ES2408132B1 - PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF THE EPIPHORE. - Google Patents

Abstract

Pharmaceutical composition for the treatment of epiphora. # The invention relates to therapeutic compositions for the treatment of epiphora and, more specifically with compositions comprising a TRPM8 receptor antagonist.

Description

Pharmaceutical composition for epiphora treatment

Technical Field of the Invention

The invention relates to therapeutic compositions for the treatment of epiphora and, more specifically, to compositions comprising a TRPM8 antagonist.

Background of the Invention

The degree of moisture of the ocular surface and of the external mucous membranes remains stable thanks to the continuous production of aqueous fluid by exocrine glands. The interruption of this process determines the development of ocular, oral and vaginal dryness syndromes, which are highly prevalent, especially in the elderly (Moss, SE, et al. 2008. Optom.Vis.Sci. 85: 668-674; Barker, KE & Savage, NW 2005. Aust.Dent.J. 50: 220-223; Leiblum, SR, et al. 2009. J.Sex Med 6: 2425-2433). In the eye, the tear flow produced in the absence of emotional factors or exogenous irritant stimuli ("basal" tear secretion) is adjusted to environmental conditions and the frequency of blinking (Dartt, DA 2009. Prog.Retin.Eye Res. 28 : 155-177). Tearing also increases significantly after irritation of the ocular surface (Acosta, M. C. et al. 2004. Invest Ophthalmol.Vis.Sci. 45: 2333-2336). Irritant stimuli are detected by the mechano-nociceptor and polymodal nerve endings of the trigeminal, which are sensitive to forces of harmful intensity, harmful heat and chemical irritants, evoking pain (Belmonte, C., et al. 2004. Exp.Eye Res 78: 513-525) and reflex tearing. However, the neural structures responsible for detecting the degree of ocular dryness involved in the regulation of the basal tear rate are not known.

Regarding the syndrome of ocular dryness or xerophthalmia, this is an eye disease characterized by persistent dryness of the conjunctiva and opacity of the cornea.

There are multiple causes that can produce xerophthalmia, being more frequent in people with age. Among the diseases that produce xerophthalmia are: vitamin A deficiency, Sjögren's syndrome, rheumatoid arthritis and other rheumatic diseases, chemical or thermal burns and drugs such as atenolol, chlorpheniramine, hydrochlorothiazide, isotretinoin, ketorolac, ketotifen, levocabastine, levofloxacin, oxibutinin, oxybutynin .

Treatments used to treat xerophthalmia include corticosteroids that may be effective in the early stages of the disease, vitamin A supplements and pilocarpine which is a drug that increases tear production. Among the preparations that improve dryness (artificial tears) are used hypromellose solutions and carbomer gels that are applied on the conjunctiva. However, these treatments have clear limitations regarding their efficacy and toxicity. Therefore there is a need to provide new improved treatments for the treatment of dry eye xerophthalmia and dry mouth syndrome.

Summary of the Invention

In one aspect, the invention relates to the use of a TRPM8 antagonist for the preparation of a medicament for the treatment of epiphora.

In another aspect, the invention relates to a composition comprising at least one TRPM8 antagonist and at least one drug useful for the treatment of epiphora and, if desired, a pharmaceutically acceptable carrier.

Description of the Figures of the Invention

Figure 1. Response characteristics of cold-sensitive nerve endings of the mouse cornea. to. Nerve impulse activity (NTI) in response to cooling and heating pulses, and to menthol. The paths, from top to bottom, represent: Average trigger frequency (Mean freq., In Hz), instantaneous frequency (Inst. Freq., In Hz), direct recording of electrical activity (Imp. Ampl., In μV), temperature of the infusion solution (Temp., in ° C). b. Average trigger frequency (Mean freq., In pulses / s) of 55 cold-sensitive corneal terminations in response to a cooling ramp. The data is the mean ± the standard error. C. Distribution of cold thresholds (represented as the change from baseline temperature, -

T, in ° C) of 55 thermosensitive terminals. d. Change of the firing frequency with the temperature, determined in the same 55 cold-sensitive terminals. The frequency change (Mean freq., In pulses / s) between the threshold temperature and that at which the peak frequency is reached has been represented for each terminal. The thick line represents the average value of the 55 individual slopes. and. Electrical activity of a cold-sensitive nerve end in response to staggered cooling. The plots show the average trigger frequency (Mean freq., Hz), the instantaneous frequency (Inst. Freq., Hz), the number of pulses per burst of action potentials (Impulses / Bursa) and the temperature of the solution. perfusion (Temp., ° C). F. Average trigger frequency (Mean frequency, in pulses / s) of 10 thermosensitive terminals during staggered cooling. A significant increase in the average trigger frequency was observed both during the dynamic phase (initial 30s of each temperature step) and during the static phase (last 30s of the stage) of the stimulus, being in both cases proportional to the temperature decrease (Dynamic response: Pearson correlation coefficient = -0.98, p = 0.002; Static response: Pearson correlation coefficient = -0.96, p = 0.014).

Figure 2. Immunohistochemistry of the mouse corneal nerve fibers TRPM8-EYFP. a, b, and c are examples of nerve fillets entering the peripheral cornea to form the stromal plexus. In a, all the sensory nerve fibers of the nerve fillet have been stained with an antibody against neuron-specific class III beta-tubulin (Tuj-1), while in b only those fibers of the nerve fillet that reacted against the antibody against green fluorescent protein (GFP). c shows a double staining of immunofluorescence of the stromal nerves, with antibodies against Tuj-1 (upper panel) and against GFP (intermediate panel), as well as the superimposed image (lower panel), showing the reduced proportion of cold-sensitive fibers of among the general population of corneal sensory fibers. d, e and f show the characteristic architecture of the nerve fibers that run under the basal layer of the corneal epithelium (subbasal plexus), where they appear as straight and more or less parallel strained fibers (brush fibers) that run from the peripheral cornea to the center over long distances. In d, all subbasal plexus fibers (positive for Tuj-1) are stained, while in e, only those positive for GFP have been stained. In f a double staining against Tuj-1 (gray) and GFP (white) is shown to illustrate the shortage of axons presumably positive for TRPM8. Gyh show further increase in the areas framed in dye, respectively, showing in more detail the morphology of the subbasal arrosiated fibers, as well as their ramifications that ascend perpendicularly from the basal layers of the corneal epithelium to the more superficial ones, occasionally presenting nerve endings in the subbasal layer To show the appearance of intraepithelial nerve endings, in i (all epithelial nerve endings, Tuj-1 positive) and j (supposed cold-sensitive nerve terminals, positive for GFP) the same areas of gyh are shown, but focusing on a plane more superficial. The different morphology and the low degree of branching of presumably positive terminals for TRPM8 are also shown in k, using upright fluorescence microscopy. It shows a sweep of foci compiled in a single image to illustrate the trajectory of axons in their path from the stroma to the corneal surface, where they finally originate clusters of relatively grouped terminations (framed square, corresponding to the area shown in k). A drawing is shown in the upper right area of the figure to schematically show the location of the sensory nerves in the different areas of the cornea, and their correspondence with the different panels that make up the figure. A-C corresponds to the anterior third of the stroma, where the stromal nerve trunks are located, D-H represents the fibers of the subbasal plexus, and I-K, the nerve endings between the most superficial layers of the corneal epithelium. The scales represent 70 μm (a-c), 150 μm (d, e) or 40 μm (f-l).

Figure 3. Response to cold ramps of corneal thermosensitive nerve endings recorded in TRPM8 (- / -), TRPM8 (+/-) and TRPA1 (- / -) mice, and in wild mice. to. Average trigger frequency (in pulses / s, upper panel) during a cooling ramp (lower panel) recorded in 143 nerve endings of TRPM8 (- / -) mice (thin line), 11 terminals of TRPM8 (+/-) ( dashed line) and 12 terminals of wild mice from the same litters (solid line), pre-fused with control solution (left) and in the presence of 50 μM menthol (right). b. Medium frequency of firing of 11 thermosensitive terminations recorded in corneas of TRPA1 (- / -) mice (thin line) and in 13 corneal terminations of wild mice of the same litters (thick line), during a cooling ramp (lower panel ). Data are mean ± standard error.

Figure 4. Variation of the tear secretion rate with the corneal temperature. to. Basal tear rate, expressed as the average length of wet phenol red yarn for 2 minutes, measured in the eyes of anesthetized mice exposed to an ambient temperature of 24.8 ± 0.9ºC and 42.5 ± 0.4ºC, with an ambient humidity of 63.7 ± 0.4% and 38.2 ± 1.4%, respectively, that modified the corneal temperature to the values indicated in the figure. Full and shaded columns represent, respectively, the rate of tearing in animals exposed to a neutral (24.8 ° C) or hot (42.5 ° C) ambient temperature. The number of measurements of the successive columns is 35, 11, 23, 9 and 6 observations. ** p <0.01, *** p <0.001, Mann-Whitney test). b. Increase in the rate of tearing measured in wild animals (black columns) and TRPM8 - / - (gray columns), in response to the application for 30 s of a filter paper soaked in 1 μM capsaicin (n = 12 and 15) or 500 μM allyl isothiocyanate (AITC; n = 10 and 6). The first column shows the response to vehicle application (0.5% DMSO in saline) in wild animals (n = 6). Tear secretion measurements were performed 1 minutes after removing the paper with the drug. Statistically significant differences were found regarding the basal tear values, using the Wilcoxon test (* p <0.05, *** p <0.001). C. Average tear rate, expressed as the length of wet phenol red thread for 15 s, measured in the eyes of volunteers of both sexes at ambient temperatures of 18ºC, 25ºC and 43ºC, which produced a corneal temperature of 32.4 ± 0.4ºC, 34.2 ± 0.1ºC and 36.0 ± 0.2ºC, respectively (** p <0.01, ANOVA for repeated measurements).

Detailed description of the invention

The inventors of the present invention have discovered that, surprisingly, the TRPM8 receptor is involved in the control of tearing and that its activation using agonists thereof results in an increase in tearing. Specifically, the inventors of the present invention have described that cold fear receptors that innervate the cornea in mammals maintain a tonic firing activity at normal corneal temperature, and are exquisitely sensitive to small thermal variations of the ocular surface, such as those resulting of the evaporation of the precorneal tear film that occurs in the intervals between flickers or during exposure to dry environments. This marked sensitivity to cold is a consequence of the high expression of TRPM8 channels, which crucially determine a basal spontaneous activity and the increase in the trigger frequency in response to cooling. Likewise, the inventors have observed that elimination with genetic techniques of the TRPM8 channels reduces tear secretion by half in mice. Its partial silencing by heating the cornea also reduces tear secretion in humans.

Therefore, TRPM8 is a molecular candidate for the detection of moisture in cold thermoreceptor nerve fibers that innervate the exposed ocular surface in terrestrial animals. The data shown in this application (see example 1) indicate that in the thermosensitive corneal terminals, the TRPM8 is critical for the development of both the spontaneous activity and the one evoked by the cold that results in the production of tears. In this way, the stimulation of TRPM8 increases the stimulation of tear secretion by the cold-sensitive fibers through the activation of TRPM8.

The results obtained by the authors of the present invention open the door for the treatment of excessive tearing (epiphora) by using different agents that inhibit the TRPM8 receptor.

Second therapeutic use of the invention

In another aspect the invention relates to the use of a TRPM8 antagonist or combinations thereof to produce a medicament for the treatment or prevention of epiphora.

In another aspect the invention relates to a TRPM8 antagonist or combinations thereof for use in the treatment or prevention of epiphora.

In another aspect the invention relates to a method for the treatment of epiphora in a subject comprising the administration to said subject of a TRPM8 antagonist or combinations thereof.

The TRPM8 receptor or “Transient receptor potential cation channel subfamily M member 8”, also known as the cold and menthol receptor 1 (CMR1), is a protein that in humans is encoded by the TRPM8 gene. (Clapham DE, et al. 2005. Pharmacological Reviews 57 (4): 427–50).

TRPM8 is an ionic channel that, after being activated, allows the entry of sodium (Na +) and calcium (Ca2 +) ions into the cell so that a depolarization of the cell is generated that leads to the generation of a change in membrane potential .

The TRPM8 protein is expressed in sensory neurons and is activated by cold temperatures (below approximately 26 ° C), by chemical agents such as menthol and by voltage. TRPM8 is also expressed in the prostate, lungs, bladder without its function in these organs being known.

The human TRPM8 gene is located on chromosome 2 in the 2p37.1 region; and encodes for a protein of 1104 amino acids (NP_076985.4, SEQ ID NO: 1) encoded by the nucleotide sequence NM_024080.4 (SEQ IS NO: 2). The TRPM8 gene has six trans-membrane segments with the terms C and N terminal on the cytoplasmic side. Four subunits tetramerize to form active channels.

The term "TRPM8" as used in that description does not only refer to the human gene and protein but also to the orthologs of other species, such as dog (XP_543296.2), mouse (NP_599013.1), rat (NP_599198.2), etc.

The term "treat" or "treatment" refers to both therapeutic and prophylactic treatment or preventive measures, in which the object is to prevent or curb (reduce) an unwanted physiological change or disorder, such as dry eyes, vagina or mouth. For the purpose of this invention, beneficial or desired clinical results include, without limitation, relief of symptoms, reduction of the extent of the disease, stabilized pathological state (specifically not worsened), delay or brake of disease progression, improvement or palliation of the pathological state and remission (both partial and total), both detectable and non-detectable. Those subjects

Those in need of treatment include those subjects who already suffer from the condition or disorder, as well as those with a tendency to suffer the condition or disorder or those in which the condition or disorder is to be prevented.

The term "treatment method" means the administration to an individual in need of said treatment of a pharmaceutical composition comprising a TRPM8 agonist according to the invention. "Epiphora" is understood in the present invention by the existence of continuous tearing and excessive. This excessive production of tears may be caused by an external stimulus that acts as an irritant, for example cold exposure, contaminated environments, chemicals, foreign bodies or corneal ulcers. The epiphora can also be caused by processes that cause inflammation of the ocular surface, for example acute conjunctivitis.

On other occasions the cause is a defect in the tear drainage system, due to abnormal disposition of the eyelid (ectropion) or obstruction at the level of the nasolacrimal duct or tear sac. The obstruction of the tear system can be congenital if it is present from birth, in this case it is most often due to the imperforation of the nasolacrimal membrane. When it appears in the adult, it may be due to tear sac infection or dacryocystitis. Sometimes the origin of the epiphora is the paralysis of the facial nerve that causes weakness of the orbicularis muscle of the eyelids. Other causes of the epiphora are: Graves-Basedow disease, Ackerman syndrome, animal allergies, pollen etc, bacterial conjunctivitis and blepharitis.

Thus, in a particular embodiment, the epiphora is associated with a disease selected from: Graves-Basedow disease, corneal ulcers, Ackerman syndrome, allergies (animals, pollen etc), bacterial conjunctivitis, blepharitis, facial nerve paralysis , ectropion or obstruction at the level of the nasolacrimal duct or tear sac.

In a particular embodiment, said medicament decreases the stimulation of tear secretion by the cold-sensitive fibers by inactivating TRPM8.

In the present invention, "TRPM8 receptor antagonist" is understood as any molecule that specifically binds to the TRPM8 receptor and which, when bound, is capable of causing a decrease in the activity of the TRPM8 channel, that is to say that it decreases the flow of sodium and calcium. through the channel causing a re-polarization of the cell.

Suitable methods to detect if a given compound is a TRPM8 antagonist co-exist with those described to detect the activity of agonists of the TRPM8 receptor. There is a wide variety of assays available to detect the activity of TRPM8 receptor agonists, such as the whole-cell Patch-clamp electrophysiological experiments cited in the examples of the present invention (see Example 1), calcium microscopy methods (Bodding et al., 2007, Cell Calcium, 42, 618-628) and methods based on the detection of fluorescence on fluorometric imaging plate reader assay plate (Behrendt et al., 2004. J.Pharmacol. 141, 737 -745), among others.

Table 3 shows illustrative, non-limiting examples of TRPM8 antagonists that can be used in the present invention. Additionally, the compounds described in international patent application WO2010 / 021882 can be used.

Table 3

TRPM8 antagonists

Antagonist

Number

Antisense oligonucleotide specific for the sequence of the gene encoding TRPM8.

one

DNA enzyme specific for the sequence of TRPM8

2

Specific microRNA for the gene encoding TRPM8

3

Ribozyme specific for the gene sequence encoding TRPM-8.

4

Antagonist

Number

Specific interference RNA for the gene sequence encoding TRPM8. 5´-AGAAAUUCUCGAAUGUUCUUU-3´ (sense) (SEQ ID NO: 3) 3´- UUUCUUUAAGAGCUUACAAGA-5´ (antisense) (SEQ ID NO: 4) siRNAs for human TRPM-8. Described in Zhang L. et al. (2004. Cancer Research 64: 8365-8373). 5´-GAAAACACCCAACCTGGTCATTTC-3´ (direction) (SEQ ID NO: 5) 5´-CACCGTGCGGGGTAAAAAGCG-3´ (antisense) (SEQ ID NO: 6) siRNAS for the sequences of positions 894 and 2736 (exons 8 and 21) : 5′-UCUCUGAGCGCACUAUUCA (dTdT) -3 ′ (sense) (SEQ ID NO: 7) 5′-UAUCCGUCGGUCAUCUA (dTdT) -3 ′ (sense) (SEQ ID NO: 8) 5′-TCTCTGAGCGCACTATTCA (dTdT) -3 ′ (SEQ ID NO: 9) Position 894-912 of the sequence of TRPM8 (NM_024080.3), described in Thebault et al. (2005. Chemistry, 280: 39423-39435.)

5

Peptide capable of specifically binding TRPM8 and inhibiting its activity

6

Antibody capable of specifically binding TRPM8 and inhibiting the activity of said channel

7

NN O HNN Cl BCTC N- (4-tert-butyl-phenyl) -4- (3-chloropyridin-2-yl) tetrahydropyrazine-1 (2H) -carboxamide

8

NN O HN F F F N Cl CTPC (2R) -4- (3-chloro-2-pyridinyl) -2-methyl-N- [4- (trifluoromethyl) phenyl] -1-piperazine-carboxamide

9

N N S O HN N Cl thio-BCTC N- (4-tert-butyl-phenyl) -4- (3-chloropyridin-2-yl) tetrahydropyrazine-1 (2H) - (thio) carboxamide

10

Antagonist

Number

HN O HNN Br SB-452533 N- (2-bromophenyl) -N ’- (2- [ethyl (3-methylphenyl) amino] ethyl) urea

eleven

OR

N N O

12

O SKF96365 1- [2- (4-methoxyphenyl) -2- [3- (4-methoxyphenyl) propoxy] ethyl-1H-imidazole

N N Cl Cl O Cl Econazol 1- [2 - [(4-chlorophenyl) methoxy] -2- (2,4-dichlorophenyl) ethyl] -1H-imidazole

13

Antagonist

Number

NN Cl Clotrimazole 1 - [(2-chlorophenyl) diphenylmethyl] -1H-imidazole

14

N H O OH O ACA N- (p-amylcinnamoil) anthranilic acid

fifteen

O N S O H2N AMTB N- (3-aminopropyl) -2 - {[(3-methylphenyl) methyl] oxy} -N- (2-thienylmethyl) -benzamide

16

Antagonist

Number

N HN SHO HO Cl Capsazepine N- [2- (4-chlorophenyl) ethyl] -1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide

17

N N Phenanthroline

18

O HN MAD1d N - [(1R, 2S, 5R) -2-Isopropyl-5-methylcyclohexyl] biphenyl-4-carboxamide

19

N H O O MAD2e 4-tert-Butylphenyl (1R, 2S, 5R) -2-isopropyl-5-methylcyclohexylcarbamate

twenty

BCTC CTPC compound

Cold IC50 (μM) 0.68 ± 0.06a (CI) 0.54 ± 0.04a (EP) N / A IC50 menthol (μM) 0.47 ± 0.01b (CI) 0.34 ± 0.04b (EP) 0.143 ± 0.019f (EP) 0.131 ± 0.014f (EP)

uncle-BCTC

N / A 3.5 ± 1.1c (FL)

SB-452533 SKF96365

N / A 1.0 ± 0.2a (CI) 0.8 ± 0.1a (EP) 0.571 ± 0.077f (EP) 3 ± 1b (CI)

Econazol

0.42 ± 0.07d (CI) N / A

Clotrimazole

8 ± 1d (CI) 1.2e (EP)

ACA AMTB Capsazepine Phenanthrolina MAD1d

N / A N / A 12 ± 2d (CI) 100 ± 20a (CI) 180 ± 20a (EP) N / A 3.9g (FL) 6.23 ± 0.02h (FL) 18 ± 1c (FL) N / A 0.02 ± 0.002i (SF)

MAD2e N / A 0.1 ± 0.02i (SF)

Table 4. Summary of TRPM8 antagonist. Potential inhibitor of different cold native TRPM8 channel inhibitors or at 100 μM of menthol measured using calcium microscopy (IC), "fluorometric imaging plate reader assay" (FL), spectrofluorimeter (SF) or patch-clamp electrophysiology (EP). The data are from: a {Malkia, et al. 2007 J Physiol. 581 (Pt 1): 155-74.}; bmodified by {Madrid, 2006 J Neurosci. 26 (48): 12512-25.}; c {Behrendt, et al. 2004 Br J Pharmacol. 141 (4): 737-45}; d {Malkia, et al. 2009 Mol Pain. 5:62.}; e {Meseguer, et al. 2008. J Neurosci. 28 (3): 576-86.}; f {Weil, 2005 Mol Pharmacol. 68 (2): 518

27.}; g {Kraft, et al. 2006 Br J Pharmacol. 148 (3): 264-73}, h {Lashinger, et al. 2008 Am J Physiol Renal Physiol. 295 (3): F803-10.}, I {Ortar, et al. 2010 Bioorg Med Chem Lett. 20 (9): 2729-32}. Whole cell patch-clamp data is given as +80 mVa, b, +50 mVe, or −70 mVf. Note: AMTB results were obtained with icilin instead of menthol. The results for MAD1d and MAD2e were obtained with 20 μM menthol at 22 ° C.

Antisense oligonucleotides

In a particular embodiment, a specific antisense oligonucleotide is used to inhibit the expression of the gene encoding TRPM8, for example, by inhibiting the transcription and / or translation of the nucleic acid encoding TRPM8 (whose activity is desired to inhibit). Antisense oligonucleotides can be attached to their potential target by conventional base complementarity, or, for example, in the case of binding to double stranded DNA, through specific interactions in the major groove of the double helix. For use in the present invention, a construct comprising an antisense oligonucleotide can be distributed, for example, as an expression plasmid that, when transcribed in the cell, produces RNA that is complementary to at least a single part of the cellular mRNA. which encodes TRPM8. Alternatively, the antisense construct is an oligonucleotide probe that is generated ex vivo and that, when introduced into the cell, produces inhibition of gene expression by hybridizing with mRNA and / or genomic sequences of the target nucleic acid. Such oligonucleotide probes are preferably modified oligonucleotides, which are resistant to endogenous nucleases, for example, exonucleases and / or endonucleases, and which are therefore stable in vivo. Illustrative nucleic acid molecules for use as antisense oligonucleotides include phosphoramidate, phosphothionate and methylphosphonate DNA analogs (see, for example, US5176996, US5264564 and US5256775). Additionally, for a review of the general approaches to construct oligomers useful in antisense therapy see, for example, Van der Krol et al., BioTechniques 6: 958-976, 1988; and Stein et al., Cancer Res 48: 2659-2668, 1988.

With respect to the antisense oligonucleotide, oligodeoxyribonucleotide regions derived from the translation initiation site are preferred, for example, between -10 and +10 of the target gene. The antisense approaches involve the design of oligonucleotides (either DNA or RNA) complementary to the mRNA encoding the target polypeptide. Antisense oligonucleotides will bind to mRNA transcripts and prevent translation.

Complementary oligonucleotides could also be used either to the 5 ’or 3’ untranslated, non-coding regions of a gene in an antisense approach to inhibit the translation of that mRNA. Oligonucleotides complementary to the 5 ’untranslated region of the mRNA should include the complement of the AUG initiation codon. Oligonucleotides complementary to mRNA coding regions are less effective translation inhibitors but could also be used according to the invention. If they are designed to hybridize with the 5 ', 3' or coding region of the mRNA, the antisense nucleic acids should be at least 6 nucleotides in length and preferably be less than about 100 and more preferably less than about 50, 25, 17 or 10 nucleotides in length.

Preferably, in vitro studies should be performed to quantify the ability of antisense oligonucleotides to inhibit gene expression. Advantageously, said studies will use controls that distinguish between antisense gene inhibition and non-specific biological effects of oligonucleotides. It is also preferred that these studies compare the levels of the target RNA or protein with those of an internal control of RNA or protein. The results obtained using the antisense oligonucleotides can be compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide be approximately the same length as the oligonucleotide to be tested and that the oligonucleotide sequence differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.

The antisense oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single chain or double chain. The oligonucleotide can be modified in the base, in the sugar or in the phosphate skeleton, for example, to improve the stability of the molecule, its hybridization capacity etc. The oligonucleotide may include other bound groups, such as peptides (for example, to direct them to host cell receptors) or agents to facilitate transport across the cell membrane (Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556, 1989; Lemaitre et al., Proc. Natl. Acad. Sci. 84: 648-652, 1987; WO88 / 09810) or the blood brain barrier (WO89 / 10134), intercalating agents (Zon, Pharm. Res. 1988. 5: 539549). For this purpose, the oligonucleotide may be conjugated to another molecule, for example, a peptide, a transport agent, a hybridization triggered cutting agent, etc.

In some cases, it may be difficult to achieve intracellular concentrations of the antisense oligonucleotide sufficient to suppress the translation of endogenous mRNAs. Thus, a preferred approach uses a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter.

Alternatively, the expression of the target gene can be reduced by directing deoxyribonucleotide sequences complementary to the regulatory region of the gene (i.e., the promoter and / or enhancers) to form triple helix structures that prevent transcription of the gene in the target cells in the body (Helene et al, Anticancer Drug Des. 6 (6): 569-84, 1991).

In certain embodiments, the antisense oligonucleotides are antisense morpholinos.

DNA Enzymes

In another particular embodiment, a specific DNA enzyme is used to inhibit the expression of the gene encoding TRPM8. DNA enzymes incorporate some of the mechanistic characteristics of both antisense oligonucleotide technologies and ribozyme technologies. DNA enzymes are designed to recognize a particular nucleic acid target sequence (in this case, the sequence encoding TRPM8), similar to the antisense oligonucleotide; however, similar to ribozyme, they are catalytic and specifically cut the target nucleic acid.

Ribozymes

In another particular embodiment, a specific ribozyme designed to catalytically cut transcripts of a target mRNA is used to prevent the translation of mRNAs encoding TRPM8 whose activity is to be inhibited. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cut of RNA [for a review see Rossi, 1994. Current Biology 4: 469-471]. The sequence of ribozyme molecules preferably includes one or more sequences complementary to the target mRNA, and the well-known sequence responsible for mRNA cutting or a functionally equivalent sequence [see, for example, US5093246].

Ribozymes used in the present invention include hammerhead ribozymes, endoribonuclease RNAs, etc. [Zaug et al., 1984. Science 224: 574-578].

Ribozymes may be composed of modified oligonucleotides (for example, to improve stability, targeting, etc.) and should be distributed to cells expressing the target gene in vivo. A preferred method of distribution involves using a DNA construct that "encodes" the ribozyme under the control of a strong constitutive promoter of pol III or pol II, so that the transfected cells will produce sufficient amounts of the ribozyme to destroy the endogenous target messengers. and inhibit translation. Since ribozymes, contrary to other antisense molecules, are catalytic, a lower intracellular concentration is required to be effective.

MicroRNAs

In another particular embodiment, a specific microRNA is used for the sequence encoding TRPM8. As is known, a microRNA (miRNA or miRNA) is a single-stranded RNA, between 21 and 25 nucleotides in length, and that has the ability to regulate the expression of other genes through various processes, using it the ribointerference path.

RNAi

In another particular embodiment, an interference RNA (RNAi) is used, such as a small interference RNA (siRNA) specific for the sequence encoding TRPM8 whose activity is to be inhibited.

Small interference RNAs or siRNAs (siRNAs) are agents capable of inhibiting the expression of a target gene by RNA interference. An siRNA can be chemically synthesized, or, alternatively, it can be obtained by in vitro transcription or it can be synthesized in vivo in the target cell. Typically, siRNAs consist of a double strand of RNA between 15 and 40 nucleotides in length, which may contain a 3 ’and / or 5’ protruding region of 1 to 6 nucleotides. The length of the protuberant region is independent of the total length of the siRNA molecule. SiRNAs act by degradation or post-transcriptional silencing of the target messenger.

The siRNAs can be called shRNA (short hairpin RNA), characterized in that the antiparallel chains that form the siRNA are connected by a loop or hairpin region. The shRNAs may be encoded by plasmids or viruses, particularly retroviruses, and be under the control of promoters such as the U6 promoter of RNA polymerase III.

In a particular embodiment, the siRNAs that can be used in the present invention are substantially homologous to the mRNA of the gene encoding TRPM8 or to the genomic sequence encoding said protein. By "substantially homologous" is meant that they have a sequence that is sufficiently complementary or similar to the target mRNA, so that the siRNA is capable of causing the degradation thereof by RNA interference. Suitable siRNAs to cause such interference include siRNAs formed by RNA, as well as siRNAs containing different chemical modifications such as:

-

SiRNA in which the bonds between nucleotides are different from those that appear in nature, such as phosphorothioate bonds;

-

RNA chain conjugates with a functional reagent, such as a fluorophore;

-

modifications of the ends of the RNA chains, in particular the 3 ′ end by modification with different hydroxyl functional groups in 2 ’position;

-

nucleotides with modified sugars such as O-alkylated moieties in 2 ’position such as 2’-O-methylribose or 2’-O-fluorosibose;

-

nucleotides with modified bases such as halogenated bases (for example 5-bromouracil and 5-iodouracil), alkylated bases (for example 7-methylguanosine).

The siRNAs and siRNAs that can be used in the present invention can be obtained using a series of techniques known to the person skilled in the art. The region of the nucleotide sequence encoding TRPM8 that is taken as the basis for designing the siRNAs is not limiting and may contain a region of the coding sequence (between the initiation codon and the termination codon) or, alternatively, may contain sequences of the 5 'or 3' untranslated region, preferably between 25 and 50 nucleotides in length and in any position in a 3 'sense position with respect to the initiation codon. One way to design an siRNA involves the identification of the AA (N19) TT motifs, where N can be any nucleotide in the sequence encoding TRPM8, and the selection of those with a high G / C content. If this motif is not found, it is possible to identify the motif NA (N21), where N can be any nucleotide.

In a particular embodiment, the TRPM8 inhibitor is a specific RNAi for TRPM8, such as a specific RNAi selected from the group consisting of the specific RNAi shown in Table 3 (5) [SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9].

Inhibitor peptides

In another particular embodiment, a TRPM8 inhibitor peptide is used to prevent said protein from exerting any of its functions, in particular, the passage of sodium and calcium ions through the channel.

The term "inhibitor peptide", as used herein, refers to those peptides capable of binding to TRPM8 and inhibiting its activity as explained above, that is, preventing sodium and calcium ions from passing through the channel. of TRPM8.

       Inhibitory antibodies

In another particular embodiment, a TRPM8 inhibitor antibody is used to prevent said protein from exerting any of its functions, in particular, that sodium and calcium ions pass through the channel. Thus, an "inhibitor" antibody of TRPM8 as used herein refers to an antibody that is capable of binding TRPM8 specifically and inhibiting the passage of sodium and calcium ions through the channel. Antibodies can be prepared using any of the methods that are known to the person skilled in the art. Once antibodies with TRPM8 binding capacity have been identified, those capable of inhibiting the activity of this protein will be selected using an inhibitor agent identification assay [see, for example, Metz; S. et al. 2008. J.Biol.Chem. 283: 5985-5995].

In the present invention, the term "antibody" has to be interpreted broadly and includes polyclonal, monoclonal, multispecific antibodies and fragments thereof (F (ab ') 2, Fab), provided they are capable of recognizing the antigen of Of interest, it is capable of specifically binding to the TRPM8 receptor or to the extracellular domain of said receptor. Examples of antibodies that may be employed in the context of the present invention are, for example and without limitation, polyclonal antibodies, monoclonal antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, etc.

Polyclonal antibodies are originally heterogeneous mixtures of antibody molecules produced in the serum of animals that have been immunized with an antigen. They also include monospecific polyclonal antibodies obtained from heterogeneous mixtures, for example, by column chromatography with peptides of a single epitope of the antigen of interest.

A monoclonal antibody is a homogeneous population of antibodies specific for a single epitope of the antigen. These monoclonal antibodies can be prepared by conventional techniques already described, for example in Köhler and Milstein [Nature, 1975; 256: 495-397] or Harlow and Lane ["Using Antibodies. A Laboratory Manual ”by E. Harlow and D. Lane, Editor: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; 1998 (ISBN 978-0879695439)].

A chimeric antibody is a monoclonal antibody constructed by cloning or recombination of antibodies from different animal species. In a typical but non-limiting configuration of the invention, the chimeric antibody includes a part of a monoclonal antibody, generally the variable region (Fv) that includes sites for recognition and binding to the antigen, and the other part corresponding to a human antibody, generally the part that includes the constant region and the adjacent constant.

A fully human antibody is an antibody or antibodies that have been produced in transgenic animals with a human immune system or by in vitro immunization of human immune cells (including both genetic and traditional immunization with or without adjuvants and pure or not antigen; or by any method of exposure of the antigen to the immune system) or by native / synthetic libraries produced from human immune cells. These antibodies can be obtained and selected from transgenic animals (eg mice) in which human immunoglobulin genes have been cloned and which are immunized with the target antigen (in the present invention with the TRPM8 receptor). These antibodies can be obtained by selection of variable single chain (scFv) or human antigen-binding (Fab) regions presented in phage display libraries and subsequent cloning and grafting in a human antibody or by any other production method and presentation (display) known by the person skilled in the art, of the libraries generated by cloning the variable regions of both chains and subsequent combination / mutation of these to generate antibody libraries.

A humanized antibody is a monoclonal antibody constructed by cloning and grafting the hypervariable complementarity determining regions (CDR) of a murine monoclonal antibody into a human antibody replacing its own hypervariable CDR regions.

Chemical compounds

In another particular embodiment, a chemical compound is used that decreases the activity of TRPM8 when contacted with said protein. Illustrative, non-limiting examples of said chemical compounds include the compounds mentioned in Table 3 (8 to 20) and include BCTC, CTPC, thio-BCTC, SB-452533, SKF96365, Econazol, Clotrimazole, ACA, AMTB, Capsazepine, Phenanthroline, MAD1d and MAD2e.

In a particular embodiment, the TRPM8 antagonist used in the second use of the invention is an antagonist selected from: antisense oligonucleotide specific for the gene sequence encoding TRPM8, DNA enzyme specific for the TRPM8 sequence, gene specific microRNA encoding TRPM8, ribozyme specific for the gene sequence encoding TRPM-8, interference RNA specific for the gene sequence encoding TRPM8, peptide capable of specifically binding to TRPM8 and inhibiting its activity, antibody capable of specifically binding to TRPM8 and inhibit the activity of said channel, BCTC (N- (4-tert-butyl-phenyl) -4- (3-chloropyridin-2-yl) tetrahydropyrazine-1 (2H) -carboxamide), CTPC ((2R) -4- (3-Chloro-2-pyridinyl) -2-methylN- [4- (trifluoromethyl) phenyl] -1-piperazine-carboxamide), thio-BCTC (N- (4-tert-butyl-phenyl) -4- (3 -chloropyridin-2-yl) tetrahydropyrazine-1 (2H) - (thio) carboxamide), SB-452533 (N- (2-bromophenyl) -N '- (2- [ethyl (3-methylfe nil) amino] ethyl) urea), SKF96365 (1- [2- (4-methoxyphenyl) -2- [3- (4-methoxyphenyl) propoxy] ethyl-1H-imidazole), Econazol (1- [2 - [( 4-chlorophenyl) methoxy] 2- (2,4-dichlorophenyl) ethyl] -1H-imidazole), Clotrimazole (1 - [(2-chlorophenyl) diphenylmethyl] -1H-imidazole), ACA (N- (pamilcinnamoil) anthranilic acid ), AMTB (N- (3-aminopropyl) -2 - {[(3-methylphenyl) methyl] oxy} -N- (2-thienylmethyl) -benzamide), Capsazepine (N- [2- (4-chlorophenyl) ethyl ] -1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide), Phenanthroline, MAD1d (N - [(1R, 2S, 5R) -2-Isopropyl-5- methylcyclohexyl] biphenyl-4-carboxamide), MAD2e (4-tert-Butylphenyl (1R, 2S, 5R) -2-isopropyl-5-methylcyclohexylcarbamate).

In another particular embodiment, the TRPM8 antagonist is a specific antagonist for TRPM8.

Second composition of the invention

In another aspect the invention relates to a composition (hereinafter second composition of the invention) comprising at least one TRPM8 antagonist and at least one drug useful for the treatment of the epiphora and, if desired, a pharmaceutically acceptable carrier.

The term "TRPM8 antagonist" has been described above and is used in the same manner in relation to the second composition of the invention.

As used herein, "pharmaceutically acceptable carrier" includes additives, such as preservatives, excipients, fillers, wetting agents, binders, disintegrants, buffers that may be present in the compositions of the invention. Such additives may be, for example, magnesium and calcium carbonates, carboxymethyl cellulose, starches, sugars, gums, magnesium or calcium stearate, coloring or flavoring agents. There is a wide variety of pharmaceutically acceptable additives for pharmaceutical dosage forms and the selection of appropriate additives is a routine matter for those skilled in the art of pharmaceutical formulation.

Administration of the composition of the invention can be carried out by different routes, for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, intradermally, intranasally or intrabronchially, and can be administered locally or systemically or directly to the target site. A review of the different routes of administration of active ingredients, of the excipients to be used and their manufacturing procedures can be found in the Treaty of Farmacia Galenica, C. Faullí i Trillo, Luzán 5, S.A. of Editions, 1993 and in Remington’s Pharmaceutical Sciencies (A.R. Gennaro, Ed.), 20th edition, Williams & Wilkins PA, USA (2000).

The dosage regimen will be determined by the doctor and the clinical factors. As is well known in medicine, the dosages depend on many factors that include the physical characteristics of the patient (age, size, sex), the route of administration used, the severity of the disease, the particular compound employed and the pharmacokinetic properties of the individual.

Preferred excipients or carriers for use in the present invention include sugars, starches, celluloses, gums and proteins. In a particular embodiment, the pharmaceutical composition of the invention will be formulated in a solid dosage pharmaceutical form (for example tablets, capsules, dragees, granules, suppositories, sterile crystalline or amorphous solids that can be reconstituted to provide liquid forms etc.), liquid (for example solutions, suspensions, emulsions, elixirs, lotions, ointments etc.) or semi-solid (gels, ointments, creams and the like). The pharmaceutical compositions of the invention can be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intrarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteric, topical, sublingual or rectal. A review of the various forms of administration of active ingredients, of the excipients to be used and their manufacturing procedures can be found in the Treaty of Farmacia Galenica, C. Faulí i Trillo, Luzán 5, S.A. of Editions, 1993 and in Remington´s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 20th edition, Williams & Wilkins PA, USA (2000).

The term "epiphora" has been described in detail previously and is used in the same way in the context of the present composition, thus indicating the epiphora that appears as a symptom in different types of disorders such as Graves-Basedow disease, ulcers in the cornea, Ackerman syndrome, allergies (animals, pollen etc), bacterial conjunctivitis, blepharitis, facial nerve paralysis, ectropion or obstruction at the level of the nasolacrimal duct or tear sac.

In a particular embodiment, the TRPM8 antagonist used in the second composition of the invention is an antagonist selected from: antisense oligonucleotide specific for the sequence of the gene encoding TRPM8, DNA enzyme specific for the sequence of TRPM8, microRNA specific for the gene encoding TRPM8, ribozyme specific for the gene sequence encoding TRPM-8, interference RNA specific for the gene sequence encoding TRPM8, peptide capable of specifically binding to TRPM8 and inhibiting its activity, antibody capable of specifically binding to TRPM8 and inhibit the activity of said channel, BCTC (N- (4-tert-butyl-phenyl) -4- (3-chloropyridin-2-yl) tetrahydropyrazine-1 (2H) -carboxamide), CTPC ((2R) - 4- (3-Chloro-2-pyridinyl) -2-methyl-N- [4- (trifluoromethyl) phenyl] -1-piperazine-carboxamide), thio-BCTC (N- (4-tert-butyl-phenyl) -4- (3-Chloropyridin-2yl) tetrahydropyrazine-1 (2H) - (thio) carboxamide), SB-452533 (N- (2-bromophenyl) -N '- (2- [ethyl ( 3-methylphenyl) amino] ethyl) urea), SKF96365 (1- [2- (4-methoxyphenyl) -2- [3- (4-methoxyphenyl) propoxy] ethyl-1H-imidazole), Econazol (1- [2- [(4-chlorophenyl) methoxy] 2- (2,4-dichlorophenyl) ethyl] -1H-imidazole), Clotrimazole (1 - [(2-chlorophenyl) diphenylmethyl] -1H-imidazole), ACA (N- (pamilcinnamoil acid) ) anthranilic), AMTB (N- (3-aminopropyl) -2 - {[(3-methylphenyl) methyl] oxy} -N- (2-thienylmethyl) -benzamide), Capsazepine (N- [2- (4-chlorophenyl) ) ethyl] -1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide), Fenantroline, MAD1d (N - [(1R, 2S, 5R) -2-Isopropyl- 5-methylcyclohexyl] biphenyl-4-carboxamide), MAD2e (4-tert-Butylphenyl (1R, 2S, 5R) -2-isopropyl-5-methylcyclohexylcarbamate).

Drugs useful for the treatment of epiphora are known to those skilled in the art such as antibiotics, as well as the compositions described in documents CN101612199A, WO08066644, RU2305517C, CN1775261A, CN1775263A, CN1565501A, CN1199617A and JP57179121A.

In another aspect, the invention relates to the use of a second composition according to the invention for the preparation of a medicament for the treatment of epiphora. In another aspect, the invention relates to a second composition according to the invention for use in the treatment of epiphora. In a third aspect, the invention relates to a method for the treatment of epiphora in a subject comprising the administration to said subject of a second composition of the invention.

The following Example illustrates the invention and should not be considered as limiting the scope thereof.

EXAMPLE

To functionally define the thermal sensitivity and coding capacity of the thermosensitive nerve terminals, the nerve impulse activity (NTI) in the cornea of wild mice was recorded in vitro (Brock, JA, et al., 1998. J. Physiol 512 : 211-217). Thermoreceptor nerve endings were identified by their spontaneous firing of nerve impulses at 34 ° C whose frequency increased with cooling and was silenced upon reheating, as well as by their response to the application of menthol (Figure 1A) (Schafer, K., et al., 1986. J.Gen. Physiol 88: 757-776). 72% of cold-sensitive endings also responded to heat pulses (paradoxical response) (Long, R. R. 1977. J. Neurophysiol. 40: 489-502). (Figure 1A) and 100nM capsaicin (65%).

The spontaneous trigger frequency of cold-sensitive nerve endings at baseline temperature (34 ° C) was 4.0 ± 0.4 impulses / s (n = 55). The cold terminals fired spontaneously, as many unique potentials as, occasionally, in bursts of two or more action potentials at regular intervals. The cold pulses from 34ºC to 20ºC evoked a pulse discharge that progressively increased with the reduction of the temperature, reaching a peak frequency and decreasing the firing frequency or silencing later, upon reaching the lower temperature values (Figures 1 A and 1 B). The time course of the average trip frequency and the temperature decrease during the cooling ramps, recorded in a total of 55 cold-sensitive terminals, is shown in Figure 1 B. The temperature thresholds required to evoke an increase in the Trigger frequency during the cooling ramps were less than 2ºC (mean value: -1.5 ± 0.2ºC, Figure 1 C). Around 30% of the thermosensitive units significantly increased their firing frequency with temperature decreases of 1ºC or even less. The average value of the peak frequency during the cooling ramp was 37.6 ± 1.8 imp / s (n = 55).

The thermal sensitivity of the corneal cold thermoreceptors, expressed as the change in the trigger frequency for each centigrade degree of temperature decrease during a continuous cooling ramp, varied between the units registered. The average slope was 6.3 ± 1.0 impulses / s / ºC (n = 55) (red line in Figure 1D). The capacity of the thermosensitive terminals to encode sustained temperature values was also assessed, carrying out temperature steps of -2.5ºC, between 34ºC and 24ºC (Figure 1 E). The initial decrease in temperature produced a transient increase in the trigger frequency, which was then adapted to a new stable value. Cold sensitive corneal terminals encoded static temperature over a wide temperature range. Responses to low temperatures were characterized by an increase in the trigger frequency; a marked change in the firing pattern, which happened to be in bursts in most cases (Figure 1 E; Table 1), and a transitory modification of the shape of the registered potentials (Brock, JA, et al., 1998. J. Physiol 512: 211-217). Figure 1F summarizes the average static and dynamic trigger frequency changes of 10 thermoreceptors and shows the remarkable sensitivity of these corneal sensory receptors to small temperature changes.

Menthol (50μM), an activator of well-known cold sensitive afferent sensory endings, (Schafer, K., et al., 1986. J.Gen. Physiol. 88: 757-776). increased spontaneous activity in 98% of the studied terminations (Figure 1 A) (from 3.4 ± 0.3 imp / s to 18.4 ± 1.4 imp / s, n = 44; p <0.001, peer test). The percentage of terminals that had a burst pattern in bursts at 34 ° C was almost ten times higher during menthol infusion (44%) than before treatment (5%). The menthol sensitized the response of the terminals to cooling: in the presence of menthol, the cooling ramps evoked a greater increase in the firing frequency, reaching the peak frequency at higher temperatures (control: 26.7 ± 0.5 ºC; menthol: 28.6 ± 0.5 ºC, n = 44; p <0.001, peer test), and also silenced at higher temperatures (control: 23.8 ± 0.4 ºC; menthol: 25.2 ± 0.4 ºC, n = 44; p <0.05, peer test) . BCTC is a potent reversible blocker of TRPM8 ion channels in vitro (Madrid, R. et al. 2006. J Neurosci. 26: 12512-12525, Jordt, S. E. et al. 2004. Nature 427: 260265). The effect of a saturating concentration of BCTC (10μM) on sensory endings was studied. The spontaneous activity and the increases in firing frequency evoked by cold and menthol gradually decreased and were almost completely silenced after perfusion with BCTC for 90 minutes. These effects of BCTC were also observed in TRPA1 (- / -) mice. The decrease in activity was partially reversible after removing the BCTC and was not observed in the absence of the drug, suggesting that the activity of the thermosensitive terminals depends primarily on TRPM8 channels. Unlike the stimulating effect of menthol, the specific agonist TRPA1, allyl-isothiocyanate (AITC, 100μM) (Bandell, M. et al. 2004. Neuron 41: 849-857) tested in 28 cold terminals, did not modify the spontaneous activity nor evoked by the cooling in 24 of them. Only 4 sensory units showed a significant increase in firing frequency during AITC perfusion at baseline temperature (data not shown), suggesting the absence or reduced expression of TRPA1 channels in most cold-sensitive corneal terminals. Together, these results support the existence, previously suggested, of an overlap in the expression of TRPM8 and TRPA1 channels (Story, G. M. et al. 2003. Cell 112: 819-829).

The IKD is a voltage-activated potassium current, shaker type, which acts as a brake on excitability against the activation of sensory neurons induced by temperature decreases, contributing to determine the thermal threshold of sensory thermoreceptors (Madrid, R ., et al. 2009. J.Neurosci. 29: 3120-3131). The IKD 4-amino-pyridine current blocker (4-AP, 100μM) was used to explore whether this current affected the thermal sensitivity of corneal cold nerve endings. In three of the nine units studied, spontaneous activity increased significantly after perfusion with 4-AP, but no parallel changes were found in the cold threshold of these terminals. In contrast, the peak frequency during the cooling response decreased by 39 ± 6% in all terminals treated with 4-AP, compared to the control values (p <0.001, n = 9). Globally, these data suggest that the thermal threshold of the cold nerve endings of the mouse cornea is not determined, at least significantly, by the Kv1 shaker potassium channels, and that these terminals belong to cold thermoreceptor neurons of low threshold (Belmonte, C., et al. 2009. Exp Brain Res. 196: 13-30).

Next, the presence, morphology and density of nerve fibers that presumably express the cold sensitive channel TRPM8 in the mouse cornea were analyzed, using genetically modified mice to express the green fluorescent protein (EYFP) under sequence control TRMP8 regulators (TRPM8-EYFP mice) (Figure 2). Nerve fibers presumably positive for TRPM8 were distributed homogeneously throughout the cornea. The stromal nerve trunks extended along the outer third of the cornea (Figure 2 A-C). Approximately one in nine stromal axons was positive for TRPM8 (Figure 2C). Once the stromal nerve plexus branches penetrated Bowman's foil, located between the stroma and the corneal epithelium, they branched into several subbasal nerve fibers (ribbon fibers) that ran parallel to each other toward the center of the cornea, immediately below the basal cells of the epithelium (Figure 2 DH). Some subbasal fibers positive for TRPM8 already gave nerve endings in the subbasal plexus itself (Figure 2H, arrowhead). However, most of them gave collaterals that ascended to the most superficial layers of the corneal epithelium, ending as asymmetric clusters (Figure 2 I-K). Unlike non-fluorescent nerve fibers, which presumably were polymodal and mechanonocytoptor fibers, positive axons for TRPM8 branched sparingly in the epithelium, to end up in the more superficial epithelial layers as a reduced number of brush terminals (Figure 2 JL) . Both subbasal tape fibers and intraepithelial nerve endings showed, in all cases, an arrosary morphology (Figure 2 H-K). Using double immunofluorescence staining against green fluorescent protein (GFP) and against neurofilaments, it was determined that the supposed cold thermoreceptor fibers represented about 12% of the total number of subbasal fibers and approximately 10% of superficial intraepithelial nerve endings ( see Methods section; Figure 2 C, F, K, L).

The contribution of TRPM8 channels to cold sensitivity was then determined, exploring nerve impulse discharge evoked by cooling in TRPM8 (- / -) - EGFP ki mutant mice (Dhaka, A. et al. 2007. Neuron 54 : 371-378). An attempt was made to record the electrical activity of the thermosensitive cold terminations in 12 TRPM8 (- / -) corneas. However, in the hundreds of registration attempts, in which the entire corneal surface was repeatedly explored with the registration pipette, only spontaneous activity was recorded in 14 nerve endings (Figure 3A, dotted line). In these cases, the spontaneous activity recorded was low frequency (0.6 ± 0.2 imp / s, n = 14) and only one of the TRPM8 terminals (- / -) obtained an increase in the trigger frequency during the ramp of cooling, with a temperature threshold of 28.4ºC and a peak frequency during the 3 imp / s ramp obtained at 24.7ºC. Menthol is a potent activator of TRPM8 channels (McKemy, D. D., et al. 2002. Nature 416: 52-58). The 50μM menthol infusion did not induce any increase in the trigger frequency, either at the baseline temperature of 34 ° C or during the cooling ramps (n = 6) (Figure 3 A, punctuated line). This result is consistent with the existence of a dependence between spontaneous and cold-evoked activity, and the expression of TRPM8 in cold-sensitive terminals. Capsaicin (100nM), tested in 6 of the active units registered in TRPM8 corneas (- / -), evoked the firing of a few pulses in two terminals (5 and 8 pulses, respectively), and a vigorous discharge with a duration of more than 30 s in another one of them.

In contrast to the great inhibition of spontaneous and cold-evoked activity, observed in TRPM8 (- / -) mice, the corneal nerve endings of TRPA1 (- / -) mice (Kwan, KY et al. 2006. Neuron. 50: 277-289) showed a trigger frequency and a cold response similar to those of wild mice (Figure 3B). These results confirm that TRPA1 channels are not critical molecular determinants for cooling sensitivity of cold-sensitive corneal nerve endings. The results are also consistent with previous results of our group, obtained in sensory trigeminal neurons in culture Madrid, R., et al. (J.Neurosci., 2009, 29: 3120-3131). To exclude the possibility that thermosensitive nerve endings were absent in the corneas of TRPM8 (- / -) mice, or had an altered morphology, they were stained with anti-GFP antibodies, in order to visualize the morphology of nerve fibers expressing truncated TRPM8 channels (Dhaka, A., et al. 2008. J.Neurosci. 28: 566-575. The distribution of stained fibers in different regions of the corneal circumference was variable for an animal TRPM8 (- / - ) to another However, the general morphology of the sub-basal tape fibers and the clusters of intraepithelial nerve endings was in all cases similar to those of the fibers of the TRPM8-EYFP mice, and no significant differences were found. in the total density of nerve endings, which excludes the possibility that the absence of activity and cold response observed in electrophysiology experiments was due to the fact that the recording pipette It did not reach the endings.

Additionally, the activity evoked by the cold in the corneas of TRMP8 (+/-) mice was explored, in which presumably the response to cooling should be reduced due to a lower expression of TRPM8 channels.30 In these animals, 30% of the terminals had spontaneous activity but lacked response to cold and menthol, unlike wild animals, in which it was exceptional (7%) to find units without cold sensitivity that presented spontaneous activity. Among active nerve endings, the mean spontaneous activity at 34 ° C was significantly lower than in the TRPM8 (+ / +) animals (2.2 ± 0.4 imp / s vs. 4.4 ± 0.9 imp / s; n = 11; p = 0.025, Mann-Whitney test), while the temperature threshold measured during the cold ramps, that is, the temperature necessary to evoke an increase in the trigger frequency, was set at lower value temperatures (30.6 ± 0.5ºC vs. 32.7 ± 0.4 ° C; p = 0.004, Mann-Whitney test), and the peak frequency during cooling was also significantly lower in the corneas of TRMP8 mice (+/-) than in those of wild mice (19.4 ± 3.4 imp / s vs. 33.8 ± 3.5 imp / s; p = 0.008, Student's t) (Figure 3 A, continuous stroke). In TRMP8 (+/-) mice, menthol (50μM) increased the trigger frequency at baseline, cold threshold and peak frequency, although significantly less than in TRMP8 (+ / +) animals (Figure 3 A , continuous stroke).

Next, it was stated that if tear production is associated with the neural activity of cold-tempering fibers, the basal tear flow should be reduced in TRPM8 (- / -) animals, in which spontaneous activity is practically absent. . Figure 4 A shows that this is the case. The volume of tear fluid, expressed as the length of thread impregnated with phenol red that changes color when moistened with tears over a period of 2 minutes, measured in TRPM8 mice (- / -) in basal conditions (1.5 ± 0.2 mm , n = 23) was significantly lower than that measured in wild mice (3.7 ± 0.4 mm, n = 35; p <0.001). In contrast, the basal lacrimation of the TRPA1 (- / -) mice was not significantly different from the average in wild animals (Figure 4A, black columns). On the other hand, the topical application of capsaicin (1μM) and AITC (500μM), two agents that activate the TRPV1 and TRPA1 channels in polymodal nociceptors, significantly increased tear flow in both eyes of TRPM8 (- / -) and wild mice , while the vehicle of both drugs had no significant effect (Figure 4B).

Next, we tried to confirm if the relationship between corneal temperature and basal tear rate was also present in humans, for which the tear rate was measured in 11 young volunteers (28.9 ± 1.8 years) exposed for 10 minutes , in different sessions, at ambient temperatures of 18ºC, 25ºC and 43ºC with a constant humidity of 32%. Exposure to these temperatures brought the corneal surface temperature to values of 32.4 ± 0.4ºC, 34.2 ± 0.1ºC and 36.0 ± 0.2ºC, respectively. The temperature values measured on the corneal surface were significantly different from each other (p <0.001, ANOVA for repeated measurements). On the contrary, only with the ambient temperature of 43ºC the tear rate was significantly reduced (17.1 ± 1.4 mm, compared to 22.8 ± 1.2 mm at 25ºC or 23.2 ± 2.3 mm at 18ºC; p = 0.006) (Figure 4C). When subjects were exposed to 43 ° C (corneal temperature 36.0 ± 0.1 ° C) with an ambient humidity of 62.5%, the reduction in the rate of tearing was the same (17.6 ± 2.4 mm) as in the case of humidity of 32%. These experiments were shaken in wild mice and anesthetized TRPM8 (- / -), placing them in the same environmental conditions used in human experiments, that is, exposing them to high and neutral ambient temperatures under conditions of constant ambient humidity. In wild mice, when the corneal temperature rose to 36.4 ± 0.2 ° C (n = 11), tearing decreased to 1.8 ± 0.4 mm (Figure 4 A), while in a neutral temperature environment, the corneal temperature was 27.2 ± 0.1 (n = 6) and the tear rate was 4.6 ± 0.8 mm (p = 0.017, Mann-Whitney test). In contrast, in TRPM8 (- / -) mice exposed to similar conditions, the tear flow rate was not significantly modified by varying the temperature of the corneal surface (Figure 4A).

Translation of the English terms present in the Sequence List

The term "Artificial Sequence" present in the Sequence List means "Artificial Sequence".

Claims (11)

one.

Use of a TRPM8 antagonist for the preparation of a drug for the treatment of epiphora.

2.

Use according to claim 1, wherein the epiphora is associated with a disease selected from: Graves-Basedow disease, corneal ulcers, Ackerman syndrome, allergies (animals, pollen etc), bacterial conjunctivitis, blepharitis, paralysis of the facial nerve, ectropion or obstruction at the level of the nasolacrimal duct or tear sac.

3.

Use according to any one of claims 1 or 2, wherein said antagonist decreases the stimulation of tear secretion by the cold-sensitive fibers by inactivating TRPM8.

Four.

Use according to any one of claims 1 to 3, wherein the TRPM8 antagonist is selected from the TRPM8 antagonists shown in Table 3.

5.

Use according to claim 4, wherein the TRPM8 antagonist is selected from the group consisting of: an antisense oligonucleotide specific for the sequence of the gene encoding TRPM8, DNA enzyme specific for the sequence of TRPM8, microRNA specific for the gene encoding TRPM8 , ribozyme specific for the gene sequence encoding TRPM-8, RNA interference specific for the gene sequence encoding TRPM8, peptide capable of specifically binding to TRPM8 and inhibiting its activity, antibody capable of specifically binding to TRPM8 and inhibiting the activity of said channel, BCTC (N- (4-tert-butyl-phenyl) -4- (3-chloropyridin-2-yl) tetrahydropyrazine-1 (2H) -carboxamide), CTPC ((2R) -4 (3 -chloro-2-pyridinyl) -2-methyl-N- [4- (trifluoromethyl) phenyl] -1-piperazine-carboxamide), thio-BCTC (N- (4-tert-butyl-phenyl) 4- (3- chloropyridin-2-yl) tetrahydropyrazine-1 (2H) - (thio) carboxamide), SB-452533 (N- (2-bromophenyl) -N '- (2- [ethyl (3-methylphenyl) amino] ethyl) urea), SKF96 365 (1- [2- (4-methoxyphenyl) -2- [3- (4-methoxyphenyl) propoxy] ethyl-1H-imidazole), Econazol (1- [2 - [(4-chlorophenyl) methoxy] -2- (2,4-Dichlorophenyl) ethyl] -1H-imidazole), Clotrimazole (1 - [(2chlorophenyl) diphenylmethyl] -1H-imidazole), ACA (N- (p-amylcinnamoyl) anthranilic acid), AMTB (N- (3 -aminopropyl) -2 {[(3-methylphenyl) methyl] oxy} -N- (2-thienylmethyl) -benzamide), Capsazepine (N- [2- (4-chlorophenyl) ethyl] -1,3,4,5 -tetrahydro7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide), Fenantroline, MAD1d (N - [(1R, 2S, 5R) -2-Isopropyl-5methylcyclohexyl] biphenyl-4-carboxamide), MAD2e (4- tert-Butylphenyl (1R, 2S, 5R) -2-isopropyl-5-methylcyclohexylcarbamate).

6.

Composition comprising at least one TRPM8 antagonist and at least one drug useful for the treatment of the epiphora and, if desired, a pharmaceutically acceptable carrier.

7.

Composition according to claim 6, wherein the TRPM8 antagonist is selected from the TRPM8 antagonists shown in Table 3.

8.

Composition according to claims 6 or 7, wherein the TRPM8 antagonist is selected from the group consisting of: an antisense oligonucleotide specific for the sequence of the gene encoding TRPM8, DNA enzyme specific for the sequence of TRPM8, microRNA specific for the gene that encodes TRPM8, ribozyme specific for the gene sequence encoding TRPM-8, interference RNA specific for the gene sequence encoding TRPM8, peptide capable of specifically binding to TRPM8 and inhibiting its activity, antibody capable of specifically binding to TRPM8 and inhibit the activity of said channel, BCTC (N- (4-tert-butyl-phenyl) -4- (3-chloropyridin-2-yl) tetrahydropyrazine-1 (2H) -carboxamide), CTPC ((2R) -4 - (3-Chloro-2-pyridinyl) -2-methyl-N- [4- (trifluoromethyl) phenyl] -1-piperazine-carboxamide), thio-BCTC (N- (4-tertbutyl-phenyl) -4- ( 3-chloropyridin-2-yl) tetrahydropyrazine-1 (2H) - (thio) carboxamide), SB-452533 (N- (2-bromophenyl) -N '(2- [ethyl (3-methylphenyl) amino] ethyl) urea), SKF96365 (1- [2- (4-methoxyphenyl) -2- [3- (4-methoxyphenyl) propoxy] ethyl-1Himidazole), Econazol (1- [2 - [(4-chlorophenyl) methoxy] -2- (2,4-dichlorophenyl) ethyl] -1H-imidazole), Clotrimazole (1 - [(2chlorophenyl) diphenylmethyl] -1H-imidazole), ACA (N- (p-amylcinnamoyl) anthranilic acid), AMTB (N - (3-aminopropyl) -2 {[(3-methylphenyl) methyl] oxy} -N- (2-thienylmethyl) -benzamide), Capsazepine (N- [2- (4-chlorophenyl) ethyl] -1.3, 4,5-tetrahydro7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide), Phenanthroline, MAD1d (N - [(1R, 2S, 5R) -2-Isopropyl-5methylcyclohexyl] biphenyl-4-carboxamide), MAD2e (4-tert-Butylphenyl (1R, 2S, 5R) -2-isopropyl-5-methylcyclohexylcarbamate).

9.

Use of a composition according to any of claims 6 to 8, for the preparation of a medicament for the treatment of epiphora.

10.

Use according to claim 9, wherein the epiphora is associated with one or more of the selected diseases of Graves-Basedow disease, corneal ulcers, Ackerman syndrome, allergies (animals, pollen etc), bacterial conjunctivitis, blepharitis , facial nerve paralysis, ectropion or obstruction at the level of the nasolacrimal duct or tear sac.

Figure 1 Figure 1 (cont.) Figure 1 (cont.) Figure 1 (cont.) Figure 2 Figure 3 Figure 4A Figure 4B Figure 4C  SEQUENCE LIST <110> UNIVERSITY MIGUEL HERNÁNDEZ DE ELCHE SUPERIOR SCIENTIFIC RESEARCH COUNCIL <120> PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF THE EPIPHORE <130> P6082ES01 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 1104 <212> PRT <213> Homo sapiens <400> 1 Met Ser Phe Arg Ala Ala Arg Leu Ser Met Arg Asn Arg Arg Asn Asp1 5 10 15 Thr Leu Asp Ser Thr Arg Thr Leu Tyr Be Ser Ala Be Arg Ser Thr 20 25 30 Asp Leu Ser Tyr Ser Glu Ser Asp Leu Val Asn Phe Ile Gln Ala Asn35 40 45 Phe Lys Lys Arg Glu Cys Val Phe Phe Thr Lys Asp Ser Lys Ala Thr50 55 60 Glu Asn Val Cys Lys Cys Gly Tyr Ala Gln Ser Gln His Met Glu Gly65 70 75 80 Thr Gln Ile Asn Gln Ser Glu Lys Trp Asn Tyr Lys Lys His Thr Lys85 90 95 Glu Phe Pro Thr Asp Wing Phe Gly Asp Ile Gln Phe Glu Thr Leu Gly100 105 110 Lys Lys Gly Lys Tyr Ile Arg Leu Ser Cys Asp Thr Asp Ala Glu Ile115 120 125 Leu Tyr Glu Leu Leu Thr Gln His Trp His Leu Lys Thr Pro Asn Leu130 135 140 Val Ile Ser Val Thr Gly Gly Ala Lys Asn Phe Ala Leu Lys Pro Arg145 150 155 160 Met Arg Lys Ile Phe Ser Arg Leu Ile Tyr Ile Gln Ser Lys Gly165 170 175 Wing Trp Ile Leu Thr Gly Gly Thr His Tyr Gly Leu Met Lys Tyr Ile180 185 190 Gly Glu Val Val Arg Asp Asn Thr Ile Ser Arg Ser Ser Glu Glu Asn195 200 205 Ile Val Wing Ile Gly Ile Wing Trp Gly Met Val Ser Asn Arg Asp210 215 220 Thr Leu Ile Arg Asn Cys Asp Wing Glu Gly Tyr Phe Leu Wing Gln Tyr225 230 235 240 Leu Met Asp Asp Phe Thr Arg Asp Pro Leu Tyr Ile Leu Asp Asn Asn245 250 255 His Thr His Leu Leu Leu Val Asp Asn Gly Cys His Gly His Pro Thr260 265 270 Val Glu Ala Lys Leu Arg Asn Gln Leu Glu Lys Tyr Ile Ser Glu Arg275 280 285 Thr Ile Gln Asp Ser Asn Tyr Gly Gly Lys Ile Pro Ile Val Cys Phe290 295 300 Gln Wing Gly Gly Gly Lys Glu Thr Leu Lys Ile Wing Asn Thr Ser Ile305 310 315 320 Lys Asn Lys Ile Pro Cys Val Val Val Glu Gly Ser Gly Gln Ile Ala325 330 335 Asp Val Ile Wing Ser Leu Val Glu Val Glu Asp Wing Leu Thr Ser Ser340 345 350 Wing Val Lys Glu Lys Leu Val Arg Phe Leu Pro Arg Thr Val Ser Arg355 360 365 Leu Pro Glu Glu Glu Thr Glu Ser Trp Ile Lys Trp Leu Lys Glu Ile370 375 380 Leu Glu Cys Ser His Leu Leu Thr Val Ile Lys Met Glu Glu Ala Gly385 390 395 400 Asp Glu Ile Val Ser Asn Ala Ile Ser Tyr Ala Leu Tyr Lys Ala Phe405 410 415 Ser Thr Ser Glu Gln Asp Lys Asp Asn Trp Asn Gly Gln Leu Lys Leu420 425 430 Leu Leu Glu Trp Asn Gln Leu Asp Leu Wing Asn Asp Glu Ile Phe Thr435 440 445 Asn Asp Arg Arg Trp Glu Ser Ala Asp Leu Gln Glu Val Met Phe Thr450 455 460 Wing Leu Ile Lys Asp Arg Pro Lys Phe Val Arg Leu Phe Leu Glu Asn465 470 475 480 Gly Leu Asn Leu Arg Lys Phe Leu Thr His Asp Val Leu Thr Glu Leu485 490 495 Phe Ser Asn His Phe Ser Thr Leu Val Tyr Arg Asn Leu Gln Ile Ala500 505 510 Lys Asn Ser Tyr Asn Asp Ala Leu Leu Thr Phe Val Trp Lys Leu Val515 520 525 Wing Asn Phe Arg Arg Gly Phe Arg Lys Glu Asp Arg Asn Gly Arg Asp530 535 540 Glu Met Asp Ile Glu Leu His Asp Val Ser Pro Ile Thr Arg His Pro545 550 555 560 Leu Gln Wing Leu Phe Ile Trp Wing Ile Leu Gln Asn Lys Lys Glu Leu565 570 575 Ser Lys Val Ile Trp Glu Gln Thr Arg Gly Cys Thr Leu Wing Wing Leu580 585 590 Gly Ala Ser Lys Leu Leu Lys Thr Leu Ala Lys Val Lys Asn Asp Ile595 600 605 Asn Wing Wing Gly Glu Ser Glu Glu Leu Wing Asn Glu Tyr Glu Thr Arg610 615 620 Wing Val Glu Leu Phe Thr Glu Cys Tyr Ser Ser Asp Glu Asp Leu Ala625 630 635 640 Glu Gln Leu Leu Val Tyr Ser Cys Glu Ala Trp Gly Gly Ser Asn Cys645 650 655 Leu Glu Leu Wing Val Glu Wing Thr Asp Gln His Phe Ile Wing Gln Pro660 665 670 Gly Val Gln Asn Phe Leu Ser Lys Gln Trp Tyr Gly Glu Ile Ser Arg675 680 685 Asp Thr Lys Asn Trp Lys Ile Ile Leu Cys Leu Phe Ile Ile Pro Leu690 695 700 Val Gly Cys Gly Phe Val Ser Phe Arg Lys Lys Pro Val Asp Lys His705 710 715 720 Lys Lys Leu Leu Trp Tyr Tyr Val Ala Phe Phe Thr Ser Pro Phe Val725 730 735 Val Phe Ser Trp Asn Val Val Phe Tyr Ile Phe Leu Leu Leu Phe740 745 750 Wing Tyr Val Leu Leu Met Asp Phe His Ser Val Pro His Pro Pro Glu755 760 765 Leu Val Leu Tyr Ser Leu Val Phe Val Leu Phe Cys Asp Glu Val Arg770 775 780 Gln Trp Tyr Val Asn Gly Val Asn Tyr Phe Thr Asp Leu Trp Asn Val785 790 795 800 Met Asp Thr Leu Gly Leu Phe Tyr Phe Ile Wing Gly Ile Val Phe Arg805 810 815 Leu His Be Be Asn Lys Be Be Leu Tyr Be Gly Arg Val Ile Phe820 825 830 Cys Leu Asp Tyr Ile Ile Phe Thr Leu Arg Leu Ile His Ile Phe Thr835 840 845 Val Ser Arg Asn Leu Gly Pro Lys Ile Ile Met Leu Gln Arg Met Leu850 855 860 Ile Asp Val Phe Phe Phe Leu Phe Leu Phe Ala Val Trp Met Val Ala865 870 875 880 Phe Gly Val Wing Arg Gln Gly Ile Leu Arg Gln Asn Glu Gln Arg Trp885 890 895 Arg Trp Ile Phe Arg Ser Val Ile Tyr Glu Pro Tyr Leu Wing Met Phe900 905 910 Gly Gln Val Pro Ser Asp Val Asp Gly Thr Thr Tyr Asp Phe Ala His915 920 925 Cys Thr Phe Thr Gly Asn Glu Ser Lys Pro Leu Cys Val Glu Leu Asp930 935 940 Glu His Asn Leu Pro Arg Phe Pro Glu Trp Ile Thr Ile Pro Leu Val945 950 955 960 Cys Ile Tyr Met Leu Ser Thr Asn Ile Leu Leu Val Asn Leu Leu Val965 970 975 Wing Met Phe Gly Tyr Thr Val Gly Thr Val Gln Glu Asn Asn Asp Gln980 985 990 Val Trp Lys Phe Gln Arg Tyr Phe Leu Val Gln Glu Tyr Cys Ser Arg995 1000 1005 Leu Asn Ile Pro Phe Pro Phe Ile Val Phe Ala Tyr Phe Tyr Met1010 1015 1020 Val Val Lys Lys Cys Phe Lys Cys Cys Cys Lys Glu Lys Asn Met1025 1030 1035 Glu Ser Ser Val Cys Cys Phe Lys Asn Glu Asp Asn Glu Thr Leu 1040 1045 1050 Wing Trp Glu Gly Val Met Lys Glu Asn Tyr Leu Val Lys Ile Asn1055 1060 1065 Thr Lys Ala Asn Asp Thr Ser Glu Glu Met Arg His Arg Phe Arg1070 1075 1080 Gln Leu Asp Thr Lys Leu Asn Asp Leu Lys Gly Leu Leu Lys Glu1085 1090 1095 Ile Ala Asn Lys Ile Lys1100 <210> 2 <211> 5621 <212> DNA <213> Homo sapiens <400> 2 aagaaaatcc tgcttgacaa aaaccgtcac ttaggaaaag atgtcctttc gggcagccag 60 gctcagcatg aggaacagaa ggaatgacac tctggacagc acccggaccc tgtactccag 120 cgcgtctcgg agcacagact tgtcttacag tgaaagcgac ttggtgaatt ttattcaagc 180 aaattttaag aaacgagaat gtgtcttctt taccaaagat tccaaggcca cggagaatgt 240 gtgcaagtgt ggctatgccc agagccagca catggaaggc acccagatca accaaagtga 300 gaaatggaac tacaagaaac acaccaagga atttcctacc gacgcctttg gggatattca 360 gtttgagaca ctggggaaga aagggaagta tatacgtctg tcctgcgaca cggacgcgga 420 aatcctttac gagctgctga cccagcactg gcacctgaaa acacccaacc tggtcatttc 480 tgtgaccggg ggcgccaaga acttcgccct gaagccgcgc atgcgcaaga tcttcagccg 540 gctcatctac atcgcgcagt ccaaaggtgc ttggattctc acgggaggca cccattatgg 600 cctgatgaag tacatcgggg aggtggtgag agataacacc atcagcagga gttcagagga 660 gaatattgtg gccattggca tagcagcttg gggcatggtc tccaaccggg acaccctcat 720 caggaattgc gatgctgagg gctatttttt agcccagtac cttatggatg acttcacaag 780 agatccactg tatatcctgg acaacaacca cacacatttg ctgctcgtgg acaatggctg 840 tcatggacat c ccactgtcg aagcaaagct ccggaatcag ctagagaagt atatctctga 900 caagattcca gcgcactatt actatggtgg caagatcccc attgtgtgtt ttgcccaagg 960 gagactttga aggtggaaaa aagccatcaa tacctccatc aaaaataaaa ttccttgtgt 1020 ggtggtggaa ggctcgggcc agatcgctga tgtgatcgct agcctggtgg aggtggagga 1080 tgccctgaca tcttctgccg tcaaggagaa gctggtgcgc tttttacccc gcacggtgtc 1140 ccggctgcct gaggaggaga ctgagagttg gatcaaatgg ctcaaagaaa ttctcgaatg 1200 ttaacagtta ttctcaccta ttaaaatgga agaagctggg gatgaaattg tgagcaatgc gctctataca 1260 catctcctac aagccttcag caccagtgag caagacaagg ataactggaa 1320 tgggcagctg aagcttctgc tggagtggaa ccagctggac ttagccaatg atgagatttt 1380 caccaatgac cgccgatggg agtctgctga ccttcaagaa gtcatgttta cggctctcat 1440 aaaggacaga cccaagtttg tccgcctctt tctggagaat ggcttgaacc tacggaagtt 1500 tctcacccat gatgtcctca ctgaactctt ctccaaccac ttcagcacgc ttgtgtaccg 1560 atcgccaaga gaatctgcag attcctataa tgatgccctc ctcacgtttg tctggaaact 1620 ggttgcgaac ttccgaagag gcttccggaa ggaagacaga aatggccggg acgagatgga 1680 catagaactc cacgacgtg t ctcctattac tcggcacccc ctgcaagctc tcttcatctg 1740 ggccattctt cagaataaga aggaactctc caaagtcatt tgggagcaga ccaggggctg 1800 cactctggca gccctgggag ccagcaagct tctgaagact ctggccaaag tgaagaacga 1860 catcaatgct gctggggagt ccgaggagct ggctaatgag tacgagaccc gggctgttga 1920 gagtgttaca gctgttcact gcagcgatga agacttggca gaacagctgc tggtctattc 1980 ctgtgaagct tggggtggaa gcaactgtct ggagctggcg gtggaggcca cagaccagca 2040 tttcatcgcc cagcctgggg tccagaattt tctttctaag caatggtatg gagagatttc 2100 aagaactgga ccgagacacc agattatcct gtgtctgttt attataccct tggtgggctg 2160 tcatttagga tggctttgta agaaacctgt cgacaagcac aagaagctgc tttggtacta 2220 tgtggcgttc ttcacctccc ccttcgtggt cttctcctgg aatgtggtct tctacatcgc 2280 cttcctcctg ctgtttgcct acgtgctgct catggatttc cattcggtgc cacacccccc 2340 cgagctggtc ctgtactcgc tggtctttgt cctcttctgt gacagtggta gatgaagtga 2400 cgtaaatggg gtgaattatt ttactgacct gtggaatgtg atggacacgc tggggctttt 2460 ttacttcata gcaggaattg tatttcggct ccactcttct aataaaagct ctttgtattc 2520 tggacgagtc attttctgtc tgg actacat tattttcact ctaagattga tccacatttt 2580 tactgtaagc agaaacttag gacccaagat tataatgctg cagaggatgc tgatcgatgt 2640 gttcttcttc ctgttcctct ttgcggtgtg gatggtggcc tttggcgtgg ccaggcaagg 2700 gatccttagg cagaatgagc agcgctggag gtggatattc cgttcggtca tctacgagcc 2760 ctacctggcc atgttcggcc aggtgcccag tgacgtggat ggtaccacgt atgactttgc 2820 ccactgcacc ttcactggga atgagtccaa gccactgtgt gtggagctgg atgagcacaa 2880 cctgccccgg ttccccgagt ggatcaccat ccccctggtg tgcatctaca tgttatccac 2940 caacatcctg ctggtcaacc tgctggtcgc catgtttggc tacacggtgg gcaccgtcca 3000 ggagaacaat gaccaggtct ggaagttcca gaggtacttc ctggtgcagg agtactgcag 3060 ccgcctcaat atccccttcc ccttcatcgt cttcgcttac ttctacatgg tggtgaagaa 3120 gtgcttcaag tgttgctgca aggagaaaaa catggagtct tctgtctgct gtttcaaaaa 3180 tgaagacaat gagactctgg catgggaggg tgtcatgaag gaaaactacc ttgtcaagat 3240 gccaacgaca caacacaaaa cctcagagga aatgaggcat cgatttagac aactggatac 3300 aaagcttaat gatctcaagg gtcttctgaa agagattgct aataaaatca aataaaactg 3360 tatgaactct aatggagaaa aatctaat ta tagcaagatc atattaagga atgctgatga 3420 acaattttgc tatcgactac taaatgagag attttcagac ccctgggtac atggtggatg 3480 attttaaatc accctagtgt gctgagacct tgagaataaa gtgtgtgatt ggtttcatac 3540 ttgaagacgg atataaagga agaatatttc ctttatgtgt ttctccagaa tggtgcctgt 3600 ttctctctgt gtctcaatgc ctgggactgg aggttgatag tttaagtgtg ttcttaccgc 3660 ctcctttttc ctttaatctt atttttgatg aacacatata taggagaaca tctatcctat 3720 gaataagaac ctggtcatgc tttactcctg tattgttatt ttgttcattt ccaattgatt 3780 ctctactttt cccttttttg tattatgtga ctaattagtt ggcatattgt taaaagtctc 3840 tcaaattagg ccagattcta aaacatgctg cagcaagagg accccgctct cttcaggaaa 3900 tttctcagga agtgttttca tgcttcttac ctgtcagagg aggtgacaag gcagtctctt 3960 gctctcttgg actcaccagg ctcctattga aggaaccacc cccattccta aatatgtgaa 4020 aagtcgccca aaatgcaacc ttgaaaggca ctactgactt tgttcttatt ggatactcct 4080 cttattattt ttccattaaa aataatagct ggctattata gaaaatttag accatacaga 4140 gatgtagaaa gaacataaat tgtccccatt accttaaggt aatcactgct aacaatttct 4200 ggatggtttt tcaagtctat tttttttcta tg tatgtctc aattctcttt caaaatttta 4260 tcatactaca cagaatgtta tatatacttt ttatgtaagc tttttcactt agtattttat 4320 caaatatgtt tttattatat tcatagcctt cttaaacatt atatcaataa ttgcataata 4380 ggcaacctct agcgattacc ataattttgc tcattgaagg ctatctccag ttgatcattg 4440 ggatgagcat ctttgtgcat gaatcctatt gctgtatttg ggaaaatttt ccaaggttag 4500 attccaataa atatctattt attattaaat attaaaatat ctatttatta ttaaaaccat 4560 ttataaggct ttttcataaa tgtatagcaa ataggaatta ttaacttgag cataagatat 4620 gagatacatg aacctgaact attaaaataa aatattatat ttaaccctta gtttaagaag 4680 aagtcaatat gcttatttaa atattatgga tggtgggcag atcacttgag gtcaggagtt 4740 ctggccaaca cgagaccagc tggcaaaacc acatctctac taaaaataaa aaaattagct 4800 gggtgtggtg gtgcactcct gtaatcccag ctactcagaa ggctgaggta caagaattgc 4860 tggaacctgg gaggcggagg ttgcagtgaa ccaagattgc accactgcac tccagccggg 4920 gagactccga gtgacagagt ctgaaaataa ataaataaat aaataaataa ataaataaat 4980 attatggatg gtgaagggaa tggtatagaa ttggagagat tatcttactg aacacctgta 5040 gtcccagctt tctctggaag tggtcgtatt tgagcag gat gtgcacaagg caattgaaat 5100 gcccataatt agtttctcag ctttgaatac actataaact cactggctga aggaggaaat 5160 tttagaagga agctactaaa agatctaatt tgaaaaacta caaaagcatt aactaaaaaa 5220 gtttattttc cttttgtctg ggcagtagtg aaaataacta ctcacaacat tcactatgtt 5280 tgcaaggaat taacacaaat aaaagatgcc tttttactta aacaccaaga cagaaaactt 5340 gcccaatact gagaagcaac ttgcattaga gagggaactg ttaaatgttt tcaacccagt 5400 tcatctggtg gatgtttttg caggttactc tgagaatttt gcttatgaaa aatcattatt 5460 tttagtgtag ttcacaataa tgtattgaac atacttctaa tcaaaggtgc tatgtccttg 5520 tgtatggtac taaatgtgtc ctgtgtactt ttgcacaact gagaatcctg cagcttggtt 5580 taatgagtgt gttcatgaaa taaataatgg aggaattgtc a 5621 <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> RNA interference <400> 3 agaaauucuc gaauguucuu u 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> RNA interference <400> 4 uuucuuuaag agcuuacaag a 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RNA interference <400> 5 gaaaacaccc aacctggtca tttc 24 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RNA interference <400> 6 caccgtgcgg ggtaaaaagc g <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RNA interference <220> <221> misc_feature <222> (21) .. (22) <223> deoxithymidine <400> 7 ucucugagcg cacuauucat t <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RNA interference <220> <221> misc_feature <222> (18) .. (19) <223> deoxithymidine <400> 8 uauccgucgg ucaucuatt <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RNA interference <220> <221> misc_feature <222> (20) .. (21) <223> deoxithymidine <400> 9 tctctgagcg cactattcat t SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201131122 SPAIN Date of submission of the application: 08.09.2010 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

TO

WO 2007017092 A1 (BAYER HEALTHCARE AG et al.) 02.02.2007, claims. 1-10

TO

US 2008214654 A1 (LAMPE THOMAS et al.) 04.09.2008, claims. 1-10

TO

WO 2010021882 A2 (JANSSEN PHARMACEUTICA NV et al.) 25.02.2010, page 4, lines 21-30. 1-10

TO

WO 03099278 A1 (ALTANA PHARMA AG et al.) 04.12.2003, claims. 1-10

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 04.17.2013

Examiner H. Aylagas Cancio Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201131122 CLASSIFICATION OBJECT OF THE APPLICATION A61K31 / 16 (2006.01) A61K31 / 4164 (2006.01) A61K31 / 435 (2006.01) A61K31 / 517 (2006.01) A61K31 / 55 (2006.01) A61P27 / 02 (2006.01) Minimum documentation sought (classification system followed by classification symbols) A61P, A61K Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, WPI, NPL, MEDLINE, BIOSIS, EMBASE, XPESP, XPESP2 State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201131122 Date of Completion of Written Opinion: 04.17.2013 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims 1-10 Claims IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims 1-10 Claims IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201131122 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

WO 2007017092 A1 (BAYER HEALTHCARE AG et al.) 02.15.2007

D02

US 2008 214654 A1 (LAMPE THOMAS et al.) 04.09.2008

D03

WO 2010021882 A2 (JANSSEN PHARMACEUTICA NV et al.) 02.25.2010

D04

WO 03099278 A1 (ALTANA PHARMA AG et al.) 04.12.2003

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present application refers to the use of a TRPM8 antagonist (transient receptor potential cation channel subfamily melastatin member 8) for the preparation of a medicament for the treatment of epiphora (excess tear secretion) associated with certain diseases such as blepharitis, bacterial conjunctivitis, Ackerman syndrome, allergies, etc., and a composition comprising a TRPM8 antagonist and at least one drug useful for the treatment of epiphora. Documents D1 and D2 refer to benzyloxy phenylmethylamide derivatives antagonists of CMR-1 (cold menthol receptor 1) also called TRPM8. They are used in the treatment of urological diseases, urinary incontinence, benign prostatic hyperplasia, etc. Document D3 refers to the use of TRPM8 antagonists in the treatment of diseases affected by the modulation of TRPM8 receptors such as pain, diseases that lead to such pain, and vascular or pulmonary dysfunctions (see page 4, lines 21-30). Document D4 refers to the use of roflumilast in the treatment of eye diseases, including the epiphora (see claim 3) The use of this group of TRPM8 antagonist compounds in the treatment of epiphora is not deduced from any of the cited documents or any combination thereof. Therefore, the matter corresponding to claims 1-10 of the present application has novelty and inventive activity according to articles 6.1 and 8.1 of the L.P. State of the Art Report Page 4/4