# Patent

## Title
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.

## Background
This application is a divisional of co-pending U.S. application Ser. No. 13/336,147, filed Dec. 23, 2011, which is a divisional of U.S. application Ser. No. 11/628,419 filed Oct. 3, 2007, now U.S. Pat. No. 8,088,356, which is a national phase application under 35 U.S.C. §371 of International Application No. PCT/EP2005/006061 filed Jun. 6, 2005, which claims priority to European Application No. 04013266.4 filed Jun. 4, 2004, the entire text and figures of which disclosures are incorporated herein by reference without disclaimer.


The sequence listing that is contained in the file named “UGENP0012USD2_ST25.txt”, which is 219 KB (as measured in Microsoft Windows®) and was created on Apr. 2, 2014, is filed herewith by electronic submission and is incorporated by reference herein.
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.
In this specification, a number of documents is cited. The disclosure of these documents, including manufacturer's manuals, is herewith incorporated by reference in its entirety.
Hearing impairment is a widespread and severe sensory deficit. It is the third most prevalent major chronic disability in the over 65-year-old age group, but also found in younger persons. Slightly more than 1 percent of people under the age of 17 have hearing loss, the prevalence rises to 12 percent between the ages of 45 and 64, to 24 percent between the ages of 65 and 74, and up to 39 percent for ages over 75. There are three major causes of hearing loss: noise-dependent hearing loss, drug-associated hearing loss and age-associated hearing loss. Interestingly, there appears to be a common mechanism to three major causes of hearing loss, namely destruction of sensory epithelium and cochlear neurons through reactive oxygen species. In terms of treatment, no efficient drug treatment or prophylaxis of hearing loss are available at this point and the only option at present is the use of hearing aids. This situation is further aggravated by the limited understanding of the molecular processes involved in hearing loss and the scarcity of suitable molecular targets for therapeutic intervention.
The inner ear is a highly complex structure involved in hearing and balancing. The conversion of sound into electrical signals occurs within the cochlea, in the organ of Corti, and the electrical signals are conducted by the axons of spiral ganglion neurons to the brain. The linear movement of the head is sensed by the otolith organs (utricle and saccule) and the rotation movements by the ampullas of the semicircular canals. The signals generated in the vestibular system are transmitted by the vestibular ganglion neurons to the central nervous system.
Hearing impairment due to loss of cochlear function occurs frequently, if not invariably over lifetime. Noise and ototoxic chemicals may lead to a precocious, rapid hearing loss, while age itself leads to a more insidious, chronic loss of hearing. Research over the last decades has identified reactive oxygen species (ROS1) as the major factor mediating hearing loss [1]. ROS is generated within the cochlea after exposure to ototoxic drugs (e.g. cisplatin [2, 3], aminoglycoside antibiotics [3]) or to noise [4]. Signs of oxidative stress, such as DNA damage and lipid peroxidation, have been documented in vivo in response to those challenges [5, 6], as well as in cochlear aging [7]. The vestibular system is also damaged by ototoxic drugs [8, 9] in a process that includes excessive ROS production [10, 11]. 1 The abbreviations used are: bp, base pair; DPI, diphenylene iodonium; DUOX, dual domain oxidase; 5-FU, 5-Fluorouracil; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; gp91phox, 91-kDa glycoprotein subunit of the phagocyte NADPH oxidase; NOX, NADPH oxidase; NOXA1, NOX activator 1; NOXO1, NOX organizer 1; PMA, phorbol 12-myristate 13-acetate; PCR, polymerase chain reaction; ROS, reactive oxygen species; RT-PCR, reverse transcription-PCR; SOD, superoxide dismutase.
While the role of oxidative stress in inner ear damage is well established, its source is poorly understood. A role of non-enzymatic generation of ROS by ototoxic compounds has been suggested [12]. The possibility that a superoxide-generating enzyme could be localized within the inner ear, and thereby account for the oxidative damage of this organ, has received little attention.
Over the last decade, it has been proven that the expression of superoxide-generating NADPH oxidases is not restricted to phagocytes. Beside the well-known catalytic subunit of the phagocyte NADPH oxidase, gp91phox/NOX2 (for review see [13]), six other superoxide-producing enzymes have been identified in mammals [14, 15]. For most NOX and DUOX enzymes, a predominant tissue localization has been described, e.g. colon epithelium for NOX1 [16, 17], kidney cortex for NOX4 [18], lymphoid organs and testis for NOX5 [19], and the thyroid gland for DUOX1 and DUOX2 [20, 21]. For NOX3, with the exception of some very low level expression in the embryonic kidney [22], no convincing tissue localization had been found so far.
Our knowledge of the activation mechanisms of members of the NOX/DUOX family varies considerably among individual enzymes. NOX1 and gp91phox/NOX2 are subunit-dependent enzymes that need to assemble with an activator subunit (NOXA1 and p67phox, respectively) and an organizer subunit (NOXO1 and p47phox, respectively) to generate superoxide [23-26]. NOX5, DUOX1 and DUOX2, on the other hand, have N-terminal Ca2+-binding motifs (EF-hand domains), and so far one of them, NOX5, has been shown to be activated by increased Ca2+ concentration [27]. The mechanism of NOX4 activation is less clear. There are indications that it might be a constitutively active enzyme [18].
Tinnitus, also referred to as phantom hearing, is a common and in some instances invalidating medical complaint. Presently, the pathophysiology of the disease is poorly understood and there is not proven causative treatment available. There is however evidence that reactive oxygen species might play a role in the pathophysiology of tinnitus (Neri S. Tinnitus and oxidative stress in a selected series of elderly patients. Arch Gerontol Geriatr. 2002; 35 Suppl:219-23) and there are at least some reports that suggest a beneficial effect of antioxidant medication such as Gingko extract on the course of the disease (e.g. Schneider D et al. Gingko biloba (Rokan) therapy in tinnitus patients and measurable interactions between tinnitus and vestibular disturbances. Int Tinnitus J. 2000; 6(1):56-62). Thus, NOX3 might also be involved in the pathophysiology of tinnitus and the use of a NOX3 modulator or inhibitor is an interesting new concept for the treatment of tinnitus.
US-A1 20040001818 and WO-A1 0230453 describe methods of inhibiting angiogenesis, endothelial cell migration or endothelial cell proliferation using NADPH oxidase inhibitors.
EP-A2 1410798 describes a pharmaceutical composition comprising and uses of inhibitors of the production or the release of reactive oxygen metabolites (ROMs) and of compounds effective to scavenge ROMs. The uses are directed to the manufacture of a medicament for the treatment of Adult Respiratory Distress Syndrome (ARDS); ischemia or reperfusion injury, infectious disease, autoimmune or inflammatory diseases, and neurodegenerative diseases. Compounds effective to inhibit enzymatic ROM production or release comprise NADPH oxidase inhibitors.
EP-A2 0914821 relates to a method for diagnosis of atherosclerosis involving measurement of NADPH oxidase activity.
WO-A2 9719679 describes the use of NADPH oxidase inhibitors for the manufacture of a medicament for prevention of atherosclerosis.
US-A1 20040009901 relates to a method of treating a mammal having an autoimmune condition involving NADPH oxidase deficiency. Also, a method for identifying an agent that enhances NADPH oxidase activity is described.
WO-A2 02079224 relates to human peptides and proteins that are related to NADPH oxidase subfamily and methods for identifying modulators thereof. The proteins are described as being substantially similar to p47phox.
WO-A2 04007689 describes regulatory proteins for Nox enzymes, which are referred to as p41Nox proteins, and nucleic acid sequences encoding these proteins. Furthermore, a method for identifying a compound that modulates superoxide production is rescribed, the method involving administration of the protein. The envisaged medical indications relate to abnormal cell growth and proliferation and include cancer, prostatic hypertrophy and atherosclerosis.
NCBI Entrez protein database entry NP_056533 comprises the amino acid sequence of human NADPH oxidase 3 (NOX3). The sequence is 568 amino acids in length. The database entry recites similarity to gp91phox.
In view of the limited understanding of processes leading to hearing loss and phantom hearing, the technical problem underlying the present invention was therefore the provision of means and methods for the development of drugs for treatment of hearing loss and phantom hearing.
Accordingly, 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).
The term “modulator” designates a compound modulating the activity of a target molecule, preferably by performing one or more of the following effects: (i) the transcription of the gene encoding the protein to be modulated is modulated, (ii) the translation of the mRNA encoding the protein to be modulated is modulated, (iii) the protein performs its biochemical function with modulated efficiency in presence of the modulator, and (iv) the protein performs its cellular function with modulated efficiency in presence of the modulator. It is understood that the term “modulator” includes inhibitors and activators at all regulatory levels mentioned above.
The term “NADPH oxidase” comprises any NADPH oxidase. It includes NOX enzymes such as NOX1, NOX2, NOX3, NOX4 and NOX5 as well as DUOX enzymes such as DUOX1 and DUOX2 (see references 13 to 27).
The term “lead compound” designates a compound which is a drug candidate and which may require chemical modifications in order to optimize its pharmacological properties and eventually become a drug to be formulated as a medicament. Methods of optimization are known in the art and further detailed below.
The term “hearing loss” according to the invention embraces drug-, noise- and age-related hearing loss. Age-related hearing loss is also referred to as presbyacusis. The term “phantom hearing”, also known as “tinnitus”, is a common and in some instances invalidating medical complaint.
The term “protein” recited in the main claim extends to homologues having at least 75% sequence identity. Preferably, the sequence identity level is 80% or 85%, more preferred 90% or 95%, and yet more preferred 98% or 99%. For the purpose of determining the level of sequence identity, two nucleotide or protein sequences can be aligned electronically using suitable computer programs known in the art. Such programs comprise BLAST (Altschul et al. (1990), J. Mol. Biol. 215, 403-410), variants thereof such as WU-BLAST (Altschul & Gish (1996), Methods Enzymol. 266, 460-480), FASTA (Pearson & Lipman (1988), Proc. Natl. Acad. Sci. USA 85, 2444-2448) or implementations of the Smith-Waterman algorithm (SSEARCH, Smith & Waterman (1981), J. Mol. Biol. 147, 195-197). These programs, in addition to providing a pairwise sequence alignment, also report the sequence identity level (usually in percent identity) and the probability for the occurrence of the alignment by chance (P-value). Programs such as CLUSTALW (Higgins et al. (1994), Nucleic Acids Res. 22, 4673-4680) can be used to align more than two sequences.
The optional presence of one or more NADPH oxidase subunits relates inter alia to embodiments, wherein not only modulators exerting their effect exclusively directly on the NADPH oxidase are to be identified, but also modulators which act by interfering with the association of the NADPH oxidase with said subunit(s) are to be identified. Such modulators may be compounds binding to regions of the NADPH oxidase and/or of the subunit(s) involved in subunit association. In other words, a test compound identified by the method of the invention which interferes with association (e.g. binds to regions of the NADPH oxidase and/or of the subunit(s) involved in subunit association) is an example of a test compound according to the invention which either modulates expression and/or activity of the protein defined in the main embodiment or modulates the expression and/or activity of said subunit(s).
Also embraced by the invention is a method as defined above, wherein test compounds may be identified which modulate the expression and/or activity of both the protein defined in the main embodiment and said subunits.
In the following, the interactions of an NADPH oxidase with its subunits is exemplified for the NADPH oxidase 3 (NOX3). NOX3 activity requires the widely distributed membrane NOX subunit p22phox. However, in the absence of further, viz. cytoplasmic subunits, no high level, but only low level ROS generation occurs. In contrast in the presence of the combination of one activator subunit (either NOXA1 or p67phox/NOXA2) and one organizer subunit (either NOXO1 or p47phox/NOXO2) NOX3 is capable of generating high levels of ROS. In addition, the NOX3 activity most likely also involves the ubiquitous GTP-binding protein Rac. The interaction sites between the partners are depicted in the following scheme.




A key interaction is the binding of the activator domain of the activator subunits (amino acids 202-212 for hNOXA1 and amino acids 200-210 for hp67phox/NOXA2) to NOX3. It is not clear whether there is a direct interaction of the organizer subunits with NOX3, but there is an indirect interaction with NOX3 through binding to p22phox via the tandem SH3 domain (amino acids 158-217 and 233-289 for hNOXO1 and amino acids 156-216 and 226-286 for hp47phox/NOXO2) and through binding to an SH3 domain of the activator subunit (amino acids 402-463 for hNOXA1 and amino acids 457-513 for hp67phox/NOXA2) through its proline-rich region (amino acids 321-331 for hNOXO1 and 360-370 for hp47phox/NOXO2). The precise site of interaction between NOX3 and p22phox, as well as the sites of interaction of Rac1 with NOX3 and the activator subunits (NOXA1 or p67phox) are not known. The table below provides a compilation of the interaction sites.











binding region of the subunit
target







activator region of activator subunit
NOX3


(aa 202-212 for hNOXA1 and


aa 200-210 for hp67phox/NOXA2)


tandem SH3 domain of organizer
p22phox


subunit


(aa 158-217 and aa 233-289


for hNOXO1 and


aa 156-216 and 226-286 for


hp47phox/NOXO2)


proline-rich region of organizer subunit
SH3 domain of activator subunit


(aa 321-331 for hNOXO1 and
(aa 402-463 for hNOXA1 and


360-370 for hp47phox/NOXO2)
aa 457-513 for hp67phox/



NOXA2)









The optional determination of binding test compounds in step (b) relates to any biophysical binding assay, which may be used to identify binding test molecules prior to performing the functional assay with the binding test molecules only. Suitable biophysical binding assays are known in the art and comprise fluorescence polarization (FP) assay, fluorescence resonance energy transfer (FRET) assay and surface plasmon resonance (SPR) assay. Step (b) is particularly advantageous if said biophysical assay is more amenable to high throughput than the functional assay.
Step (c) relates to the above mentioned functional assay. Determining whether a test compound, or a binding test compound, modulates the expression of a target protein may be accomplished by measuring the expression level. In a more preferred embodiment, the expression level to be determined is the mRNA expression level. Methods for the determination of mRNA expression levels are known in the art and comprise Real Time PCR, Northern blotting and hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for transcripts encoding proteins of the NADPH oxidase family.
In another more preferred embodiment, the expression level to be determined is the protein expression level. The skilled person is aware of methods for the quantitation of proteins. Amounts of purified protein in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular protein in a mixture rely on specific binding, e.g. of antibodies. Specific detection and quantitation methods exploiting the specificity of antibodies comprise immunohistochemistry (in situ) and surface plasmon resonance. Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies.
The present invention also 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, under conditions allowing binding of said test compound to said protein; and (b) determining whether said test compound, upon contacting in step (a) modulates the expression and/or activity of said protein.
The present invention also 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, under conditions allowing binding of said test compound to said protein; (b) determining whether said test compound, upon contacting in step (a) modulates the expression and/or activity of said protein; and (c) performing clinical trials with said modulator.
In a preferred embodiment of the method of the invention, said contacting comprises contacting with one or more NADPH oxidase subunits, under conditions allowing binding of said test compounds to said subunit(s), and wherein said determining comprises determining whether said test compound modulates the expression and/or activity of said subunit(s).
In a further preferred embodiment the method further comprises, prior to step (b), the step of (b′) determining whether said test compound binds to said protein or, if present, said subunit(s), wherein said determining in step (b) is effected upon binding in step (b′). The method according to this preferred embodiment comprises both determining of whether a test compound, upon contacting in step (a), modulates expression and/or activity and the determining of whether a test compound, upon binding in step (b′), modulates expression and/or activity. The term “expression and/or activity” relate to, as defined herein above, the expression and/or activity of the protein as defined in the main embodiment and/or of said subunit(s).
Quantitation of the modulation of the activity of an NADPH oxidase may be effected by quantifying the reactive oxygen species production. Accordingly, said modulation preferably involves modulating the ROS production of said protein, and determining in step (c) comprises quantifying ROS production. Methods of quantifying ROS are known in the art and are further exemplified in Example 4 enclosed herewith.
The inventors for the first time demonstrated high-level expression of the NADPH oxidase NOX3 in the inner ear. Thereby, a protein suitable as a target for therapeutic intervention in hearing loss and phantom hearing is provided.
Vestibular and cochlear sensory epithelia develop from a common ectodermal thickening at the head region, called placode [34]. The otic placode also gives rise to the neurons that will form the inner ear ganglia [35]. The data presented in the Examples and Figures enclosed herewith suggest that the expression of NOX3 mRNA may follow this pattern.
Furthermore, the inventors demonstrated for the first time that NOX3 is a superoxide-generating enzyme. It is also demonstrated that the pattern of subunit- and stimulus-dependence that is distinct from other known NOX family NADPH oxidases. NOX3, as opposed to NOX1 and NOX2, produces low levels of superoxide upon PKC activation without the need of subunits. While the activation of phagocyte NADPH oxidase is thought to occur through PKC-dependent phosphorylation of p47phox [13], this, obviously, cannot be the mechanism of the subunit-independent activation of NOX3. At this point, there are numerous possible pathways how PKC might activate NOX3 (e.g. direct phosphorylation of NOX3, activation of the small GTPase protein Rac1, or changes in the lipid environment). The subunit-independent ROS-generation by NOX3 is of low level in the transfected cells. Given the localization of NOX3 in the inner ear, close to or within highly ROS-sensitive cells, it is tempting to speculate that low, rather than high level superoxide generation is the default mode of NOX3 function.
However, NOX3 activity can be massively enhanced by known NOX organizer and regulator/activator subunits. Searches of mouse and human genomic databases suggest that there are probably no other close homologues of p47phox and p67phox than NOXO1 and NOXA1, respectively. Thus, if NOX3 functions in a subunit-dependent manner in vivo, it would have to use subunits of other NOX enzymes. Based on PCR data shown in FIG. 2, NOX3 could potentially interact with NOXA1 and/or p47phox in the inner ear. However, it cannot be excluded that, under specific circumstances or in a very limited number of cells, other NOX subunits may also be expressed in the inner ear.
Therefore, in a preferred embodiment, said NADPH oxidase subunit(s) is/are the activating subunit(s) NOXA1 and/or p67phox/NOXA2, and/or the organising subunit(s) NOXO1 and/or p47phox/NOXO2.
In a further preferred embodiment said protein and, if present, said subunit(s) is/are comprised in a membrane preparation. Membrane preparations according to the invention may be membrane fractions obtained, for example, by centrifugation upon cell disruption. Alternatively, said membrane preparation is obtained by reconstituting the protein(s) according to the main embodiment with membrane- or micelle-forming amphiphilic lipids.
In a further preferred embodiment said protein and, if present, said subunit(s) is/are comprised in a cell transfected with a nucleic acid encoding said protein. This embodiment relates to a cellular screen.
In a further preferred embodiment of the method of the invention, said protein and, if present, said subunit(s) is/are comprised in a non-human animal. This embodiment relates to an in vivo screen. While less amenable to high throughput, the in vivo screen offers the advantage of the assessment of the disease state of the non-human animal. Accordingly, in a more preferred embodiment, the modulation of ROS production involves improving the hearing of said animal and determining in step (c) involves quantifying said hearing.
In a further preferred embodiment, prior to said contacting, (a′) an ototoxic agent and/or an agent increasing the activity and/or the expression of said protein or subunit(s), is brought into contact with said protein or subunit(s) is/are administered to said cell or said animal. Administration of an ototoxic agent and/or an agent increasing the activity and/or the expression of said protein or subunit(s) may be used as a means of modelling (at the cellular level), or inducing/enhancing (at the organismic level) the disease or disease-related conditions.
Interestingly, while there is almost no literature on the physiological function of ROS in the inner ear, there is a considerable number of studies on the pathological effect of excessive ROS production in this organ (for reviews see [1] and [4]). It has been shown in several publications that specific ototoxic drugs (such as platinum derivatives or aminoglycoside antibiotics) lead to accumulation of ROS in both the cochlea [3] and the vestibular system [8, 11, 36], and noise trauma has been demonstrated to be a prominent cause of ROS production in the cochlea [37]. A permanent increase of ROS concentration, in turn, leads primarily to the death of sensory epithelial cells, and, to a lesser extent, to the death of innervating neurons [1]. Based on the surprising observations presented herein and relating to its localization and its capacity to generate ROS, NOX3 is likely to be a major source of ROS in the inner ear. The unexpected observation that cisplatin markedly enhances NOX3-dependent superoxide production, evokes the possibility that NOX3 is a mediator of cisplatin-dependent ototoxicity. Time course and dose-response of the cisplatin-dependent NOX3 activation is compatible with the time course [2] and dose-response [38] of cisplatin toxicity to inner ear sensory cells.
In a more preferred embodiment, said ototoxic agent is selected from the group consisting of salicylates, non-steroidal antiinflammatories, antibiotics, diuretics, cytostatics, quinine derivatives and gastroprotective drugs.
Salicylates include Aspirine and methyl-salicylates.
Non-steroidal antiinflammatories include diclofenac, etocolac, fenprofen, ibuprofen, indomethacin, naproxen, piroxicam and sulindac.
Preferred antibiotics are aminoglycosides such as amikacin, gentamycin, kanamycin, neomycin, netilmicin, streptomycin and tobramycin. Further preferred antibiotics include erythromycin, vancomycin, minocycline, polymixin B, amphotericin B and capreomycin.
Exemplary diuretics according to the invention are bendroflumethazide, bumetadine, chlorthalidone, ethacrynic acid and furosemide.
Cytostatics, or antineoplastic drugs according to the invention include bleomycine, bromocriptine, carboplatinum, cisplatin, methotrexate, nitrogen mustard, vinblastin and vincristine.
Quinine derivatives, being used as antimalarial and antiarrhythmic drugs, include chloroquine phosphate, quinacrine hydrochloride and quinine sulphate.
Misoprostol is among the envisaged gastroprotective drugs.
In a preferred embodiment of the method of the invention, said NADPH oxidase is NOX3. In a further preferred embodiment said NADPH oxidase is the protein defined in claim 1.
In a further preferred embodiment, the method of the invention further comprises the step of formulating said modulator with a pharmaceutically acceptable carrier. By “pharmaceutically acceptable carrier” is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
More preferred, and prior to said formulating, the affinity, specificity and/or pharmacological properties of the modulator are optimized and/or clinical trials are performed with said modulator or the optimized modulator.
Accordingly, the present invention also 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, under conditions allowing binding of said test compound to said protein; (b) determining whether said test compound, upon contacting in step (a) modulates the expression and/or activity of said protein; and (c) performing clinical trials with said modulator.
Methods for the optimization of the pharmacological properties of compounds identified in screens, generally referred to as lead compounds, are known in the art and comprise a method of modifying a compound identified as a lead compound to achieve: (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmacokinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carbon acids, or (iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi succinates, or (iv) formation of pharmaceutically acceptable salts, or (v) formation of pharmaceutically acceptable complexes, or (vi) synthesis of pharmacologically active polymers, or (vii) introduction of hydrophilic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (ix) modification by introduction of isosteric or bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or (xiii) derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of ketones or aldehydes to Schiff's bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidines or combinations thereof; said method optionally further comprising the steps of the above described methods.
The various steps recited above are generally known in the art. They include or rely on quantitative structure-action relationship (QSAR) analyses (Kubinyi, “Hausch-Analysis and Related Approaches”, VCH Verlag, Weinheim, 1992), combinatorial biochemistry, classical chemistry and others (see, for example, Holzgrabe and Bechtold, Deutsche Apotheker Zeitung 140(8), 813-823, 2000).
Individuals to be selected for said clinical trials comprise healthy individuals, individuals with a disposition or at risk to develop hearing loss or phantom hearing and patients suffering from hearing loss or phantom hearing. Hearing loss is understood to comprise drug-, noise- and age-related hearing loss.
Moreover, the present invention also relates to a pharmaceutical composition comprising (a) an antibody, aptamer, or a fragment or derivative thereof binding specifically the protein defined in the main embodiment; (b) an antisense nucleic acid, an siRNA, or a ribozyme binding specifically a nucleic acid encoding said protein; (c) a iodonium derivative and/or a substituted catechol such as apocynin; (d) a compound comprising the fragment of SEQ ID NO: 11 from position 202 to position 212, the fragment of SEQ ID NO: 11 from position 402 to position 463, the fragment of SEQ ID NO: 15 from position 200 to position 210, the fragment of SEQ ID NO: 15 from position 457 to position 513, the fragment of SEQ ID NO: 7 from position 158 to position 217, the fragment of SEQ ID NO: 7 from position 233 to position 289, the fragment of SEQ ID NO: 7 from position 321 to position 331, the fragment of SEQ ID NO: 19 from position 156 to position 216, the fragment of SEQ ID NO: 19 from position 226 to position 286, the fragment of SEQ ID NO: 19 from position 360 to position 370; and/or (e) a nucleic acid comprising a sequence encoding any of the fragments according to (d). The fragments according to (d) are regions of the sequences of the respective SEQ ID NOs known or expected to be involved in subunit association.
Said compounds according to (d) may furthermore comprise a cell-penetrating peptide. The term “cell-penetrating peptide” relates to a peptide which is capable of entering into cells. This capability may be exploited for the delivery of fragments defined in (d) to cells.
For example, said compounds may be peptides or polypeptides comprising both a fragment as defined in (d) above and a cell-penetrating peptide. Alternatively, other means of functionally linking a fragments as defined in (d) and a cell-penetrating peptide are envisaged. Preferably, said compounds comprising both a fragment as defined in (d) above and a cell-penetrating peptide act as dominant negative cell-permeating inhibitors.
Said cell-penetrating peptides according to the invention include Tat-derived cell-penetrating peptides [46, 47], Antennapedia peptides or penetratins [48, 49] such as the peptide having the sequence Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys (SEQ ID NO: 25), peptides derived from HSV-1 VP22 [50], transportans [51], MAP peptides [52] such as the peptide with the sequence KLALKLALKALKAALKLA (SEQ ID NO: 26), signal sequence-based cell-penetrating peptides (NLS) [53], hydrophobic membrane translocating sequence (MTS) peptides [53] and arginine-rich transporters for drugs. According to an overview of cell-penetrating peptides is provided in [45], CPPs are divided into two classes: the first class consists of amphipathic helical peptides, such as transportan and model amphipathic peptide (MAP), where lysine (Lys) is the main contributor to the positive charge, while the second class includes arginine (Arg)-rich peptides, such as TAT and Antp or penetratin.
The nucleic acids according to (e) include the sequences with the SEQ ID NOs: 12, 16, 8 and 20 as well those fragments thereof which comprise a sequence encoding any of the fragments according to (d). Said nucleic acid may optionally comprise a sequence encoding a cell-penetrating peptide.
Also embraced by the present invention are pharmaceutical compositions comprising fragments of proteins orthologous or homologous to hNOXA1, hNOXO1, hp47phox/NOXO2 or hp67phox/NOXA2, whereby said fragments align with the fragments of hNOXA1, hNOXO1, hp47phox/NOXO2 or hp67phox/NOXA2 recited under (d), as are pharmaceutical compositions comprising nucleic acids encoding these aligning fragments. It is understood that these pharmaceutical compositions are considered equivalents of the above described embodiment directed to pharmaceutical compositions. Said orthologous or homologous proteins include the respective murine proteins, i.e., proteins having an amino acid sequence set forth in any one of SEQ ID NO: 13, 17, 9 or 21. The nucleic acids encoding the latter are set forth in SEQ ID NO: 14, 18, 10 and 22.
Two nucleotide or protein sequences can be aligned electronically using suitable computer programs known in the art. Such programs comprise BLAST (Altschul et al. (1990), J. Mol. Biol. 215, 403-410), variants thereof such as WU-BLAST (Altschul & Gish (1996), Methods Enzymol. 266, 460-480), FASTA (Pearson & Lipman (1988), Proc. Natl. Acad. Sci. USA 85, 2444-2448) or implementations of the Smith-Waterman algorithm (SSEARCH, Smith & Waterman (1981), J. Mol. Biol. 147, 195-197). These programs, in addition to providing a pairwise sequence alignment, also report the sequence identity level (usually in percent identity) and the probability for the occurrence of the alignment by chance (P-value). Programs such as CLUSTALW (Higgins et al. (1994), Nucleic Acids Res. 22, 4673-4680) can be used to align more than two sequences.
Furthermore embraced by the present invention are pharmaceutical compositions comprising (a) peptidomimetic compound(s) which has been obtained by using any of the fragments according to (d) as a lead compound.
Pharmaceutical compositions comprising a nucleic acid according to (e) and/or the above described equivalents thereof are also envisaged to be used for gene therapy. For this purpose, the nucleic acid may be part of an expression, a gene transfer or gene targeting vector. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Transgenic mice expressing a neutralizing antibody directed against nerve growth factor have been generated using the “neuroantibody” technique; Capsoni, Proc. Natl. Acad. Sci. USA 97 (2000), 6826-6831 and Biocca, Embo J. 9 (1990), 101-108. Suitable vectors, methods or gene-delivering systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodua, Blood 91 (1998), 30-36; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251; Verma, Nature 389 (1997), 239-242; Anderson, Nature 392 (Supp. 1998), 25-30; Wang, Gene Therapy 4 (1997), 393-400; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; U.S. Pat. No. 5,580,859; U.S. Pat. No. 5,589,466; U.S. Pat. No. 4,394,448 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein. The nucleic acid molecules according to (e) may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g. adenoviral, retroviral), electroporation, ballistic (e.g. gene gun) or other delivery systems into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention. The introduction and gene therapeutic approach should, preferably, lead to the expression of a fragment according to (d) of the invention, whereby said expressed fragment is particularly useful in the treatment, amelioration and/or prevention of hearing loss and/or phantom hearing.
Said antibody, which is monoclonal antibody, polyclonal antibody, single chain antibody, or fragment thereof that specifically binds said peptide or polypeptide also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, a F(ab2)′, Fv or scFv fragments etc., or a chemically modified derivative of any of these (all comprised by the term “antibody”). Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Köhler and Milstein, Nature 256 (1975), 495, and Galfré, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals with modifications developed by the art. Furthermore, antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodies are obtained by the phage display technique, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the peptide or polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in WO89/09622. A further source of antibodies to be utilized in accordance with the present invention are so-called xenogenic antibodies. The general principle for the production of xenogenic antibodies such as human antibodies in mice is described in, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. Antibodies to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook (1989), loc. cit.
The term “monoclonal” or “polyclonal antibody” (see Harlow and Lane, (1988), loc. cit.) also relates to derivatives of said antibodies which retain or essentially retain their binding specificity. Whereas particularly preferred embodiments of said derivatives are specified further herein below, other preferred derivatives of such antibodies are chimeric antibodies comprising, for example, a mouse or rat variable region and a human constant region.
The term “scFv fragment” (single-chain Fv fragment) is well understood in the art and preferred due to its small size and the possibility to recombinantly produce such fragments.
Preferably, the antibody, aptamer, fragment or derivative thereof according to the invention specifically binds the target protein, (poly)peptide or fragment or epitope thereof whose presence or absence is to be monitored.
The term “specifically binds” in connection with the antibody used in accordance with the present invention means that the antibody etc. does not or essentially does not cross-react with (poly)peptides of similar structures. Cross-reactivity of a panel of antibodies etc. under investigation may be tested, for example, by assessing binding of said panel of antibodies etc. under conventional conditions (see, e.g., Harlow and Lane, (1988), loc. cit.) to the (poly)peptide of interest as well as to a number of more or less (structurally and/or functionally) closely related (poly)peptides. Only those antibodies that bind to the (poly)peptide/protein of interest but do not or do not essentially bind to any of the other (poly)peptides which are preferably expressed by the same tissue as the (poly)peptide of interest, are considered specific for the (poly)peptide/protein of interest and selected for further studies in accordance with the method of the invention.
In a particularly preferred embodiment of the method of the invention, said antibody or antibody binding portion is or is derived from a human antibody or a humanized antibody.
The term “humanized antibody” means, in accordance with the present invention, an antibody of non-human origin, where at least one complementarity determining region (CDR) in the variable regions such as the CDR3 and preferably all 6 CDRs have been replaced by CDRs of an antibody of human origin having a desired specificity. Optionally, the non-human constant region(s) of the antibody has/have been replaced by (a) constant region(s) of a human antibody. Methods for the production of humanized antibodies are described in, e.g., ER-A1 0 239 400 and WO90/07861.
The term “aptamer” as used herein refers to DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected which bind nucleic acid, proteins, small organic compounds, and even entire organisms. A database of aptamers is maintained at http://aptamer.icmb.utexas.edu/.
An antisense nucleic acid according to the invention is a nucleic acid molecule complementary to a nucleic acid molecule encoding a protein according to the main embodiment which may be used for the repression of expression of said protein. The construction of small interfering RNAs (siRNAs) (see, e.g. Zamore Nat Struct Biol 2001, 8(9):746-50 or Tuschl T. CHEMBIOCHEM. 2001, 2:239-245) or of appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) which specifically cleave the (pre)-mRNA of a gene comprising a nucleic acid encoding said protein are also suitable for the repression of expression. The techniques underlying said repression of expression are well known in the art. Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke et al. (Methods in Cell Biology (1995) 50:449-460). Standard methods relating to antisense technology have also been described (Melani et al., Cancer Res. (1991) 51:2897-2901). Said nucleic acid molecules may be chemically synthesized or transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell. Such nucleic acid molecules may further contain ribozyme sequences as described above.
Iodonium derivatives or, more specifically, aryliodonium compounds include diphenylene iodonium (DPI, also referred to as iodoniumdiphenyl or iodonium biphenyl), di-2-thienyliodonium (also referred to as iodonium thiophene) and phenoxaiodonium. These compounds act as arylating agents and directly and irreversibly inhibit NOX enzymes.
Apocynin (4-hydroxy-3-methoxy-acetophenone) is a methoxy-substituted catechol and exerts its effect on NOX enzymes through the inhibition of subunit assembly.
Also embraced by the present invention are pharmaceutical compositions comprising (i) naphthoquinones such as plumbagin, acetylshikonin; (ii) inhibitors of HMG-CoA reductase including statins such as lovastatin, simvastatin, atorvastatin; (iii) gliotoxin; (iv) phenothiazines such as phenothiazine, trifluoperazine, and/or (v) a derivative of any one of (i) to (v).
Plumbagin is a naphtoquinone derived from Plumbago Zeylanica (Chitrak, an indian medicinal plant).
Gliotoxin is a metabolite of pathogenic fungi (Aspergillus and Candida spp) and has been implicated in infectious pathways. It exhibits immunosupressive action and antitumor activity and inhibits activation process of NOX2 (Yoshida et al., 2000) and the assembly of the enzyme (Tsunawaki et al., 2004). It is available from Sigma. Statins are inhibitors of HMG-CoA. They decrease plasma cholesterol and block rac-1 dependent activation of NADPH oxidases (Maack et al. 2003). Furthermore, they inhibit myristoylation of rac.
Trifluoperazine is an inhibitor of PKC/calmodulin and prevents the activation of NADPH oxidases (Seifert and Scachtele, 1988, Holland et al., 2000).
The term derivative relates to compounds having the same core or backbone structure while one or more of the substituents are modified, for example by replacing a methyl group with a trifluoromethyl group. These modifications are such that the biological/pharmacological activity is not substantially altered. Said activity may be monitored by the assays disclosed herein.
The present invention also relates to a pharmaceutical composition consisting of (a) ortho-methoxy-substituted catechols such as apocynin, acetosyringone, vanillin, vanillic acid, syringaldehyde, syringic acid; and (b) a pharmaceutically acceptable carrier, excipient or diluent.
Also provided by the present invention is a pharmaceutical composition comprising (a) an ototoxic agent; and (b) a compound selected from the group consisting of: (i) an antibody, aptamer, or a fragment or derivative thereof binding specifically the protein defined in claim 1; (ii) an antisense nucleic acid, an siRNA, or a ribozyme binding specifically a nucleic acid encoding said protein; (iii) a compound comprising the fragment of SEQ ID NO: 11 from position 202 to position 212, the fragment of SEQ ID NO: 11 from position 402 to position 463, the fragment of SEQ ID NO: 15 from position 200 to position 210, the fragment of SEQ ID NO: 15 from position 457 to position 513, the fragment of SEQ ID NO: 7 from position 158 to position 217, the fragment of SEQ ID NO: 7 from position 233 to position 289, the fragment of SEQ ID NO: 7 from position 321 to position 331, the fragment of SEQ ID NO: 19 from position 156 to position 216, the fragment of SEQ ID NO: 19 from position 226 to position 286, the fragment of SEQ ID NO: 19 from position 360 to position 370, wherein said compound may furthermore comprise a cell-penetrating peptide; (iv) a nucleic acid comprising a sequence encoding any of the fragments according to (c), wherein said nucleic acid may optionally comprise a sequence encoding a cell-penetrating peptide; (v) aryliodonium compounds such as diphenylene iodonium (DPI), di-2-thienyliodonium, phenoxaiodonium; (vi) naphthoquinones such as plumbagin, acetylshikonin; (vii) inhibitors of HMG-CoA reductase including statins such as lovastatin, simvastatin, atorvastatin; (viii) gliotoxin; (ix) phenothiazines such as phenothiazine, trifluoperazine, and/or (x) a derivative of any one of (v) to (ix). Said ototoxic agent may be any agent detailed herein above. Preferably, said ototoxic agent is a medicament, wherein said medicament causes ototoxicity as a side effect. Therefore, and in view of the disclosure of the mechanism of ototoxicity in this application, a combination therapy with a medicament with ototoxic side effect and an inhibitor of the protein defined in the main embodiment is provided. Also provided is the use of an ototoxic agent and of a compound as defined in (b) above for the manufacture of pharmaceutical composition, wherein said compound as defined in (b) prevents, alleviates or cures the ototoxic effect of said ototoxic agent.
In a preferred embodiment of said pharmaceutical composition, said ototoxic agent is an antibiotic.
In a more preferred embodiment of said pharmaceutical composition, said ototoxic agent is an aminoglycoside antibiotic, preferably gentamycin. This type of combination therapy is particularly envisaged for those regions or countries where aminoglycoside antibiotics such as gentamycin, owing to their low cost, are widely used.
The present invention also relates to the use of a modulator of the protein defined in the main embodiment for the preparation of a pharmaceutical composition for the treatment and/or prevention of hearing loss and/or phantom hearing, wherein said modulator is selected from the group consisting of (a) an antibody, aptamer, or a fragment or derivative thereof binding specifically said protein; (b) an antisense nucleic acid, an siRNA, or a ribozyme binding specifically a nucleic acid encoding said protein; (c) a known modulator of NOX3 and/or NADPH oxidases and/or electron transport proteins; (d) a compound comprising the fragment of SEQ ID NO: 11 from position 202 to position 212, the fragment of SEQ ID NO: 11 from position 402 to position 463, the fragment of SEQ ID NO: 15 from position 200 to position 210, the fragment of SEQ ID NO: 15 from position 457 to position 513, the fragment of SEQ ID NO: 7 from position 158 to position 217, the fragment of SEQ ID NO: 7 from position 233 to position 289, the fragment of SEQ ID NO: 7 from position 321 to position 331, the fragment of SEQ ID NO: 19 from position 156 to position 216, the fragment of SEQ ID NO: 19 from position 226 to position 286, the fragment of SEQ ID NO: 19 from position 360 to position 370; (e) a nucleic acid comprising a sequence encoding any of the fragments according to (d); and (f) a modulator identified by the method of any one of claims 1 to 13. The fragments according to (d) are regions of the sequences of the respective SEQ ID NOs known or expected to be involved in subunit association. Said compounds according to (d) may furthermore comprise a cell-penetrating peptide. The term “cell-penetrating peptide” is defined herein above.
The nucleic acids according to (e) include the sequences with the SEQ ID NOs: 12, 16, 8 and 20 as well those fragments thereof which comprise a sequence encoding any of the fragments according to (d). Said nucleic acid may optionally comprise a sequence encoding a cell-penetrating peptide.
Also embraced by the present invention are uses of fragments of proteins orthologous or homologous to hNOXA1, hNOXO1, hp47phox/NOXO2 or hp67phox/NOXA2, whereby said fragments align with the fragments of hNOXA1, hNOXO1, hp47phox/NOXO2 or hp67phox/NOXA2 recited under (d), as are uses of nucleic acids encoding these aligning fragments. It is understood that these uses are considered equivalents of the above described embodiment. Said orthologous or homologous proteins include the respective murine proteins, i.e., proteins having an amino acid sequence set forth in any one of SEQ ID NO: 13, 17, 9 or 21. The nucleic acids encoding the latter are set forth in SEQ ID NO: 14, 18, 10 and 22.
Furthermore embraced by the present invention are uses of (a) peptidomimetic compound(s) which has been obtained by using any of the fragments according to (d) as a lead compound.
Uses of a nucleic acid according to (e) and/or of the above described equivalents thereof are also envisaged for gene therapy.
The present invention also relates to the use of a cisplatin and/or hydrogen hexachloroplatinate for the preparation of a pharmaceutical composition for the treatment and/or prevention of tinnitus. Cisplatin and hydrogen hexachloroplatinate are activators of the protein defined the main embodiment. Surprisingly, in many incidences of tinnitus a positive response to a treatment with compounds known to induce oxidative stress in the inner ear is observed.
Also provided is a method of diagnosing hearing loss and/or phantom hearing and/or an individual's disposition or risk to develop said loss and/or said phantom hearing, comprising the steps of: (a) determining (a) polymorphism(s) in a NOX3 gene or cDNA and/or in a gene or cDNA encoding an NADPH oxidase subunit in a sample obtained from said individual; and (b) associating said polymorphism(s) with a disease state or disposition state. Preferably, said sample is a blood sample. Preferably, said NOX3 gene comprises or consists of the sequence set forth in SEQ ID NO: 23 or 24. Preferably said NOX3 cDNA (or equivalently mRNA) comprises or consists of the sequence set forth in SEQ ID NO: 2, 4 or 6. Preferably said cDNA encoding an NADPH oxidase subunit comprises or consists of the sequence set forth in any one of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20 or 22.
The term “polymorphism”, or “nucleotide polymorphism” refers to the occurrence of one or more different nucleotides or bases at a given location on a chromosome. Usually, polymorphisms are distinguished from mutations based on their prevalence. Sometimes a threshold of 1% prevalence in a population of individuals is considered for separating polymorphisms (more frequent) from mutations (less frequent). A single nucleotide polymorphism (SNP) is a polymorphism of a single nucleotide or base. The SNP database maintained at NCBI (http://www.ncbi.nlm.nih.gov/SNP/) divides SNPs into SNPs in the proximity of a known locus and such that are 5′ further away than 2 kb from the most 5′ feature of a gene and 3′ further away than 500 bases from the most 3′ feature of a gene. SNPs in the proximity of a known locus are further divided into SNPs occurring at an mRNA location and such that do not. SNPs occurring at an mRNA location comprise coding and non-coding SNPs.
It is understood that the term “polymorphism(s) in a NOX3 gene and/or in a gene encoding an NADPH oxidase subunit” embraces polymorphisms in exons, introns and regulatory regions such as promoters. Polymorphisms in exons may be determined or analysed using genomic DNA or cDNA (or equivalently mRNA). Polymorphisms in introns or regulatory regions such as promoters may be determined or analysed using cDNA (or equivalently mRNA).
Said associating of polymorphism(s) with a disease state or disposition state refers to classifying of individuals and patients. The term “classifying” refers to the assignment of individuals or patients to two or more groups or classes. In other words, individuals, previously unclassified, get labelled by their respective class. The assigned class label may refer to parameters used for classification, e.g. polymorphisms, or may refer to parameters not used for classification because their values are not known beforehand, e.g. fast or slow response to therapy. In the first case, class discovery methods, e.g. clustering may be applied, whereas in the second case predictive classification methods are used. Classification may be done manually by a trained person or by a computer program provided with the values of the parameters used for class distinction. Patients have to give informed consent. Data have to be handled and kept secret in accordance with national laws.
The present invention also provides the use of a compound binding to the protein defined in the main embodiment or to a NADPH oxidase subunit for the preparation of a diagnostic composition for the diagnosis of hearing loss and/or phantom hearing and/or an individual's disposition or risk to develop said loss and/or said phantom hearing, wherein said compound is selected from the group consisting of (a) an antibody, aptamer, or a fragment or derivative thereof binding specifically said protein; and (b) a known modulator of NOX3 and/or NADPH oxidases and/or electron transport proteins.
In a preferred embodiment of the use according to the invention, said known modulator is selected from the group consisting of iodonium derivatives, substituted catechols such as apocynin, platinum derivatives and palladium derivatives.
Known modulators to be used for the preparation of a pharmaceutical composition according to the invention are selected from the group consisting of (i) aryliodonium compounds such as diphenylene iodonium (DPI), di-2-thienyliodonium, phenoxaiodonium; (ii) ortho-methoxy-substituted catechols such as apocynin, acetosyringone, vanillin, vanillic acid, syringaldehyde, syringic acid; (iii) naphthoquinones such as plumbagin, acetylshikonin; (iv) inhibitors of HMG-CoA reductase including statins such as lovastatin, simvastatin, atorvastatin; (v) gliotoxin; (vi) phenothiazines such as phenothiazine, trifluoperazine; and (vii) a derivative of any one of (i) to (vi). Said known modulators act as inhibitors of the protein defined in the main embodiment.
Known modulators to be used for the preparation of a diagnostic composition according to the invention are selected from the known modulators to be used for the preparation of a pharmaceutical composition and cisplatin and hexachloroplatinate as well as derivatives thereof. Cisplatin and hexachloroplatinate bind and activate the protein defined in the main embodiment and are therefore specifically envisaged for the manufacture of a diagnostic composition.
Cisplatin, as demonstrated by the inventors, is a preferred platinum derivative which modulates NOX3 activity. The platinum derivative hydrogen hexachloroplatinate and palladium derivatives are known to modulate the activity of NOX2 (phagocyte NADPH oxidase). In both cases, there are indications that modulation is a direct effect on the NOX enzymes.
Also envisaged is the use of a compound binding to a nucleic acid encoding the protein defined in the main embodiment or an NADPH oxidase subunit for the preparation of a diagnostic composition for the diagnosis of hearing loss and/or phantom hearing and/or an individual's disposition or risk to develop said loss and/or said phantom hearing, wherein said compound is a nucleic acid complementary to said nucleic acid and at least 15 nucleotides in length. This embodiment is directed to oligonucleotide probes for the detection of genomic DNA or mRNA. With regard to genomic DNA, also the detection and distinction of polymorphisms is envisaged.
Preferably, said compound is detectably labelled.
More preferred, said diagnosis to be performed involves imaging of the human or animal body.
In a preferred embodiment of the method or the use of the invention, said animal is a rodent. More preferred, said rodent is mouse or rat.
In a preferred embodiment of the method or the use of the present invention, said modulator is an inhibitor.
The term “inhibitor” designates a compound lowering the activity of a target molecule, preferably by performing one or more of the following effects: (i) the transcription of the gene encoding the protein to be inhibited is lowered, (ii) the translation of the mRNA encoding the protein to be inhibited is lowered, (iii) the protein performs its biochemical function with lowered efficiency in presence of the inhibitor, and (iv) the protein performs its cellular function with lowered efficiency in presence of the inhibitor.
Compounds falling in class (i) include compounds interfering with the transcriptional machinery and/or its interaction with the promoter of said gene and/or with expression control elements remote from the promoter such as enhancers. Compounds of class (ii) comprise antisense constructs and constructs for performing RNA interference well known in the art (see, e.g. Zamore (2001) or Tuschl (2001)). Compounds of class (iii) interfere with molecular function of the protein to be inhibited, in case of an NADPH oxidase with its enzymatic activity and/or its capability to associate with NADPH oxidase subunits. Accordingly, active site binding compounds, in particular compounds capable of binding to the active site of any NADPH oxidase, are envisaged, as are compounds interfering with the association of NADPH oxidase with said subunits. More preferred are compounds specifically binding to an active site of NADPH oxidase. Also envisaged are compounds binding to or blocking substrate binding sites of NADPH oxidase. Class (iv) includes compounds which do not necessarily directly bind to NADPH oxidase, but still interfere with NADPH oxidase activity, for example by binding to and/or inhibiting the function or inhibiting expression of members of a pathway which comprises NADPH oxidase. These members may be either upstream or downstream of NADPH oxidase within said pathway.
In a preferred embodiment, the inhibitor is a low molecular weight compound. Low molecular weight compounds are compounds of natural origin or chemically synthesized compounds, preferably with a molecular weight between 100 and 1000, more preferred between 200 and 750, and even more preferred between 300 and 600.
The efficiency of the inhibitor can be quantitized by comparing the level of activity in the presence of the inhibitor to that in the absence of the inhibitor. For example, as an activity measure may be used: the change in amount of mRNA formed, the change in amount of protein formed, the change in amount of substrate converted or product formed, and/or the change in the cellular phenotype or in the phenotype of an organism.
In a preferred embodiment, the level of activity is less than 90%, more preferred less than 80%, 70%, 60% or 50% of the activity in absence of the inhibitor. Yet more preferred are inhibitors lowering the level down to less than 25%, less than 10%, less than 5% or less than 1% of the activity in absence of the inhibitor.
The present invention also relates to a nucleic acid (i) comprising or consisting of the sequence of SEQ ID NO: 6, or (ii) encoding a protein comprising or consisting of the sequence of SEQ ID NO: 5, or (iii) encoding a fragment of the protein according to (ii), wherein said fragment exhibits NADPH oxidase activity, or (iv) encoding a protein having a sequence at least 95% identical with the protein according to (ii) or with the fragment according to (iii) and exhibiting NADPH oxidase activity.
Preferably, said protein having at least 95% sequence identity with the protein according to (ii) or with the fragment according to (iii), has 98% or 99% identity with said protein or fragment.
An alternative embodiment of the invention relates to a vector comprising the above defined nucleic acid.
The vector of the present invention may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
Furthermore, the vector of the present invention may, in addition to the nucleic acids of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the artisan and may include a promoter, a splice cassette, translation initiation codon, translation and insertion site for introducing an insert into the vector. Preferably, the nucleic acid of the invention is operably linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells.
Many suitable vectors are known to those skilled in molecular biology, the choice of which would depend on the function desired and include plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering. Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook (1989), loc. cit., and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994). Alternatively, the nucleic acids and vectors of the invention can be reconstituted into liposomes for delivery to target cells. According to the invention relevant sequences can be transferred into expression vectors where expression of a particular (poly)peptide/protein is required. Typical cloning vectors include pBscpt sk, pGEM, pUC9, pBR322 and pGBT9. Typical expression vectors include pTRE, pCAL-n-EK, pESP-1, pOP13CAT.
Furthermore, a protein encoded by said nucleic acid is provided.
The present invention furthermore relates to host containing an aforementioned vector or an aforementioned nucleic acid, or an aforementioned protein. Said host may be produced by introducing said vector or nucleic acid into a host cell which upon its presence in the cell mediates the expression of a protein encoded by the nucleic acid of the invention or comprising a nucleic acid or a vector according to the invention wherein the nucleic acid and/or the encoded (poly)peptide/protein is foreign to the host cell.
By “foreign” it is meant that the nucleic acid and/or the encoded (poly)peptide/protein is either heterologous with respect to the host, this means derived from a cell or organism with a different genomic background, or is homologous with respect to the host but located in a different genomic environment than the naturally occurring counterpart of said nucleic acid. This means that, if the nucleic acid is homologous with respect to the host, it is not located in its natural location in the genome of said host, in particular it is surrounded by different genes. In this case the nucleic acid may be either under the control of its own promoter or under the control of a heterologous promoter. The vector or nucleic acid according to the invention which is present in the host may either be integrated into the genome of the host or it may be maintained in some form extrachromosomally. In this respect, it is also to be understood that the nucleic acid of the invention can be used to restore or create a mutant gene via homologous recombination.
The host can be any prokaryote or eukaryotic cell, such as a bacteria, an insect, fungal, plant or animal cell.
The term “prokaryote” is meant to include all bacteria which can be transformed or transfected with a DNA or RNA molecules for the expression of a protein of the invention. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term “eukaryotic” is meant to include yeast cells, cells of higher plant, insect cells and preferably mammalian cells. Depending upon the host employed in a recombinant production procedure, the protein encoded by the nucleic acid of the present invention may be glycosylated or may be non-glycosylated. A nucleic acid of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Furthermore, methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art (Sambrook (1989), loc. cit.).
Preferably, said host is a cell. More preferred, the host is a human cell or human cell line.
Alternatively, said host is a transgenic non-human animal.
A method for the production of a transgenic non-human animal, for example transgenic mouse, comprises introduction of a nucleic acid or vector according to the invention into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom. The non-human animal can be used in accordance with a screening method of the invention described herein. Production of transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press. The DNA of the embryonal membranes of embryos can be analyzed using, e.g., Southern blots with an appropriate probe. A general method for making transgenic non-human animals is described in the art, see for example WO 94/24274. For making transgenic non-human organisms (which include homologously targeted non-human animals), embryonal stem cells (ES cells) are preferred. Murine ES cells, such as AB-1 line grown on mitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley, Cell 62: 1073-1085 (1990)) essentially as described (Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J. Robertson, ed. (Oxford: IRL Press), p. 71-112) may be used for homologous gene targeting. Other suitable ES lines include, but are not limited to, the E14 line (Hooper et al., Nature 326: 292-295 (1987)), the D3 line (Doetschman et al., J. Embryol. Exp. Morph. 87: 27-45 (1985)), the CCE line (Robertson et al., Nature 323: 445-448 (1986)), the AK-7 line (Zhuang et al., Cell 77: 875-884 (1994) which is incorporated by reference herein). The success of generating a mouse line from ES cells bearing a specific targeted mutation depends on the pluripotence of the ES cells (i.e., their ability, once injected into a host developing embryo, such as a blastocyst or morula, to participate in embryogenesis and contribute to the germ cells of the resulting animal). The blastocysts containing the injected ES cells are allowed to develop in the uteri of pseudopregnant non-human females and are born as chimeric mice. The resultant transgenic mice are chimeric for cells having either the recombinase or reporter loci and are backcrossed and screened for the presence of the correctly targeted transgene(s) by PCR or Southern blot analysis on tail biopsy DNA of offspring so as to identify transgenic mice heterozygous for either the recombinase or reporter locus/loci.
Methods for producing transgenic flies, such as Drosophila melanogaster are also described in the art, see for example U.S. Pat. No. 4,670,388, Brand & Perrimon, Development (1993) 118: 401-415; and Phelps & Brand, Methods (April 1998) 14: 367-379.
Transgenic worms such as C. elegans can be generated as described in Mello, et al., (1991) Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. Embo J 10, 3959-70, Plasterk, (1995) Reverse genetics: from gene sequence to mutant worm. Methods Cell Biol 48, 59-80.
The invention also relates to transgenic non-human animals such as transgenic mouse, rats, hamsters, dogs, monkeys, rabbits, pigs, C. elegans and fish such as Torpedo fish comprising a nucleic acid according to the invention.
Also provided is an antibody or aptamer, or fragment or derivative thereof binding specifically to the protein encoded by said nucleic acid as is an antisense nucleic acid, an siRNA, or a ribozyme binding specifically said nucleic acid.



The Figures show:
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.
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 p67phox and p47phox cDNA from the indicated rat tissues. The first lane of each panel shows DNA size markers.
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.
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.
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.
FIG. 6: Subunit regulation of NOX3 activity. A, B, and C, HEK293 cells were transfected with different combinations of NOX3, NOXO1, NOXA1, p47phox, and p67phox, 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.
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 μM 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 μM 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 μM 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 μM cisplatin for the indicated periods of time.



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
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.
Results.
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 designed—based on these results—mouse and rat NOX3 PCR primers.
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
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 μg 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 p47phox 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 p67phox 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.
Quantitative PCR was carried out using ABI Prism 7900HT Sequence Detection System with standard temperature protocol and 2×SYBR Green PCR Master Mix reagent (Applied Biosystems, Worrington, UK) in 25 μl 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.
Results.
NOX3 is Predominantly Expressed in the Inner Ear—
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).
Expression of Cytoplasmic NOX Subunits in the Inner Ear—
NOX1 and gp91phox/NOX2 require cytoplasmic organizer subunits (NOXO1, p47phox) and activator subunits (NOXA1, p67phox) to form a functional enzyme. As NOX3 shows a high degree of homology with NOX1 and gp91phox/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 p47phox was expressed in the inner ear (FIG. 2). mRNA of the activator subunit, p67phox, and the organizer subunit, NOXO1, could be detected only at very high cycle numbers (40 PCR cycles; data not shown). Since p47phox 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.
Expression of NOX3 in Different Parts of the Cochlea—
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
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 μm thick inner ear sections.
Results.
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
Cell Culture and Transfection—
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 μg/ml), and 4 mmol/liter L-glutamine. NOX3-, NOXO1-, NOXA1-, p47phox, and p67phox 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 μg/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.
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 μM 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 μM ferricytochrome C (referred to as cytochrome C reduction technique). The O−2 production was calculated using an absorption coefficient of 21.1 mM−1 cm−1 and normalized to 107 cells [29].
Results.
NOX3-Dependent Superoxide Generation in the Absence of Subunits—
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 gp91phox/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.
Regulation of NOX3 by the Organizer and Activator Subunits of NOX1 and gp91phox/NOX2—
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, p67phox and p47phox. 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 p47phox and p67phox but devoid of NOX3, did not produce any superoxide (not shown). Interestingly p67phox alone, in the absence of p47phox, was sufficient to double the PMA-induced superoxide generation of NOX3, while p47phox in the absence of p67phox, 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).
At a least on a biochemical level, there is promiscuity among the organizer and regulator subunits: NOXO1 is able to function with p67phox, and NOXA1 with p47phox [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, p67phox, and NOX3 in HEK293 cells, led to spontaneous superoxide generation that could not be further enhanced by PMA (FIG. 6C). However, when p47phox, NOXA1, and NOX3 were expressed, superoxide production by HEK293 cells was largely PMA-dependent (FIG. 6C). Thus, the organizer subunit (p47phox versus NOXO1) determines whether NOX3 activity is PKC-dependent or independent.
Cisplatin Enhances NOX3 Activity
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 μM 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).
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 μM 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 μM, 17 hours) did not influence superoxide production (data not shown). HEK293 cells were also co-transfected with NOX3, p47phox, and p67phox, and incubated with 20 μM cisplatin for 12 hours. Cisplatin enhanced the superoxide production of NOX3-, p47phox, and p67phox-transfected cells by a factor of approximately 3.3 (FIG. 7D); this superoxide production could be blocked by addition of 5 μM DPI (not shown).
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 μM concentration, and 20 μM cisplatin had a maximal effect (FIG. 7E). The EC50 of NOX3 activation by cisplatin was 3.6+/−1.4 μM.
In order to examine the time course of NOX3 activation by cisplatin, a NOX3/NOXO1/NOXA1 transfected stable clone was incubated with 20 μM 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 t50 was 11.5+/−1.7 hours.
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## 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.