIG. 7C). Addition of PMA further increased superoxide generation, while an NADPH oxidase inhibitor, diphenylene iodonium (DPI), blocked it completely (FIG. 7A).

(37) When HEK293 cells were co-transfected with NOX3, NOXO1 and NOXA1, they produced ROS in a constitutive manner (see FIG. 6B). To investigate the effect of cisplatin under these conditions, we generated HEK293 clones stably expressing NOX3, NOXO1, and NOXA1 subunits. These clones produced superoxide constitutively and spontaneously as observed in the transient transfectants. Upon incubation with 20 M cisplatin (12 hours), a marked increase of superoxide production was detected by the luminol-amplified chemiluminescence (FIGS. 7B and C), and also by cytochrome C reduction (not shown). The superoxide generation was insensitive to PMA and could be abolished by DPI (FIGS. 7B and D). As control we investigated the effect of another chemotherapeutic drugs 5-fluorouracil, which is devoid of ototoxicity; incubation of NOX3/NOXO1/NOXA1 expressing cells with this compound (100 M, 17 hours) did not influence superoxide production (data not shown). HEK293 cells were also co-transfected with NOX3, p47.sup.phox, and p67.sup.phox, and incubated with 20 M cisplatin for 12 hours. Cisplatin enhanced the superoxide production of NOX3-, p47.sup.phox, and p67.sup.phox-transfected cells by a factor of approximately 3.3 (FIG. 7D); this superoxide production could be blocked by addition of 5 M DPI (not shown).

(38) Next the concentration and time dependency of the cisplatin effect on NOX3 activity was investigated using a NOX3/NOXO1/NOXA1 transfected stable clone. After incubating the cells with various concentrations of cisplatin for 12 hours, superoxide production was measured (FIG. 7E). Cisplatin caused an increase of NOX3-dependent ROS generation already at 1 M concentration, and 20 M cisplatin had a maximal effect (FIG. 7E). The EC.sub.50 of NOX3 activation by cisplatin was 3.6+/1.4 M.

(39) In order to examine the time course of NOX3 activation by cisplatin, a NOX3/NOXO1/NOXA1 transfected stable clone was incubated with 20 M cisplatin for various periods of time. Cisplatin enhanced NOX3 activity already after 5 hours treatment and reached its maximal effect after around 17 hours (FIG. 7F); the t.sub.50 was 11.5+/1.7 hours.

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