USE OF A MULTIFUNCTIONAL LIGAND FOR TREATING DRY EYES, MEIBOMIAN GLAND DYSFUNCTIONS AND LACRIMAL GLAND DYSFUNCTIONS

Abstract

Use of an isoquinoline multifunctional ligand, amino-7 triethoxy-4,5,6 oxo-1 dihydro-1,3 isobenzofurannyl-3)-1methoxy-8 methyl-2 methylenedioxy 6,7tetrahydro-,2,3,4isoquinoline, or tritoqualine for the treatment of dry eyes, meibomian gland dysfunctions and lacrimal gland dysfunctions.

Claims

1-7. (canceled)

8. A method of treating dry eye syndrome comprising administering to a subject in need thereof an effective amount of AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof.

9. The method according to claim 8, wherein the effective amount of AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof treats pain in dry eye syndrome.

10. The method according to claim 8, wherein the effective amount of AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof treats dry eye syndrome related to Gougerot-Sjögren's disease.

11. The method according to claim 8, wherein the effective amount of AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof treats dry eye syndrome related to autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus.

12. The method according to claim 8, wherein the AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof is administered in combination with carbomer-based humectants or hyaluronic acid-based lubricants.

13. The method according to claim 8, wherein the AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof is substituted at at least one of its methyls with a deuterated methyl.

14. The method according to claim 8, wherein the AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof is packaged in the form of an ophthalmic eye drop or ointment.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0036] FIG. 1 represents the chemical structure of tritoqualine.

[0037] FIG. 2 Activity of tritoqualine at the apical pole expressed as % of maximum effect.

[0038] FIG. 3 Activity of tritoqualine at the basal pole expressed as % of maximum effect.

[0039] FIG. 4 Effect of Tritoqualine expressed as μA/Cm.sup.2 at the apical pole on lacrimal gland epithelial cells (A=Forskolin; B=Tritoqualine; C=Inh 172).

[0040] FIG. 5 Effect of Tritoqualine expressed in μA/Cm.sup.2 at the basal pole on lacrimal gland epithelial cells (A=Forskolin; B=Tritoqualine; C=Inh 172).

[0041] FIG. 6 The lacrimal functional unit according to DARTT 2002.

[0042] FIG. 7 Affinity curve of tritoqualine on the NK 1 receptor.

[0043] FIG. 8 Inhibition percentage of the NK 1 receptor by tritoqualine.

[0044] FIG. 9 Flow cytometry analysis highlighting basophil degranulation by substance P (CD63+ and CCR3+).

[0045] FIG. 10 Inhibition of basophil degranulation by (10 μM) tritoqualine via inhibition of (10 μM) substance P action.

DETAILED DESCRIPTION

[0046] The inventors have highlighted amazing and surprising properties of tritoqualine in a human cell model of CFTR modulation without mutation.

[0047] Thus, the present invention relates to AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof for use in the treatment of diseases associated with decreased secretions of epithelial cells of the lacrimal glands and Meibomian glands.

[0048] Thus, the present invention also relates to AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof for use in the treatment of dry eye syndrome.

[0049] Thus, the present invention also relates to AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof for use in the treatment of dry eye syndrome related to Gougerot-Sjögren's disease and secondary Gougerot-Sjögren's syndromes accompanying autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus.

[0050] According to one preferred embodiment, the invention relates to AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof for use in the treatment of pain in dry eye syndrome.

[0051] According to one preferred embodiment, the invention relates to AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof for use in dry eye syndrome characterised in that it is administered in the form of an eye drop or eye ointment. According to one preferred embodiment, the invention relates to AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof for use in dry eye syndrome characterised in that it is administered in the form of an eye drop at a dose of 0.1 milligram to 5 milligrams.

[0052] According to one preferred embodiment, the invention relates to AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof for use in dry eye syndrome characterised in that it is administered in the form of an eye drop in association with humectants and lubricants based on hyaluronic acid or carbomers. According to one preferred embodiment, the invention relates to AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof for use in the treatment of dry eye syndrome characterised in that it is substituted at at least one of its methyls with a deuterated methyl.

[0053] The present invention also relates to AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof for use in improving ion transport both apically and at the basal pole of corneal, lacrimal glandular and meibomian glandular epithelial cells.

[0054] According to the preferred embodiment of the invention AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof is remarkable in that it is substituted at its methyls with deuterated methyls to improve pharmacokinetics.

[0055] According to one preferred embodiment of the invention, AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof is for use in the treatment of dry eye syndrome at a dose of 0.1 milligram to 5 milligrams/day.

[0056] According to one preferred embodiment of the invention, AMINO-7 TRIETHOXY-4,5,6 OXO-1 DIHYDRO-1,3 ISOBENZOFURANNYL-3)-1METHOXY-8METHYL-2METHYLENEDIOXY-6,7TETRAHYDRO-,2,3,4ISOQUINOLINE, or tritoqualine and pharmaceutically acceptable salts thereof also for use in the treatment of dry eye syndrome is remarkable in that it is packaged in the form of an eye drop or eye ointment.

EXAMPLES

[0057] To investigate this action on CFTR modulation, the inventors used the Ussing chamber method, invented by the Dane Hans H. Ussing in the late 1950s. This technique makes it possible to study ionic exchanges across an epithelium, as it allows the tissue of interest to be kept alive for a few hours under controlled conditions of temperature and medium. The positioning of the tissue between two half-chambers makes it possible to define an apical compartment (corresponding to the lumen of the organ) and a basolateral compartment (corresponding to the blood compartment) and to investigate exchanges between these two compartments via the tissue.

[0058] This technique is used as a routine and is particularly well adapted for a pharmacological approach to ionic transport and to search for molecules of therapeutic interest in the context of ionic secretions of epithelial cells. It consists in measuring the transepithelial current (known as the short-circuit current and noted Ise). Ise is expressed in amperes to unit area of the epithelium (Ise in μA/cm.sup.2). The cells are grown on a porous filter for 10 to 15 days at the liquid-liquid interface and then air-liquid interface in order to mimic conditions close to in vivo. The transepithelial resistance is measured regularly when culturing. The higher this resistance is (several hundred ohms), the more the epithelial tissue is junctive, polarised and therefore tight. At the air (apical side)-liquid (basolateral side) interface the epithelial cells polarise and form a tight mat which can be investigated with the Ussing chamber technique. A system with 6 Physiology Instrument® cuvettes allowing 6 experiments in parallel has been used.

[0059] The following material and molecules have been used in the study:

[0060] Amiloride: 100 μM final concentration; 100 mM stock solution, solvent water (supplier Sigma®)).

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

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

[0063] CFTR inh172: 10 μM final concentration; 10 mM stock solution, DMSO solvent (supplier Fisher®).

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

[0065] Various media and reagents including supports adapted to the Ussing chamber: Snapwell (Fisher®), culture medium (Gibco®), SVF (Gibco®), Puromycin (Gibco®), T75 culture flasks (FisherED) have been used.

[0066] Tritoqualine has a surprising action on CFTR, which is not known. No publication or patent mentions any activity of tritoqualine on CFTR of corneal epithelial cells or glandular epithelial cells.

[0067] The inventors have used USSING chambers to analyse the activity of tritoqualine on CFTR of glandular epithelial cells.

[0068] Typical protocols used for human epithelial cells expressing non-mutated CFTR:

[0069] Measurement of short circuit current in the presence of amiloride (ENaC channel inhibitor, 100 μM) followed by addition of 10 μM of the tritoqualine molecule and then addition of CFTR inh172 (10 μM, CFTR inhibitor) followed by addition of UTP (100 μM, validates the experiment by activating the calcium-sensitive Cl transport).

[0070] Measurement of short circuit current in the presence of amiloride (100 μM) and forskolin (0.05 μM activator of intracellular cAMP) then addition of 10 μM of the tritoqualine molecule then addition of CFTRinh172 (10 μM) then addition of UTP (100 μM).

[0071] Measurement of the short circuit current in the presence of CFTR Inh172 (10 μM) then addition of tritoqualine and forskolin (0.05 μM) then addition of UTP.

[0072] The inventors have prepared a 100 μM stock solution in DMSO. The compounds have been aliquoted per 100 μL and stored at −20° C.

[0073] Tritoqualine has been added to USSING cells at a dose of 10 μM in non-mutated cells FIG. 5.

[0074] The effect of tritoqualine on non-mutated epithelial cells on the apical side is significant, as the differential increases from 16 to more than 20 (expressed in μA/cm.sup.2).

[0075] The addition of forskolin changes the potential from 20 to 27 (expressed in μA/cm.sup.2).

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

[0077] Tritoqualine activates the cell potential in lacrimal gland epithelial cells expressing non-mutated CFTR. This effect is additive with that of forskolin.

[0078] The effect of tritoqualine on non-mutated lacrimal gland epithelial cells on the apical side is significant, as the differential increases from 6.5 to more than 7.5 (expressed in μA/cm.sup.2). The addition of forskolin changes the potential from 3.5 to 6.5 (expressed in μA/cm.sup.2).

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

[0080] In conclusion, tritoqualine activates ion transport in the epithelial cells of the lacrimal gland. This effect is blocked by inh172, which specifically inhibits CFTR ion transport.

[0081] Tritoqualine appears to be a molecule capable of stimulating ion transport via the non-mutated CFTR.

[0082] The effective dose of tritoqualine has then been determined on both the apical and basal poles. The result is an EC50 activity of 3.42+/−0.19 μM for the apical pole and an EC50 of 4.87+/−0.27 μM for the basal pole FIG. 2, FIG. 3.

[0083] Thus, tritoqualine can improve the lacrimal secretion function of patients with proven dry eye disease including patients with Gougerot-Sjögren's disease, but also so-called “secondary” Gougerot-Sjögren's because they are associated with specific autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus.

[0084] The inventors have also used an NK 1 agonist (substance P—Supplier: Sigma-Aldrich) to demonstrate the activity of tritoqualine on the NK 1 receptor.

[0085] Tritoqualine has been tested on the NK 1 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 March.

[0086] This test shows an affinity of tritoqualine, which is equal to 97% and a Ki equal to 1.4 10-7. FIG. 7 shows precisely the affinity curve and Ki of tritoqualine on the NK 1 receptor. FIG. 8 shows the percent of affinity with the NK 1 receptor. Tritoqualine appears as an NK 1 receptor antagonist in this experiment. It has been verified that the NK 1 receptor was involved in the basophil activation pathway by using an NK 1 agonist.

[0087] The inventors have used the Kit-Flow CAST (www.buhlmannlabs.ch/products-solutions/cellular-allergy/flow-cast/) to test the action of tritoqualine on the inhibition of basophil degranulation. The marker for degranulation is CD63. Upon basophil activation, CD63 markers bound to intracytoplasmic granules will fuse with the plasma membrane. They are then expressed on the cell surface: activated basophils thereby become CD63+. Further to CD63, another marker specific to basophils makes it possible to better target them, it is CCR3 (chemokine receptor 3). The latter is always expressed by this cell type. Thus, the degranulation of basophils is identified in the flow cytometry window as CD63+ and CCR3+.

[0088] In a first step 2 patients have been sampled. substance P has been tested at a dose of 10 μmoles. In this experiment, it is surprisingly found that CD63 is present at 70% on the basophil. FIG. 9 shows the intensity of degranulation when substance P is incubated with basophils implying that substance P caused degranulation of the basophil.

[0089] In a second step to determine whether tritoqualine also blocked substance P-induced degranulation, the following experiment has been methodically performed: tritoqualine has been incubated at a dose of 10 pmoles without the FcεRI antibody and then substance P has been put into the tubes. After 30 minutes, degranulation in each of the tubes is around 7% as with the negative control. FIG. 10 shows that tritoqualine blocks the activation of basophil degranulation caused by substance P. It can be said that tritoqualine has a surprising action on the inhibition of degranulation via its antagonistic action on the NK 1 receptor.

[0090] Thus, in the dry eye, tritoqualine would have 2 actions via the inhibition of substance P: an action on the basophil and an action on the neurological vicious circle of the dry eye. Thus tritoqualine, through its action on NK1, would have 2 complementary actions; one on the inflammation related to the basophil degranulation inhibition and another on the pain related to its action on the neurological vicious circle. Thus, the local action of tritoqualine on dry eye appears remarkable and surprising due to its 2 pharmacological actions on CFTR and substance P.