Use of multifunctional ligands for treating dry eye, meibomian gland dysfunction and lacrimal gland dysfunction

Abstract

The present invention relates to use of a multifunctional isoquinoline ligand, namely 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline, for treating dry eye, meibomian gland dysfunction and lacrimal gland dysfunction.

Description

Technical Field

[0001] The present invention relates to the use of a multifunctional ligand, in particular to the left-handed and right-handed enantiomers of (7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-isobenzofuran-3)-yl-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline for treating dry eye syndrome and meibomian gland and lacrimal gland dysfunction.

[0002] The present invention relates to the use of multifunctional ligands that are active not only at the NK1 receptor but also at the transmembrane conductance regulator (CFTR) modulator for the treatment of meibomian gland, lacrimal gland and more general dry eye syndrome, as well as pharmaceutical compositions and methods of treatment. Background Art

[0003] Dry eye syndrome was defined by the Dry Eye Symposium in 2007 as a multifactorial disease of the tears and ocular surface that causes discomfort, visual impairment, and tear film instability, with the potential for damage to the ocular surface. The ocular surface may present with more or less severe keratitis, which can lead to corneal ulcers. It is accompanied by an increase in tear film osmolarity and inflammation of the ocular surface. The etiology is diverse and different, and traditionally has been divided into reduced tear flow (e.g., Gougerot- Syndrome) caused by excessive tear evaporation and dry eye syndrome caused by excessive tear evaporation.

[0004] Meibomian gland dysfunction (MGD) is the most common cause of dry eye disease (DED). A 2011 workshop also defined MGD. Eyelid inflammation, microbial growth, associated skin diseases, and potentially serious corneal complications make MGD a complex, multifactorial disease. Meibomian gland inflammation can be considered a disease in itself. MGD is likely a heterogeneous disease resulting from the combined effects of five distinct pathophysiological mechanisms: eyelid inflammation; conjunctival inflammation; corneal damage; microbial changes; and tear film instability leading to DED. The pathogenesis of both MGD and DED can be explained by a "vicious cycle": the underlying pathophysiological mechanisms of DED and MGD interact, resulting in a double vicious cycle. This double vicious cycle contributes to the development of dry eye and its intractability.

[0005] Dry eye syndrome is a complex, multifactorial ocular surface disease that leads to a loss of tear film homeostasis and causes a variety of ocular symptoms. DED is recognized to negatively impact visual function, quality of life, and economic burden (Efficacy of topical ophthalmic medications in the treatment of dry eye: A systematic literature review, Holland EJ et al., Ocul Surf., 2019). In many patients, the disease is a chronic condition requiring long-term treatment.

[0006] The prevalence of DED is high, estimated to affect 5-50% of the adult population worldwide, and the economic burden of the disease is expected to increase as the population ages. Worldwide, ophthalmic lubricants are commonly used in the initial management of DED, but they do not address the underlying cause of the disease. Over the past two decades, potentially more effective ophthalmic drugs have been investigated for different pathophysiological pathways of DED, but these efforts have resulted in only a very small number of new drug approvals. The main approved treatments are: 0.05% cyclosporine A ophthalmic emulsion in North America ( Allergan (Irvine, CA, USA) and 5.0% lifitegrast ophthalmic solution ( Shire (Lexington, USA); European cationic emulsion ( Santen Pharmaceutical (Osaka, Japan) and 3% Diquafosol-based ophthalmic solution ( Santen Pharmaceutical, Osaka, Japan); and unit dose 2% rebamipide ophthalmic suspension ( Otsuka Pharmaceutical (Tokyo, Japan). The United States has approved (August 2018) 0.09% cyclosporine A nanomicellar formulation ( Sun Pharmaceuticals (Mumbai, India) is used to increase tear production in patients with DED. In general, these drugs can reduce ocular surface inflammation or stabilize the tear film, but it is not clear which drug is best for aqueous-deficient dry eye (ADDE) or evaporative dry eye (EDE). Sjögren's syndrome (GSS) is associated with dry eye due to progressive destruction of the lacrimal glands, which may lead to severe keratitis. GSS is a chronic autoimmune disease characterized by progressive, degenerative, inflammatory involvement of the exocrine glands, which may be associated with systemic diseases that affect the joints, skin, lungs, kidneys, or peripheral nerves to varying degrees. The pathophysiology of the 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 triggers the release of proinflammatory cytokines that maintain a chronic inflammatory state, as well as the secretion of autoantibodies, ultimately leading to epithelial cell death by apoptosis.

[0007] Regardless of the cause, common initial management of dry eye syndrome is based on:

[0008] - Correction of contributing factors (medications; environmental factors; eye drops containing preservatives, particularly quaternary ammonium compounds) whenever possible;

[0009] and replacement therapy with tear substitutes (artificial tears added to eye drops, gels, and viscoelastic solution medical devices used when these two substances are ineffective).

[0010] Once initiated, dry eye can continue to progress, even with alternative treatments, and perpetuate itself according to the aforementioned vicious cycle of inflammation, leading to progressive damage to all ocular surface tissues, including the cornea. In severe cases, dry eye can cause significant damage to the cornea (or keratitis), with a range of symptoms ranging from a foreign body sensation on the ocular surface to persistent burning pain with decreased vision. The severity of dry eye is related to the degree of keratitis, inflammatory factors, and painful ocular symptoms.

[0011] To address the vicious cycle that leads to dry eye, treatment targeting several pharmacological targets is necessary. This is where multifunctional ligands come in. As the name suggests, multifunctional ligands are active against multiple pharmacological targets. For effectiveness reasons, the multifunctional ligand's affinity for the receptor is reduced, allowing it to spread across multiple pharmacological targets in a balanced manner. The affinity of multifunctional ligands typically ranges from 100 nanomolar to 1 micromolar. The inventors have demonstrated that tritoquinline, when used as a multifunctional ligand, is active against two drug targets: CFTR and NK1.

[0012] CFTR is a multifunctional protein. It is a member of the ABC transporter superfamily and is found in all organisms (bacteria, archaea, and eukaryotes). CFTR differs from all other members of the superfamily by its function as an ion channel and the presence of a unique regulatory domain (R). In epithelial cells, CFTR forms a channel permeable to chloride and thiocyanate ions. In addition to channel regulation, other functions of CFTR have been demonstrated: ATP transport; regulation of exocytosis / endocytosis; and regulation of intracellular organelle pH.

[0013] For dry eye syndrome, it is necessary not only to restore the lacrimal gland's function of producing tears, but also to restore the corneal epithelium's function of secreting electrolytes.

[0014] A 2001 paper demonstrated that CFTR affects electrolyte secretion in an immortalized rabbit corneal epithelial cell line (Invest Ophthalmol Vis Sci., September 2001, "CFTR-mediated activation of chloride current in a rabbit corneal epithelial cell line," Al-Nakkash L.). Furthermore, CFTR has been shown to influence the secretory function of the tear gland (Invest Ophthalmol Vis Sci., January 2018, "New insights into the role of CFTR in mouse lacrimal duct function," Berczeli O.). In that paper, the authors showed that CFTR modulators could increase tear secretion in mice with normal CFTR levels.

[0015] CFTR is a cAMP / ATP-mediated ion channel that is expressed in a variety of cell types, including secretory epithelial cells, where it regulates the transmembrane flow of anions and the activity of other ion channels and proteins. In epithelial cells, the normal function of CFTR is primarily to maintain the transport of electrolytes throughout the body, including respiratory tissue, digestive tissue, and even ocular tissue. CFTR is composed of approximately 1480 amino acids, which form a protein composed of two repeating transmembrane domains, each of which contains six transmembrane helices and a nucleotide binding domain. The two transmembrane domains are connected by a larger polar regulatory domain (R), which has several phosphorylation sites that regulate channel activity and cell migration.

[0016] Chloride transport occurs through the coordinated activity of ENaC and CFTR on the apical membrane, along with Na+ and K+ ATPases and chloride channels expressed on the basolateral surface of the cell. Secondary active chloride transport on the luminal side allows chloride ions to accumulate within the cell and subsequently exit the cell passively through chloride channels, thereby transporting them from the basolateral to the apical pole. In this way, water, which would otherwise never be actively transported, is transported across the epithelium due to the transepithelial osmotic gradient created by the flow of sodium and chloride ions.

[0017] To treat the causes of dry eye syndrome that appear to be influenced by insufficient CFTR function, new therapies that can modulate and activate the normal function of CFTR are needed.

[0018] On the other hand, recent evidence suggests that some dry eye symptoms are more likely manifestations of chronic neuropathology.

[0019] Neurogenic mechanisms may play a significant role in chronic ocular surface inflammation, with manifestations likely related to the acute-to-chronic transition associated with neuropathological changes associated with repeated ocular sensory nerve injury, which are also the source of pain in dry eye.

[0020] Substance P is thought to be involved in neurogenic inflammation (Stern ME and colleagues, Pathology of dry eye: interactions between the ocular surface and the lacrimal gland. Cornea, November 1998).

[0021] Although the role of neurogenic inflammation in dry eye syndrome is not fully understood, its role is certainly crucial (J Investig Allergol Clin Immunol., September 2018, Mechanisms of neuropathic pain and itch from allergic conjunctivitis, Kuruvilla M et al).

[0022] Therefore, breaking the vicious cycle of dry eye syndrome by simultaneously regulating CFTR and inhibiting substance P may be an interesting topic. Substance P is secreted upon activation of the NK1 receptor. Multifunctional ligands such as tritoquine, as NK1 antagonists, also act as inhibitors of substance P secretion. WO2007 / 117704A2 discloses tritoquine for the treatment of immune system disorders such as conjunctivitis.

[0023] Holland et al. (Ocular Surface, Vol. 17, No. 3, July 1, 2019, pp. 412-423) describe different topical compounds used to treat dry eye syndrome.

[0024] Flores et al. (FASEB Journal, Vol. 30, No. 5, May 1, 2016, pp. 1789-1797) disclosed small molecule CFTR activators that can increase tear secretion and prevent dry eye syndromes, including, inter alia, Sjögren's syndrome, but did not disclose tritoquinline.

[0025] EP2659890A1 discloses the use of tritoquine in the treatment of fibrotic diseases, in particular cystic fibrosis. Summary of the Invention

[0026] The present invention relates to the use of multifunctional ligands, which particularly include isoquinoline-based compounds, such compounds including 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or the levorotatory and dextrorotatory enantiomers of tritoquinoline and pharmaceutically acceptable salts thereof.

[0027] The inventors have discovered that 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof have surprising properties as multifunctional ligands that exert effects on normal CFTR regulation and NK1 receptors.

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

[0029] Tritoquine is known to have antiallergic activity due to its effect on histidine decarboxylase. However, this activity is very weak and does not explain its numerous properties in various clinical conditions, including rhinitis, urticaria, eczema, and mastocytosis.

[0030] The inventors have demonstrated that triptoquine has a significant effect on two important targets for dry eye treatment: CFTR and NK1 receptors. Currently, there are many patent applications based on triptoquine, but none of them mention its therapeutic activity for dry eye syndrome.

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

[0032] Isotope-labeled compounds and salts can be used for a variety of applications. They can be suitable for pharmaceuticals and / or various types of testing, such as tissue distribution assays on substrates. For example, tritium- and / or carbon-14-labeled compounds are extremely useful for various types of testing, including substrate-based tissue distribution assays, due to their relatively simple preparation and excellent detectability. For example, deuterium-labeled products are very useful therapeutically and offer potential therapeutic advantages over non-deuterium-labeled compounds. In general, deuterium-labeled compounds and salts have greater metabolic stability than non-deuterium-labeled compounds due to isotope kinetics. This greater metabolic stability directly translates into a potentially desirable longer in vivo half-life or lower dosage. Isotope-labeled compounds and salts can generally be prepared according to the procedures described in known synthetic schemes, such as those in EP3352757. Therefore, non-deuterated methyl groups can be easily replaced with deuterated methyl groups.

[0033] Tritoquine is a white crystalline powder that is insoluble in water and slightly soluble in benzene and acetone. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Represents the chemical structure of tritoquine.

[0035] Figure 2 Shown is the activity of tritoquinline at the apical pole, expressed as a percentage of the maximal effect.

[0036] Figure 3 Shown is the activity of tritoquine at the base, expressed as a percentage of the maximal effect.

[0037] Figure 4 Shown is the effect of tritoquine on lacrimal gland epithelial cells at the apical pole, expressed as μA / cm 2 (A = Forskolin; B = Tritoquine; C = Inh 172);

[0038] Figure 5 Shown is the effect of tritoquine on lacrimal gland epithelial cells at the base, expressed as μA / cm 2 (A = Forskolin; B = Tritoquine; C = Inh 172);

[0039] Figure 6 Shown are the functional units of the lacrimal gland according to DARTT 2002.

[0040] Figure 7 Shown is the affinity curve of tritoquinline for the NK1 receptor.

[0041] Figure 8Shown is the percentage inhibition of NK1 receptors by tritoquinline.

[0042] Figure 9 Shown is a flow cytometric analysis highlighting the degranulation of basophils (CD63+ and CCR3+) by substance P.

[0043] Figure 10 Shown is that tritoquine (10 μM) inhibits basophil degranulation by inhibiting the action of substance P (10 μM). DETAILED DESCRIPTION

[0044] The inventors have highlighted the surprising and surprising properties exhibited by tritoquine in a non-mutated human cell CFTR modulation model.

[0045] The present invention therefore relates to 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof for use in the treatment of diseases associated with decreased secretion of lacrimal and meibomian gland epithelial cells.

[0046] The present invention therefore also relates to 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof for use in treating dry eye syndrome.

[0047] The present invention therefore also relates to 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-yl-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof for use in the treatment of dry eye syndrome associated with Sjögren's syndrome and secondary Sjögren's syndrome associated with autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus.

[0048] According to a preferred embodiment, the present invention relates to 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof for treating dry eye syndrome pain.

[0049] According to a preferred embodiment, the present invention relates to 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof for dry eye syndrome, characterized in that they are administered in the form of eye drops or eye ointment. According to a preferred embodiment, the present invention relates to 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof, characterized in that they are administered in the form of eye drops at a dose of 0.1 mg to 5 mg.

[0050] According to a preferred embodiment, the present invention relates to 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof for dry eye syndrome, characterized in that they are administered in the form of eye drops associated with moisturizers and lubricants based on hyaluronic acid or carbomer.

[0051] According to a preferred embodiment, the present invention relates to 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof for treating dry eye syndrome, characterized in that at least one methyl group thereof is substituted with a deuterated methyl group.

[0052] The present invention also relates to 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof for simultaneously improving apical and basal ion transport in corneal, lacrimal and meibomian gland epithelial cells.

[0053] According to a preferred embodiment of the present invention, 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof are remarkable in that they improve pharmacokinetics by replacing methyl groups with deuterated methyl groups.

[0054] According to a preferred embodiment of the present invention, 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof are used to treat dry eye syndrome at a dose of 0.1 mg to 5 mg per day.

[0055] According to a preferred embodiment of the present invention, 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof, which are also used to treat dry eye syndrome, are remarkable in that they are packaged in the form of eye drops or eye ointments.

[0056] Example

[0057] To investigate this effect on CFTR regulation, the inventors used the Ussing chamber method, invented by Dane Hans H. Ussing in the late 1950s. This technique allows the tissue under investigation to be kept alive for several hours under controlled temperature and medium conditions, thus enabling the study of transepithelial ion exchange. By placing the tissue between two half-chambers, an apical compartment (corresponding to the lumen of the organ) and a basolateral compartment (corresponding to the blood compartment) can be defined, allowing the exchange of ions between these two compartments through the tissue to be studied.

[0058] This technique is a general technique that is well suited for ion transport and pharmacological approaches to finding molecules with therapeutic value in epithelial ion secretion. The technique involves the measurement of the transepithelial current (called the short-circuit current and labeled Ise). Ise is measured in amperes per unit epithelial area (Ise is in μA / cm 2 ). The cells are grown on porous filters for 10 to 15 days, first at the liquid-liquid interface and then at the air-liquid interface, to simulate conditions close to those in vivo. During the culture process, the transepithelial electrical resistance is measured regularly. The higher the resistance (hundreds of ohms), the better the connectivity of the epithelial tissue, the higher the degree of polarization and therefore the more compact it is. At the air (apical side) and liquid (basal side) interfaces, the epithelial cells polarize and form a compact cushion that can be studied using the Ussing chamber technique. Among them, a 6-well plate with 100 nm iontophoresis was used. The cuvette system can be used to perform 6 parallel experiments.

[0059] The following materials and molecules were used in this study:

[0060] Amiloride: 100 μM final concentration, 100 mM stock solution, aqueous solvent (supplier: );

[0061] Forskolin: 0.05 μM final concentration, 1 mM stock solution, DMSO solvent (supplier: );

[0062] Genistein: 30 μM final concentration, 30 mM stock solution, DMSO solvent (supplier: );

[0063] CFTR inhl72: 10 μM final concentration, 10 mM stock solution, DMSO solvent (supplier: );

[0064] UTP: 100 μM final concentration, 100 mM stock solution, DMSO solvent (supplier: ).

[0065] In addition, various culture media and reagents have been used, including Snapwell supports adapted for Usin chambers. Culture medium SVF Puromycin T75 culture flasks (FisherED).

[0066] Tritoquine has a surprising and unknown effect on CFTR. There are no publications or patents mentioning any activity of tritoquine on CFTR in corneal epithelial cells or glandular epithelial cells.

[0067] The inventors used a Ussing chamber to analyze the activity of tritoquine on CFTR in glandular epithelial cells.

[0068] A typical protocol for human epithelial cells expressing non-mutated CFTR is as follows:

[0069] - short-circuit current was measured in the presence of amiloride (ENaC channel inhibitor, 100 μM), followed by the addition of 10 μM of the tritoquine molecule, then by the addition of CFTR inhl72 (10 μM, CFTR inhibitor), and then by the addition of UTP (100 μM, experimentally verified by activating calcium-sensitive chloride channels);

[0070] - short-circuit current was measured in the presence of amiloride (100 μM) and forskolin (0.05 μM, activator of intracellular cAMP), followed by the addition of 10 μM of the tritoquine molecule, then of CFTRinh172 (10 μM), and then of UTP (100 μM);

[0071] - Short-circuit currents were measured in the presence of CFTR Inh172 (10 μM), followed by the addition of tritoquine and forskolin (0.05 μM), and then again with the addition of UTP.

[0072] The inventors prepared 100 μM stock solutions using DMSO as solvent. The compounds were aliquoted in 100 μL units and stored at -20°C.

[0073] Tritoquine was added to the non-mutant cells in the Uxin chamber at a dose of 10 μM ( Figure 5 ).

[0074] As the difference increases from 16 to more than 20 (in μA / cm 2 Therefore, the effect of tritoquine on the apical non-mutant epithelial cells is more significant.

[0075] The addition of forskolin caused the potential to change from 20 to 27 (in μA / cm 2 express).

[0076] The addition of Inh172 completely blocked the cell potential.

[0077] Tritoquine activates the cell potential of lacrimal epithelial cells expressing non-mutated CFTR, an effect that is additive with that of forskolin.

[0078] As the difference increases from 6.5 to more than 7.5 (in μA / cm 2 Therefore, the effect of tritoquine on the apical non-mutant epithelial cells is more significant. The addition of forskolin changes the potential from 3.5 to 6.5 (in μA / cm 2 express).

[0079] The addition of Inh172 completely blocked the cell potential.

[0080] In conclusion, tritoquine activates ion transport in lacrimal gland epithelial cells, an effect that can be blocked by inhl72, a specific inhibitor of CFTR ion transport.

[0081] Tritoquine appears to be a molecule capable of stimulating ion transport by non-mutated CFTR.

[0082] After that, the effective dose of tritoquine at the apical and basal sites was determined. The results showed that the EC50 activity at the apical site was 3.42±0.19μM, and the EC50 at the basal site was 4.87±0.27μM ( Figure 2 and Figure 3 ).

[0083] CONCLUSIONS: Tritoquine improves tear gland function in patients with established dry eye, including not only those with Sjögren's syndrome but also those with a form of Sjögren's syndrome known as "secondary" because it is associated with certain autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus.

[0084] The inventors further utilized NK1 agonist (Substance P supplier: -Aldrich) confirmed the activity of tritoquine on the NK1 receptor.

[0085] Among others, the effect of tritoquinline on the NK1 receptor was tested according to the method described by Heuillet et al. (Characterization of the human NK1 tachykinin receptor in the astrocytoma cell line U373MG, Heuillet et al., Neurochemistry, March 1993).

[0086] The test results showed that the affinity of tritoquine was equal to 97% and Ki was equal to 1.4×10 -7 . Figure 7 The affinity curve and Ki of tritoquinline for NK1 receptor are accurately shown. Figure 8 Shown are the percent affinity for the NK1 receptor. In this experiment, tritoquine appears to act as an NK1 receptor antagonist. The NK1 receptor has been shown to participate in basophil activation pathways using NK1 agonists.

[0087] The inventors used Kit-Flow CAST (www.buhlmannlabs.ch / products-solutions / cellular-allergy / flow-cast / ) to test the inhibitory effect of tritoquinol on basophil degranulation. The degranulation marker is CD63. After basophil activation, the CD63 marker bound to the cytoplasmic granules will fuse with the plasma membrane. Subsequently, both are expressed on the cell surface, causing the activated basophils to become CD63+. In addition to CD63, CCR3 (chemokine receptor 3) is another specific marker for basophils, which can achieve better targeting of basophils. The latter is always expressed by this type of cell. Therefore, basophil degranulation can be determined as CD63+ and CCR3+ in the flow cytometer window.

[0088] In a first step, samples were taken from two patients and tested at a dose of 10 μM for substance P. A surprising finding in this experiment was the presence of CD63 on basophils in 70% of the cells. Figure 9 Shown are the degranulation intensity when substance P was incubated with basophils, and the results indicate that substance P induces degranulation of basophils.

[0089] In a second step, to determine whether triptoquinoline also blocks substance P-induced degranulation, the following experiment was systematically performed: triptoquinoline was incubated at a dose of 10 μM in the absence of FcεRI antibody, and then the tubes were added with substance P. After 30 minutes, the percentage of degranulation in each tube was approximately 7%, consistent with the negative control. Figure 10 The results showed that tritoquine blocked the activation of basophil degranulation caused by substance P. It can be said that tritoquine has a surprising degranulation inhibitory effect due to its antagonism of NK1 receptors.

[0090] Therefore, it is clear that tritoquinoline, due to its inhibition of substance P, should have two effects on dry eye: an effect on basophils and an effect on the dry eye vicious cycle. Therefore, tritoquinoline, through its effect on NK1, should have two complementary effects: one on inflammation related to the inhibition of basophil degranulation; the other on pain related to its effect on the neurological vicious cycle. Therefore, tritoquinoline exhibits a remarkable and surprising local effect on dry eye due to its dual pharmacological effects on CFTR and substance P.

Claims

1. Use of 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof in the preparation of a medicament for treating dry eye syndrome.

2. The use according to claim 1, wherein The 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof are used for preparing drugs for treating pain in dry eye syndrome.

3. The use according to claim 1, wherein The 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof are used for preparing a drug for treating dry eye syndrome associated with Sjögren's syndrome.

4. The use according to claim 1, wherein The 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof are used for preparing a drug for treating dry eye syndrome associated with autoimmune diseases.

5. The use according to claim 1, wherein The 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and a pharmaceutically acceptable salt thereof are used in combination with a carbomer-based moisturizer or a hyaluronic acid-based lubricant.

6. The use according to any one of claims 1 to 5, wherein The 1-(7-amino-4,5,6-triethoxy-1-oxo-1,3-dihydro-3-isobenzofuran)-8-methoxy-2-methyl-6,7-methylenedioxy-2,3,4-tetrahydroisoquinoline or tritoquinoline and pharmaceutically acceptable salts thereof are packaged in the form of ophthalmic eye drops or ointments.

Images