Use of multifunctional ligands to treat dry eye, meibomian gland dysfunction, and lacrimal gland dysfunction.

Abstract

The present invention relates to the use of the isoquinoline multifunctional ligand amino-7 triethoxy-4,5,6 oxo-1 dihydro-1,3 isobenzofuranyl-3)-1 methoxy-8 methyl-2 methylenedioxy-6,7 tetrahydro-2,3,4 isoquinoline or tritoqualine for the treatment of dry eye, meibomian gland dysfunction and lacrimal gland dysfunction.

Description

Technical Field

[0001] The present invention relates to the use of multifunctional ligands, and more specifically, to the treatment of dry eye syndrome and the dysfunction of the meibomian and lacrimal glands, 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide or to the use of the levorotatory and dextrorotatory enantiomers of torquatrine.

[0002] The present invention relates to the use of multifunctional ligands having activity not only against the NK1 receptor but also against regulators of the transmembrane conductance regulator (CFTR) for the treatment of meibomian and lacrimal gland dysfunction, and more generally dry eye syndrome, as well as pharmaceutical compositions and treatment methods.

Background Art

[0003] Dry eye syndrome was defined by the 2007 Dry Eye Workshop as a multifactorial disease of the tears and ocular surface that causes symptoms of discomfort, visual disturbances, and instability of the tear film with potential damage to the ocular surface. The ocular surface develops varying degrees of keratitis, which can lead to corneal ulcers. It is accompanied by an increase in the osmotic pressure of the tear film and inflammation of the ocular surface. There are many different etiologies, and conventionally, dry eye syndrome caused by a decrease in tear flow (e.g., Gougerot-Sjogren syndrome) is distinguished from dry eye syndrome caused by excessive evaporation of tears.

[0004] Meibomian gland dysfunction (MGD) is the most common cause of dry eye disease (DED). A 2011 workshop also defined MGD. Inflammation of the eyelids, microbial growth, associated skin disorders, and potentially serious corneal complications make MGD a complex, multifactorial disorder. Meibomian gland inflammation can be considered a disease in itself. MGD appears to be a heterogeneous disease resulting from a combination of five different pathophysiological mechanisms: inflammation of the eyelids, conjunctivitis, corneal disorders, microbiological changes, and DED resulting from tear film instability. The pathophysiology of both MGD and DED can be explained by a "vicious cycle," where the underlying pathophysiological mechanisms of DED and MGD influence each other, resulting in a double vicious cycle. This double vicious cycle causes dry eye and makes its treatment difficult.

[0005] Dry eye syndrome (DED) is a complex, multifactorial ocular surface disorder that leads to a loss of tear film homeostasis and causes a variety of ocular symptoms. The negative impact of DED on visual function, quality of life, and economic burden is well recognized (Non-Patent Literature 1). In many patients, the disorder is chronic and requires long-term treatment.

[0006] DED has a high prevalence, estimated to affect 5-50% of the adult population worldwide, and its economic burden is projected to increase with age. Globally, ophthalmic lubricants are often used for the initial management of DED, but they do not address the underlying causes of the disease. Over the past 20 years, potentially more effective ophthalmic pharmacological agents targeting different distinct pathophysiological pathways of DED have been studied, but these efforts have resulted in the approval of only a few new drugs. The main approved therapeutic agents include 0.05% cyclosporine A ophthalmic emulsion (Restasis®; Allergan, Irvine, California, USA) and 5.0% lifitegrast eye drops (Xiidra®; Shire, Lexington, USA) in North America; cationic emulsion (Ikervis®; Santen Pharmaceutical, Osaka, Japan) in Europe; and 3% diquafosol eye drops (Diquas®; Santen Pharmaceutical, Osaka, Japan) and 2% rebamipide suspension eye drops (Mucosta®; Otsuka Pharmaceutical, Tokyo, Japan) in Asia. In the United States (August 2018), a nanomicelle formulation of cyclosporine A 0.09% (Cequa®; Sun Pharmaceuticals, Mumbai, India) has been approved for the purpose of increasing tear production in patients with DED. Overall, these drugs reduce inflammation on the ocular surface or stabilize the tear film, but it is unclear which drug is best suited for patients with aqueous deficient dry eye (ADDE) or evaporative dry eye (EDE). Gougerot-Sjögren's syndrome (SGS) is characterized by xerophthalmoplegia due to progressive destruction of the lacrimal glands, which can lead to severe keratitis. GSS is a chronic autoimmune disease characterized by progressive, degenerative, and inflammatory involvement of the exocrine glands and may be associated with systemic diseases affecting various parts of the joints, skin, lungs, kidneys, or peripheral nerves. The pathophysiology of this disease is characterized by infiltration of the salivary and lacrimal glands by CD4+ T cells and B cells.The local activation and proliferation of these lymphocytes induces the release of pro-inflammatory cytokines that maintain a chronic inflammatory state, as well as the secretion of autoantibodies, ultimately leading to apoptosis and death of epithelial cells.

[0007] The usual initial management of dry eye syndrome, regardless of the etiology, is based on the following: - Correction of promoting factors (drugs, environmental factors, eye drops containing preservatives (especially quaternary ammonium compounds)) as much as possible; and - Replacement therapy with tear substitutes (artificial tears in the form of viscoelastic solutions in medical devices used after the other two methods have failed).

[0008] Once dry eye develops, it progresses despite supplemental therapy and becomes self-persistent, following the concept of a vicious cycle of inflammation in which damage to all ocular surface tissues, including the cornea, progresses. In severe cases, dry eye can lead to significant corneal damage (or keratitis), causing a range of symptoms from a foreign body sensation or burning sensation on the surface of the eye to persistent pain accompanied by decreased vision. The severity of dry eye is related to the degree of keratitis, the inflammatory component, and painful eye symptoms.

[0009] To treat the vicious cycle that causes dry eye, it is necessary to treat several pharmacological targets. This is achieved by multifunctional ligands. As the name suggests, multifunctional ligands exert activity against multiple pharmacological targets. In order to exert their effect, multifunctional ligands have low affinity to receptors and are able to diffuse in a balanced manner to multiple pharmacological targets. This affinity is often between 100 nanomolar (nM) and 1 micromolar (μM) concentrations. The inventors have demonstrated that tritoqualin behaves as a multifunctional ligand and is active against two drug targets, CFTR and NK1.

[0010] CFTR is a multifunctional protein. The CFTR protein is a member of the ABC transporter superfamily and is found in all domains of life (bacteria, archaea, and eukaryotes). CFTR is distinguished from all other members of this superfamily by its role as an ion channel and the presence of its unique regulatory domain (R). CFTR forms a permeable channel for chloride and thiocyanate ions in epithelial cells. It also explains other functions independent of channel regulation: ATP transport, regulation of exocytosis / endocytosis, and pH regulation of intracellular organelles.

[0011] In dry eye syndrome, it is necessary not only to restore tear production from the lacrimal glands but also to restore electrolyte secretion from the corneal epithelium.

[0012] A paper published in 2001 showed that CFTR is involved in electrolyte secretion in immortalized rabbit corneal epithelial cell lines (Non-Patent Literature 2). Furthermore, it also affects lacrimal gland secretion (Non-Patent Literature 3). In this paper, the authors show that a CFTR modulator can increase tear secretion in mice with normal CFTR.

[0013] CFTR is a cAMP / ATP-mediated anion channel expressed in various cell types, including secretory epithelial cells, that regulates the flow of anions across the membrane and controls the activity of other ion channels and proteins. In epithelial cells, normal CFTR function is essential for maintaining systemic electrolyte transport, including in the respiratory and digestive tracts, as well as in ocular tissues. CFTR is composed of approximately 1480 amino acids, each forming a protein consisting of a repeating transmembrane domain containing six transmembrane helices and a nucleotide-binding domain. The two transmembrane domains are linked by a large polarity-regulating domain [R] containing several phosphorylation sites that control channel activity and cellular trafficking.

[0014] Chloride transport is mediated by the coordinated activity of ENaC and CFTR present in the apical membrane, as well as Na+ and K+ ATPases and Cl- channels expressed on the basolateral surface of the cell. Secondary active chloride transport in the lumen leads to intracellular chloride accumulation, which can then be passively transported out of the cell via Cl- channels, thereby facilitating transport from the basolateral to the apical pole. Consequently, water, which is likely not actively transported, is transported across the epithelium according to the transepithelial osmotic gradient created by the sodium and chloride flows.

[0015] To treat the causes of dry eye syndrome, which are thought to be due to insufficient CFTR function, it is necessary to find novel therapies that regulate and activate normal CFTR function.

[0016] On the other hand, recent evidence suggests that some symptoms of dry eye may be better manifested by chronic neuropathy.

[0017] Neurogenic mechanisms may play a significant role in chronic inflammation of the ocular surface. Symptoms may be related to repeated damage to ocular sensory nerves, leading to an acute-to-chronic transition associated with neuropathological changes. These neuropathic changes are also the cause of dry eye pain.

[0018] Substance P is thought to be involved in this neurogenic inflammation. (Stern ME et al., Non-Patent Literature 4)

[0019] Although neurogenic inflammation in dry eye syndrome is not fully understood, it certainly plays an important role (Non-Patent Literature 5).

[0020] Therefore, it is interesting to perform both CFTR regulation and substance P inhibition in order to break the vicious cycle of dry eye syndrome. Substance P is secreted after activation of the NK1 receptor. Multifunctional ligands typified by tritoqualine act as NK1 antagonists and are thus inhibitors of substance P secretion. Patent Document 1 discloses tritoqualine in the treatment of immune system diseases such as conjunctivitis.

[0021] Holland et al. (Non-Patent Document 6) describe various topical compounds for the treatment of dry eye syndrome.

[0022] Flores et al. (Non-Patent Document 7) disclose low molecular weight CFTR activators that increase tear secretion and particularly prevent dry eye syndrome including Sjogren's syndrome, but do not disclose tritoqualine.

[0023] Patent Document 2 discloses the use of tritoqualine in the treatment of fibrotic diseases, particularly cystic fibrosis.

Prior Art Documents

Patent Documents

[0024]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0025]

Non-Patent Document 1

Non-Patent Document 2

[0026] The present invention relates to the use of multifunctional ligands, and in particular, 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalideThe present invention relates to the use of multifunctional ligands containing isoquinoline compounds, including levorotatory and dextrorotatory enantiomers of tritoquarin and pharmaceutically acceptable salts thereof.

[0027] The inventors of this invention, 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Alternatively, we discovered the remarkable properties of tritoqualin and its pharmaceutically acceptable salts as multifunctional ligands that regulate normal CFTR and act on the NK1 receptor.

[0028] The present invention also relates to pharmaceutical compositions comprising at least one of the compounds described herein and / or at least one pharmaceutically acceptable salt thereof, wherein the compositions may further comprise at least one other active pharmaceutical ingredient and / or at least one excipient. The present invention also relates to a method for treating dry eye syndrome and Gougerot-Sjögren's disease, comprising administering at least one of the compounds described herein and / or at least one pharmaceutically acceptable salt thereof to a subject in need as part of a pharmaceutical composition optionally comprising at least one additional ingredient.

[0029] Tritoqualine is known to possess anti-allergic activity due to its inhibitory effect on histidine decarboxylase. However, this activity is very weak and does not explain the numerous properties it exhibits for various clinical symptoms, including rhinitis, urticaria, dermatitis, and mastocytosis.

[0030] The inventors have demonstrated that tritoqualin has a crucial effect on CFTR and NK1 receptors, two important targets for treating dry eye. Although many patents have been filed based on tritoqualin, none mention its activity in treating dry eye syndrome.

[0031] Tritoqualin is a benzylisoquinoline with a molecular weight of 500. This compound can be modified or substituted with a compound containing either 14 carbon atoms or a deuterated compound.

[0032] Isotope-labeled compounds and salts can be used in a variety of ways. They may be suitable for various types of tests, such as tissue distribution studies on drugs and / or substrates. For example, tritium and / or carbon-14 labeled compounds are relatively easy to prepare and have excellent detectability, making them particularly useful for various types of tests, such as substrate-based tissue distribution studies. For example, deuterium-labeled products are therapeutically useful and have potential therapeutic advantages over undeuterium-labeled compounds. In general, deuterium-labeled compounds and salts can have higher metabolic stability than undeuterium-labeled compounds due to kinetic isotope effects. High metabolic stability directly leads to extended half-lives and lower doses in vivo, which can be desirable. Isotope-labeled compounds and salts can generally be prepared according to procedures described in known synthetic schemes, such as those described in EP3352757. Therefore, it is easy to replace undeuterated methyl with deuterated methyl.

[0033] Tritoqualin is a white crystalline powder. It is insoluble in water; and sparingly soluble in benzene and acetone. [Brief explanation of the drawing]

[0034] [Figure 1] This shows the chemical structure of tritoqualin. [Figure 2] The activity of tritoqualin at the apical pole, expressed as a percentage of the maximum effect. [Figure 3] The activity of tritoqualin at the basal pole, expressed as a percentage of the maximum effect. [Figure 4] The effect of tritoqualin on lacrimal gland epithelial cells at the apical pole, expressed in μA / cm2 (A = forskolin; B = tritoqualin; C = Inh 172). [Figure 5]The effect of tritoqualin on lacrimal gland epithelial cells at the basal pole, expressed as μA / cm2 (A = forskolin; B = tritoqualin; C = Inh 172). [Figure 6] Tear film functional units according to DARTT 2002. [Figure 7] Affinity curve of tritoqualin for the NK1 receptor. [Figure 8] Percentage of NK1 receptor inhibition by tritoqualin. [Figure 9] Flow cytometry analysis showing basophil degranulation by substance P (CD63+ and CCR3+). [Figure 10] Inhibition of basophil degranulation by tritoqualin (10 μM) by inhibiting the action of substance P (10 μM). [Modes for carrying out the invention]

[0035] The inventors revealed surprising and unexpected properties of tritoqualin in a human cell model of CFTR regulation without mutations.

[0036] Therefore, the present invention is for use in the treatment of diseases associated with decreased secretions from the epithelial cells of the lacrimal glands and meibomian glands. 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or relating to tritoqualin, and its pharmaceutically acceptable salts.

[0037] Therefore, the present invention also provides for use in the treatment of dry eye syndrome. 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or relating to tritoqualin, and its pharmaceutically acceptable salts.

[0038] Therefore, the present invention is for use in the treatment of dry eye syndrome associated with Gougerot-Sjögren's disease and secondary Gougerot-Sjögren's syndrome occurring in conjunction with autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus. 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or relating to tritoqualin, and its pharmaceutically acceptable salts.

[0039] According to one preferred embodiment, the present invention is for use in treating pain in dry eye syndrome, 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or relating to tritoqualin, and its pharmaceutically acceptable salts.

[0040] According to one preferred embodiment, the present invention is for use in dry eye syndrome 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or, relating to tritoqualin, and its pharmaceutically acceptable salts, characterized in that it is administered in the form of eye drops or eye ointment. According to one preferred embodiment, the present invention is for use in dry eye syndrome 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Alternatively, the present invention relates to tritoqualin, or a pharmaceutically acceptable salt thereof, characterized in that it is administered in the form of eye drops in doses of 0.1 milligrams to 5 milligrams.

[0041] According to one preferred embodiment, the present invention is for use in dry eye syndrome 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Alternatively, the present invention relates to tritoqualin, or a pharmaceutically acceptable salt thereof, and is characterized by being administered in the form of eye drops in association with hyaluronic acid or carbomer-based moisturizers and lubricants.

[0042] According to one preferred embodiment, the present invention is for use in the treatment of dry eye syndrome. 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide The present invention relates to tritoqualin or a pharmaceutically acceptable salt thereof, characterized in that at least one methyl group is substituted with methyl deuterated.

[0043] The present invention is also for use in improving ion transport at both the apical and basal poles of epithelial cells of the cornea, lacrimal gland, and meibomian gland. 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or relating to tritoqualin, and its pharmaceutically acceptable salts.

[0044] According to a preferred embodiment of the present invention, 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalideIt is noteworthy that tritoquarin, or its pharmaceutically acceptable salts, are substituted with deuterium-substituted methyl groups at their methyl sites to improve pharmacokinetics.

[0045] According to a preferred embodiment of the present invention, 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Alternatively, tritoqualin, or its pharmaceutically acceptable salts, are intended for use in the treatment of dry eye syndrome at doses of 0.1 to 5 milligrams per day.

[0046] According to a preferred embodiment of the present invention, for use in the treatment of dry eye syndrome 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Alternatively, tritoqualin and its pharmaceutically acceptable salts are noteworthy in that they are packaged in the form of eye drops or eye ointments. [Examples]

[0047] To study this effect on CFTR regulation, the inventors used the Ussing chamber method, invented in the late 1950s by the Dane Hans H. Ussing. This technique allows for the study of ion exchange mediated by epithelium, as it can maintain the target tissue alive for several hours under controlled temperature and culture medium conditions. By positioning the tissue between two half-chambers, the apical compartment (corresponding to the organ lumen) and the basolateral compartment (corresponding to the blood compartment) are defined, allowing for the study of exchange between these two compartments through the tissue.

[0048] This technique is routinely used and is particularly well-suited for pharmacological approaches to ion transport, as well as for exploring therapeutically interesting molecules related to ion secretion from epithelial cells. It involves measuring transcutaneous current (also called short-circuit current, denoted by Ise). Ise is the amperage (μA / cm²) per unit area of ​​epithelium. 2It is expressed as (Ise) in this case. To mimic conditions close to in vivo, cells are cultured on a porous filter for 10-15 days at the liquid-liquid interface and then at the gas-liquid interface. During culture, transepithelial resistance is measured periodically. The higher this resistance value (hundreds of ohms), the more continuous, polarized, and therefore dense the epithelial tissue is. At the gas (apical)-liquid (basolateral) interface, epithelial cells are polarized and form a tight (dense) mat, which can be examined using the Ussing chamber method. We used a system with six Physiologie Instrument® cuvettes that allowed us to perform six experiments in parallel.

[0049] The following materials and molecules were used in the study: Amyloride: Final concentration 100 μM; 100 mM stock solution, aqueous solvent (Supplier: Sigma®). Forskolin: Final concentration 0.05 μM; 1 mM stock solution, DMSO solvent (supplier: Sigma®). Genistein: Final concentration 30 μM; 30 mM stock solution, DMSO solvent (supplier: Sigma®). CFTR inh172: Final concentration 10 μM; 10 mM stock solution, DMSO solvent (supplier: Fisher®). UTP: Final concentration 100 μM; 100 mM stock solution, DMSO solvent (supplier: Sigma®).

[0050] We used various media and reagents, including supports suitable for the Ussing chamber: Snapwell (Fisher®), culture medium (Gibco®), SVF (Gibco®), puromycin (Gibco®), and T75 culture flask (Fisher®).

[0051] Tritoqualin has a remarkable effect on CFTR, which is previously unknown. There are no publications or patents that mention the activity of tritoqualin on CFTR in corneal epithelial cells or glandular epithelial cells.

[0052] The inventors used a USSING chamber to analyze the activity of tritoqualin on CFTR in glandular epithelial cells.

[0053] Standard protocol used for human epithelial cells expressing non-mutant CFTR: - Measurement of short-circuit currents upon addition of 10 μM tritoqualin molecules in the presence of amiloride (ENaC channel inhibitor, 100 μM), followed by the addition of CFTR inh172 (10 μM, CFTR inhibitor), followed by the addition of UTP (100 μM, to validate the experiment by activating calcium-sensitive Cl transport). - Measurement of short-circuit current upon addition of 10 μM tritoqualin molecules, followed by addition of CFTR inh172 (10 μM), and then UTP (100 μM), in the presence of amiloride (100 μM) and forskolin (0.05 μM, an activator of intracellular cAMP). - Measurement of short-circuit current with the addition of tritoqualin and forskolin (0.05 μM) in the presence of CFTR Inh172 (10 μM), followed by the addition of UTP.

[0054] The inventors prepared a 100 μM stock solution in DMSO. The compound was aliquoted in 100 μL portions and stored at -20°C.

[0055] Tritoqualin was added to USSING cells at a dose of 10 μM compared to non-mutant cells (Figure 5).

[0056] The effect of tritoqualin on non-mutated epithelial cells at the apical end was a difference of 16 to over 20 (μA / cm²). 2 The increase (represented by ) is significant.

[0057] Forskolin addition is 20 to 27 (μA / cm²). 2 The potential is changed to (represented by ).

[0058] Adding Inh172 completely blocks the cell potential.

[0059] Tritoqualin activates the cell potential in lacrimal gland epithelial cells expressing non-mutant CFTR. This effect is additive with that of forskolin.

[0060] The effect of tritoqualin on non-mutated lacrimal gland epithelial cells at the apical end was a difference of 6.5 to over 7.5 (μA / cm²). 2 The increase (represented by ) is significant. The addition of forskolin increased from 3.5 to 6.5 (μA / cm²). 2 The potential is changed to (represented by ).

[0061] Adding Inh172 completely blocks the cell potential.

[0062] In conclusion, tritoqualin activates ion transport in lacrimal gland epithelial cells. This effect is inhibited by inh172, which specifically inhibits ion transport in CFTR.

[0063] Tritoqualin is thought to be a molecule that can promote ion transport via non-mutant CFTR.

[0064] Next, the effective dose of tritoqualin was determined at both the apical and basal poles. The results showed an EC50 activity of 3.42 ± 0.19 μM at the apical pole and an EC50 of 4.87 ± 0.27 μM at the basal pole (Figures 2 and 3).

[0065] Therefore, tritoqualin can improve tear secretion function in patients with confirmed dry eye conditions, including not only patients with Gougerot-Sjögren's disease but also patients with so-called "secondary" Gougerot-Sjögren's syndrome associated with certain autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.

[0066] The inventors also demonstrated the activity of tritoqualin against the NK1 receptor using an NK1 agonist (substance P source: Sigma-Aldrich).

[0067] Tritoqualin was tested in the NK1 receptor according to the method described by Heuillet et al. (Characterization of a human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG. Heuillet E et al. Neurochem. 1993 Mar).

[0068] This test showed affinity for tritoqualin, which was equal to 97%, with a Ki of 1.4 × 10⁶. -7 This is equivalent to Figure 7, which accurately shows the affinity curve and Ki of tritoqualin to the NK1 receptor. Figure 8 shows the percentage of affinity to the NK1 receptor. Tritoqualin appears to be an NK1 receptor antagonist in this experiment. The involvement of the NK1 receptor in the basophil activation pathway has been confirmed by using NK1 agonists.

[0069] The inventors tested the effect of tritoqualin on the inhibition of basophil degranulation using the CAST Kit-Flow (www.buhlmannlabs.ch / products-solutions / cellular-allergy / flow-cast / ). The marker for degranulation is CD63. When basophils are activated, the CD63 marker, bound to granules in the cytoplasm, fuses with the cytoplasmic membrane. They are then expressed on the cell surface: as a result, activated basophils become CD63+. In addition to CD63, another basophil-specific marker (CCR3 (chemokine receptor 3)) can be used for better targeting. The latter is always expressed by this type of cell. Therefore, basophil degranulation is identified as CD63+ and CCR3+ in the flow cytometry window.

[0070] In the first step, samples were collected from two patients. Substance P was tested at a dose of 10 μmol. Surprisingly, this experiment found that CD63 was present in 70% of basophils. Figure 9 shows the intensity of degranulation when Substance P was incubated with basophils, indicating that Substance P caused degranulation of basophils.

[0071] In the second step, the following experiment was systematically performed to determine whether tritoqualin inhibits substance P-induced degranulation: Tritoqualin was incubated at a dose of 10 μmol without FcεRI antibody, and then substance P was added to the tubes. After 30 minutes, degranulation in each tube was approximately 7%, similar to the negative control. Figure 10 shows that tritoqualin inhibits the activation of basophil degranulation induced by substance P. Tritoqualin appears to have a remarkable effect in suppressing degranulation through its antagonistic action at the NK1 receptor.

[0072] Therefore, in dry eye, tritoqualin is thought to have two effects through the inhibition of substance P: an effect on basophils and an effect on the neurological vicious cycle of dry eye. In other words, tritoqualin has two complementary effects through its effect on NK1: one is an effect on inflammation related to the suppression of basophil degranulation, and the other is an effect on the neurological vicious cycle. Thus, due to the two pharmacological effects of tritoqualin on CFTR and substance P, the local effect of tritoqualin on dry eye is noteworthy and remarkable. Finally, preferred embodiments of the present invention are described in separate sections. [Embodiment 1] For use in the treatment of dry eye syndrome 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or tritoquarin and its pharmaceutically acceptable salts. [Embodiment 2] Embodiment 1, for use in treating pain in dry eye syndrome 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalideOr tritoquarin and its pharmaceutically acceptable salts. [Embodiment 3] Embodiment 1, for use in the treatment of dry eye syndrome associated with Gudjelo-Sjögren's disease. 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or tritoquarin and its pharmaceutically acceptable salts. [Embodiment 4] Embodiment 1, for use in the treatment of dry eye syndrome associated with autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus. 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or tritoquarin and its pharmaceutically acceptable salts. [Embodiment 5] For use as described in any of Embodiments 1 to 4, in combination with a carbomer-based humectant or a hyaluronic acid-based lubricant. 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or tritoquarin and its pharmaceutically acceptable salts. [Embodiment 6] A method for treating dry eye syndrome, characterized in that at least one methyl molecule is substituted with deuterated methyl 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or tritoquarin and its pharmaceutically acceptable salts. [Embodiment 7] The use of any of Embodiments 1 to 6, characterized by being packaged in the form of eye drops or eye ointment. 7-Amino-4,5,6-Triethoxy-3-(5,6,7,8-Tetrahydro-4-methoxy-6-methyl-1,3-Dioxolo[4,5-g]isoquinoline-5-yl)phthalide Or tritoquarin and its pharmaceutically acceptable salts.

Claims

1. A composition for use in the treatment of dry eye syndrome, comprising tritoqualin represented by the following formula, or a pharmaceutically acceptable salt thereof. 【Chemistry 1】

2. The composition according to claim 1, for use in treating pain in dry eye syndrome.

3. The composition according to claim 1, for use in the treatment of dry eye syndrome associated with Gudjelau-Sjögren's disease.

4. The composition according to claim 1, for use in the treatment of dry eye syndrome associated with autoimmune diseases.

5. A composition according to any one of claims 1 to 4, to be used in combination with a carbomer-based humectant or a hyaluronic acid-based lubricant.

6. A composition for use in the treatment of dry eye syndrome, comprising tritoqualin represented by the following formula or a pharmaceutically acceptable salt thereof, wherein at least one methyl group is substituted with methyl deuteride. 【Chemistry 2】

7. The composition according to any one of claims 1 to 6, packaged in the form of eye drops or eye ointment.

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