Use of Sigma-1 receptor agonist compounds

Abstract

Compositions and methods for the prevention, inhibition, and/or treatment of progressive fibrosis present in various fibroproliferative disorders. Embodiments relate to the use of Sigma-1 receptor agonists for use in the treatment of prevention of progressive fibrosis characterized by the over proliferation of ECM producing cells, e.g. myofibroblasts and by the excessive deposition of ECM components in a medical or disease condition. Preferred Sigma-1 agonists are disclosed.

Claims

1. A method for the treatment of progressive fibrosis in a progressively fibrotic eye tissue of a subject by preventing, reversing or inhibiting fibrotic remodeling of the extracellular matrix, said method comprising administering to the subject an S1R agonist compound in an effective amount sufficient for preventing, reversing or inhibiting fibrotic remodeling of the extracellular matrix to an area of the eye affected by progressive fibrosis, said S1R agonist compound having the following formula II: ##STR00032## wherein Q.sub.1 is a Cl or F or a methyl-halogen selected from CH.sub.2F, CHF.sub.2CF.sub.3, CH.sub.2C.sub.1, CHCl.sub.2, CCl.sub.3, or a methoxy Q.sub.2 is H, Cl or F, R.sub.6 is selected from a substituted or unsubstituted C(1-6) alkyl, C(1-6) alkoxy, C(1-6) alkoxy C(1-6) alkyl, C(5-10) aryl, Y is CH or O, wherein if Y is O then R.sub.4 is not present, if Y is CH then R.sub.4 is H, methyl or ethyl, R.sub.3 is H, methyl or ethyl, or R.sub.3 and R.sub.4 together with the Y-C2 alkyl moiety which they are attached to, may form a saturated or partially unsaturated cyclic group comprising 0 to 2 heteroatom(s) or R.sub.4 and R.sub.3 together form a C(2-4) alkyl bridge, R.sub.1 and R.sub.2 are independently H, methyl or ethyl, or a pharmaceutically acceptable salt thereof.

2. The method according to claim 1 wherein Q.sub.1 is a methyl-halogen selected from CHF.sub.2, CF.sub.3, CHCL.sub.2 or CCL.sub.3, Q.sub.2 is H, R.sub.6 is selected from a substituted or unsubstituted C(1-6) alkoxyalkyl (or C(1-6) dialkyl-ether) or C(1-2) alkoxy C(2-5) alkyl, Y is CH or O, wherein if Y is O then R.sub.4 is not present, if Y is CH then R.sub.4 is H, methyl or ethyl, R.sub.3 is H or methyl.

3. The method according to claim 1 wherein said compound is fluvoxamine.

4. The method according to claim 1 wherein the subject has a fibroproliferative eye disorder.

5. The method according to claim 4 wherein said fibroproliferative eye disorder is an eye disease in the anterior segment of the eye.

6. The method according to claim 4 wherein said fibroproliferative eye disorder is glaucoma.

7. The method according to claim 6 wherein glaucoma is selected from the group consisting of open angle glaucoma, closed angle glaucoma, primary glaucoma and secondary glaucoma.

8. The method according to claim 4 wherein said fibroproliferative eye disorder is an eye disease related with a fibrosis in the trabecular meshwork.

9. The method according to claim 1 wherein said S1R agonist is administered in a formulation of eye drops.

10. The method according to claim 1 wherein said S1R agonist is administered in a formulation of ointment.

11. The method according to claim 2 wherein the subject has a fibroproliferative eye disorder which is glaucoma and said S1R agonist is administered in a formulation of eye drops.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A-D. Sigma-1 receptor (S1R) expression in various models: in vitro in myofibroblasts (FIG. 1A); in vivo in proximal tubules (FIG. 1B) and whole kidney samples (FIG. 1C) of diabetic rats and also in (FIG. 1D) renal biopsies of patients diagnosed with obstructive uropathy. SIR is also co-localized with -smooth muscle actin (SMA) (FIG. 1D). SIR was stained with red (Alexa Fluor 543 on FIGS. 1A-D), while SMA were stained with green (Alexa Fluor 488 on FIGS. 1A-D). Nucleus is stained blue with Hoechst. (Pictures were evaluated Zeiss Axiovert, confocal laser-scanning microscope, 40, 63, 100 magnification, respectively).

(2) FIGS. 2A-C. Sigma-1 receptor (S1R) compounds [fluvoxamine (FIG. 2A), NE-100 (FIG. 2A) SA-4503 (FIG. 2B), PRE-084 (FIG. 2C)] are not cytotoxic in myofibroblasts. After 24-hours treatment with the S1R compounds in different concentrations (1, 3, 5, 10, 20 M/L), cell viability was measured by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay on 96-well plate (410.sup.3 cells/well). (Bars represent meanSEM)

(3) FIGS. 3A-C. Sigma-1 receptor (S1R) agonist compounds [fluvoxamine (FIG. 3A), fluvoxamine+NE-100 (FIG. 3A), SA-4503 (FIG. 3B), PRE-084 (FIG. 3C)] inhibits PDGFB-induced cell proliferation. Myofibroblast proliferation was induced by 10 ng/mL PDGFB in 6-well plates (610.sup.6 cells/well). To investigate the effect of S1R agonists parallel to PDGF-induction a group of cells was treated with the said compounds in different concentrations (1, 3, 5, 10, 20 M/L). Subsequently cells were incubated for 24 hours at 37 C. then cell proliferation assay (MTT) was performed. Solvent treated cells served as controls. (Bars represent meanSEM)

(4) FIGS. 4A-B. Sigma-1 receptor (S1R) agonist compound fluvoxamine minimizes TGF-induced collagen-1 (FIG. 4A) and collagen-3 (FIG. 4B) production of myofibroblasts on a time-dependent manner. Collagen-1 and collagen-3 production was induced by 0.5 nM TGF- in 6-well plates (610.sup.6 cells/well). To investigate the effect of S1R agonist fluvoxamine a group of cells was treated with 20 M/L fluvoxamine parallel to TGF-induction. Subsequently cells were incubated for 48 hours at 37 C. then quantitative RT-PCR was performed. Solvent treated cells served as controls. (Bars represent meanSEM)

(5) FIGS. 5A-C. Sigma-1 receptor (S1R) agonist compounds [fluvoxamine (FIG. 5A), SA-4503 (FIG. 5B), PRE-084 (FIG. 5C)] inhibit TGF-induced extracellular matrix (ECM) production. In 6-well plates of NRK49F myofibroblasts (610.sup.6 cells/well) TGF- (1 nM) induced production of fibrillar components of the extracellular matrix was measured by Sirius Red staining. To investigate the effect of S1R agonist compounds a group of cells was treated for 48 hours with the various S1R agonists. Solvent treated cells served as controls. (Bars represent meanSEM)

(6) FIGS. 6A-H. Sigma-1 receptor (S1R) agonist fluvoxamine decreases diabetes induced tubulointerstitial fibrosis in the kidney of diabetic rats

(7) FIGS. 6A-H show the development of tubulointerstital fibrosis in the kidney sections of Streptozotocin-(65 mg/bwkg iv.) induced type 1 diabetic rats treated per os with (D): vehicle (isotonic saline); (D+7FLU): fluvoxamine (20 mg/bwkg/day) for 7 weeks or (D+FLU): fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes,) or (D+FLU2): fluvoxamine (2 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes. Additional groups were also treated per os with NE-100, a specific antagonist of S1R; (D+FLU+NE-100): fluvoxamine+NE-100 (20 mg/bwkg/day+1 mg/bwkg/day) for two weeks or (D+FLU2+NE-100): fluvoxamine+NE-100 (2 mg/bwkg/day+1 mg/bwkg/day) for two weeks from the 5.sup.th week of diabetes. Masson's trichrome staining of kidney sections was performed and the fibrotic area per total area was calculated. Average volumetric mesangial matrix expansion given relative to the total area in case of animal groups (6/A 1-7) (Bars represent MeanSEM, n=8-10/group, 20 magnification; scale bar 100 m). Specifically:

(8) FIG. 6A shows Control non-diabetic rats treated with isotonic saline, as vehicle only

(9) FIG. 6B shows Diabetic rats treated with isotonic saline, as vehicle only

(10) FIG. 6C shows Diabetic rats treated fluvoxamine (20 mg/bwkg/day) for 7 weeks

(11) FIG. 6D shows Diabetic rats treated fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes

(12) FIG. 6E shows Diabetic rats treated fluvoxamine (20 mg/bwkg/day)+NE-100 (1 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes

(13) FIG. 6F shows Diabetic rats treated fluvoxamine (2 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes

(14) FIG. 6G shows Diabetic rats treated fluvoxamine (2 mg/bwkg/day)+NE-100 (1 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes

(15) FIG. 6H shows Average volumetric tubulointerstitial fibrosis given relative to the total area in case of animal groups

(16) FIGS. 7A-H. Sigma-1 receptor (S1R) agonist compound fluvoxamine decreases diabetes induced mesangial matrix expansion in the kidney of diabetic rats

(17) FIGS. 7A-H show the development of mesangial matrix expansion in the kidney sections of Streptozotocin-(65 mg/bwkg iv.) induced type 1 diabetic rats treated per os with (D): vehicle (isotonic saline); D+7FLU fluvoxamine (20 mg/bwkg/day) for 7 weeks or (D+FLU): fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes,) or (D+FLU2): fluvoxamine (2 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes. Additional groups were also treated per os with NE-100, a specific antagonist of S1R; (D+FLU+NE-100): fluvoxamine+NE-100 (20 mg/bwkg/day+1 mg/bwkg/day) for two weeks or (D+FLU2+NE-100): fluvoxamine+NE-100 (2 mg/bwkg/day+1 mg/bwkg/day) for two weeks from the 5.sup.th week of diabetes. Kidney sections were stained with PAS reagent and mesangial fractional volume values (Vv) are defined by the ratio of mesangial area/glomerular tuft area. The mesangial area is determined by assessment of PAS-positive and nucleus-free areas in the mesangium (Bars represent MeanSEM, n=8-10/group, 20 magnification; scale bar50 m). Specifically:

(18) FIG. 7A shows Control non-diabetic rats treated with isotonic saline, as vehicle only

(19) FIG. 7B shows Diabetic rats treated with isotonic saline, as vehicle only

(20) FIG. 7C shows Diabetic rats treated fluvoxamine (20 mg/bwkg/day) for 7 weeks

(21) FIG. 7D shows Diabetic rats treated fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes

(22) FIG. 7E shows Diabetic rats treated fluvoxamine (20 mg/bwkg/day)+NE-100 (1 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes

(23) FIG. 7F shows Diabetic rats treated fluvoxamine (2 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes

(24) FIG. 7G shows Diabetic rats treated fluvoxamine (2 mg/bwkg/day)+NE-100 (1 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes

(25) FIG. 7AH shows Average volumetric mesangial matrix expansion given per glomeruli (glom) for animal groups of FIGS. 7A-G

(26) FIGS. 8A-E. Sigma-1 receptor (S1R) agonist compound fluvoxamine treatment decreases diabetes induced fibronectin accumulation in the kidney of diabetic rats

(27) FIGS. 8A-E show fibronectin accumulation in the kidney sections of Streptozotocin-(65 mg/bwkg iv.) induced type 1 diabetic rats treated per os with (D): vehicle (isotonic saline); or (D+FLU): fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes) or with (D+FLU+NE-100): fluvoxamine+specific S1R antagonist NE-100 (20 mg/bwkg/day+1 mg/bwkg/day) for two weeks from the 5.sup.th week of diabetes. Kidney sections were stained for fibronectin and the positive area/glomeruli was calculated for the sections (Bars represent MeanSEM, n=8-10/group, 20 magnification; scale bar50 m). Specifically:

(28) FIG. 8A shows Control non-diabetic rats treated with isotonic saline, as vehicle only

(29) FIG. 8B shows Diabetic rats treated with isotonic saline, as vehicle only

(30) FIG. 8C shows Diabetic rats treated fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes

(31) FIG. 8D shows Diabetic rats treated fluvoxamine (20 mg/bwkg/day)+NE-100 (1 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes

(32) FIG. 8E shows Average volumetric fibronectin positive area (given per total area) for animal groups of

(33) FIGS. 8A-D

(34) FIGS. 9A-E. Sigma-1 receptor (S1R) agonist compound fluvoxamine treatment decreases diabetes induced extracellular matrix (ECM) production in the kidney of diabetic rats

(35) FIGS. 9A-E show accumulation of fibrillar ECM components in the kidney sections of Streptozotocin-(65 mg/bwkg iv.) induced type 1 diabetic rats treated per os with (D): vehicle (isotonic saline); (D+7FLU): fluvoxamine (20 mg/bwkg/day) for 7 weeks or (D+FLU): fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes. Kidney sections were stained with 0.1% Sirius Red and the fractional volume values (Vv) are defined by the ratio of Sirius red-positive per total area. (Bars represent MeanSEM, n=8-10/group, 20 magnification; scale bar50 m). Specifically:

(36) FIG. 9A shows Control non-diabetic rats treated with isotonic saline, as vehicle only

(37) FIG. 9B shows Diabetic rats treated with isotonic saline, as vehicle only

(38) FIG. 9C shows Diabetic rats treated fluvoxamine (20 mg/bwkg/day) for 7 weeks

(39) FIG. 9D shows Diabetic rats treated fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes

(40) FIG. 9E shows Average volumetric Sirius Red positive area given per total area for animal groups of FIGS. 9A-D

(41) FIG. 10. Sigma-1 receptor (S1R) agonist compound fluvoxamine treatment decreases diabetes induced -smooth muscle (SMA) protein level in the kidney of diabetic rats

(42) FIG. 10 demonstrates the protein level of SMA in kidney homogenates of Streptozotocin-(65 mg/bwkg iv.) induced type 1 diabetic rats treated per os with (D): vehicle (isotonic saline); (D+7FLU): fluvoxamine (20 mg/bwkg/day) for 7 weeks or (D+FLU): fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes,) or (D+FLU2): fluvoxamine (2 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes. Additional groups were also treated per os with NE-100, a specific antagonist of S1R; (D+FLU+NE-100): fluvoxamine+NE-100 (20 mg/bwkg/day+1 mg/bwkg/day) for two weeks or (D+FLU2+NE-100): fluvoxamine+NE-100 (2 mg/bwkg/day+1 mg/bwkg/day) for two weeks from the 5.sup.th week of diabetes (Bars represent MeanSEM, n=8-10/group). Upper panel shows representative picture of western blot of SMA.

(43) FIGS. 11A-E. Sigma-1 receptor (S1R) agonist compound fluvoxamine treatment minimizes tubulointerstitial fibrosis in the kidney after unilateral ureteral obstruction (UUO)

(44) FIGS. 11A-E shows the development of tubulointerstital fibrosis in kidneys of six-week old mice 7 days after having UUO. Mice were treated once daily by oral gavage for one week with vehiculum (UUO), or with fluvoxamine (20 mg/bwkg/day) or with fluvoxamine+S1R antagonist NE-100 (1 mg/bwkg/day). Kidney sections were stained for Masson's trichrome and the ration of Masson positive/total area was calculated (Bars represent MeanSEM, n=6/group, 20 magnification; scale bar 100 m). Specifically:

(45) FIG. 11A shows Sham operated, control mice treated with vehicle only

(46) FIG. 11B shows Mice with UUO treated with vehicle only

(47) FIG. 11C shows Mice with UUO treated with vehicle only treated with fluvoxamine (20 mg/bwkg/day) for one week

(48) FIG. 11D shows Mice with UUO treated with vehicle only treated with fluvoxamine (20 mg/bwkg/day) +NE-100 (1 mg/bwkg/day) for one week

(49) FIG. 11E shows Average volumetric tubulointerstitial fibrosis given by Masson stained area per total area for animal groups of FIGS. 11-A-D.

(50) FIG. 12. Sigma-1 receptor (S1R) agonist compound fluvoxamine treatment minimizes -smooth muscle actin (SMA) production in the kidney after unilateral ureteral obstruction (UUO).

(51) FIG. 12 demonstrates the protein level of SMA in kidney homogenates of six-week old mice 7 days after having UUO. Mice were treated once daily by oral gavage for one week with vehicle (UUO), or with fluvoxamine (20 mg/bwkg/day). (Bars represent MeanSEM, n=6/group). Upper panel shows representative picture of western blot of SMA.

(52) FIGS. 13A-E. S1R agonist compound fluvoxamine treatment ameliorates interstitial fibrosis of the lung in a rat model of bleomycin-induced lung fibrosis.

(53) FIGS. 13A-E represent the development of lung fibrosis two weeks after the intratracheal injection of bleomycin in rats treated with vehicle (with or without sham operation) or with fluvoxamine (20 mg/bwkg/day) or with fluvoxamine+S1R antagonist NE-100 (1 mg/bwkg/day) for three weeks. Masson trichrome staining of tissue sections was performed Bars represent MeanSEM, n=6/group). Specifically:

(54) FIG. 13A shows Control non-bleomycin injected, sham operated rats treated with vehicle only

(55) FIG. 13B shows Bleomycin injected rats treated with vehicle only

(56) FIG. 13C shows Bleomycin injected rats treated with fluvoxamine (20 mg/bwkg/day) for two weeks

(57) FIG. 13D shows Bleomycin injected rats treated with fluvoxamine (20 mg/bwkg/day)+NE-100 (1 mg/bwkg/day) for three weeks

(58) FIG. 13E shows Average Masson-stained fibrotic pixels relative to all pixels for animal groups of FIGS. 13A-D

(59) FIG. 14. Sigma-1 receptor (S1R) agonist compound fluvoxamine treatment diminishes -smooth muscle (SMA) production in a rat model of bleomycin-induced lung fibrosis.

(60) FIG. 14 demonstrates the protein level of SMA in lung homogenates of rats treated with vehicle (with or without sham operation) or with fluvoxamine (20 mg/bwkg/day) or with fluvoxamine+S1R antagonist NE-100 (1 mg/bwkg/day) for three weeks after the intratracheal injection of bleomycin. (Bars represent MeanSEM, n=6/group). Upper panel shows representative picture of western blot of SMA.

(61) It should be noted that the term fibroblast is used to indicate myofibroblast in the figure legends.

(62) FIGS. 15A-C. Sigma-1 receptor (S1R) agonists suspend platelet-derived growth factor (PDGF)-induced cell proliferation in human trabecular meshwork-5 (HTM-5) cells.

(63) (FIG. 15A) MTT assay of 20 ng/mL platelet-derived growth factor (PDGF), 20 ng/mL PDGF+10 M fluvoxamine (FLU), 20 ng/mL PDGF+15 M FLU treatment for 24 h in human trabecular meshwork-5 (HTM-5) cells. (FIG. 15B) MTT assay of 20 ng/mL PDGF, 20 ng/mL PDGF+5 M sigma-1 receptor (S1R) agonist SA-4503 (SA), 20 ng/mL PDGF+10 M SA, 20 ng/mL PDGF+15 M SA treatment for 24 h in HTM-5 cells. (FIG. 15C) MTT assay of 20 ng/mL PDGF, 20 ng/mL PDGF+5 M S1R agonist PRE-087 (PRE), 20 ng/mL PDGF+10 M PRE, 20 ng/mL PDGF+15 M PRE treatment for 24 h in HTM-5 cells. All data are shown as meanSEM; n=6/group. *p<0.05 vs. Control; ***p<0.001 vs. Control; p<0.05 vs. PDGF; p<0.01 vs. PDGF; p<0.001 vs. PDGF.

(64) FIGS. 16A-C. S1R receptor agonist fluvoxamine reduces fibrotic markers in HTM-5 cells.

(65) FIG. 16A. Total RNA of FN1 (fibronectin) expression determined in human trabecular meshwork-5 (HTM-5) cells treated with 20 ng/ml platelet-derived growth factor (PDGF) (grey bar) and with 20 ng/ml PDGF plus 5 M fluvoxamine (FLU) (white bar).

(66) FIG. 16B. Total RNA of COL1A1 (collagen1a1) expression determined in human trabecular meshwork-5 (HTM-5) cells treated with 20 ng/ml PDGF (grey bar) and with 20 ng/ml PDGF plus 5 M fluvoxamine (FLU) (white bar).

(67) FIG. 16C. Total RNA MMP2 (matrix metalloproteinase 2) expression determined in human trabecular meshwork-5 (HTM-5) cells treated with 20 ng/ml PDGF (grey bar) and with 20 ng/ml PDGF plus 10 M FLU (white bar).

(68) In each of FIGS. 16A, 16B and 16C total RNA was represented by comparison with RN18S mRNA as internal control from the same samples. All data are shown as meansSEMs; n=6/group. .sup. p<0.05 vs. PDGF; .sup. p<0.01 vs. PDGF.

(69) FIGS. 17A-C. Effect of PDGF and SIR agonist FLU on actin cytoskeleton rearrangement in HTM-5 cells.

(70) Representative pictures of phalloidin immunostained human trabecular meshwork-5 (HTM-5) cells treated with platelet-derived growth factor (PDGF) or with PDGF plus fluvoxamine (FLU); 1000 magnification; yellow: F-actin; blue: nucleus; scale bar=100 m

(71) FIG. 17A. HTM-5 cells, untreated (Control);

(72) FIG. 17B. HTM-5 cells, treated for 24 h with 20 ng/mL PDGF;

(73) FIG. 17C HTM-5 cells, treated for 24 h with 20 ng/mL PDGF and 10 M FLU (PDGF+FLU).

(74) FIGS. 18A-C. Effect of PDGF and SIR agonist FLU on actin cytoskeleton rearrangement in HTM-5 cells.

(75) Representative pictures of phalloidin immunostained human trabecular meshwork-5 (HTM-5) cells treated with platelet-derived growth factor (PDGF) or with PDGF plus fluvoxamine (FLU); 6000 magnification; yellow: F-actin; blue: nucleus; scale bar=50 m FIG. 18A. HTM-5 cells, untreated (Control);

(76) FIG. 18B. HTM-5 cells, treated for 24 h with 20 ng/mL PDGF;

(77) FIG. 18C HTM-5 cells, treated for 24 h with 20 ng/mL PDGF and 10 M FLU (PDGF+FLU).

DETAILED DESCRIPTION OF THE INVENTION

(78) Progressive fibrosis is a common pathological response in many medical conditions. According to some estimates almost the half of all deaths is attributed to progressive organ fibrosis in the western world. For example, chronic Kidney Diseases (CKD) affect the 8-16% of the population worldwide and the number of them is continuously increasing mainly due to the increasing number of the diabetic patients.

(79) Progressive fibrosis is initiated by the sustained production of growth factor, proteolytic enzymes, angiogenic factors and/or fibrogenic cytokines, leading to progressive and excessive production of ECM components. In cases of progressive fibrosis when this process is not regulated to cease or reverse, the accumulation (and contraction) of ECM results in the expansion and stiffening of the interstitium that surrounds parenchymal units and disrupts their physiological function [Klingberg F et al. J Pathol. 229(2), 298-309 (2013)].

(80) Thus, in conditions of injuries or disturbed tissue or organ homeostasis usually accompanied by inflammation, resident and infiltrating immune cells secrete cytokines and growth factors, like platelet-derived growth factor (PDGF), transforming growth factor- (TGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), connective tissue growth factor (CTGF), glucose, angiontensin II, or aldosterone and various interleukins (IL-1alpha, IL-1, IL-4, IL-8, IL-13). These mediators facilitate formation and accumulation of SMA expressing and ECM-producing myofibroblasts.

(81) Myofibroblasts and the ECM in which they reside are critical components of the progressive fibrotic process. The ECM is actually a functional tissue whose components possess not only scaffolding characteristics, but also growth factor, mitogenic, and other bioactive properties. Progressive fibrosis oftentimes is leading to organ dysfunction and increased morbidity or mortality, and it is also associated with a kind of dysregulation of the tissue processes in disturbed tissue homeostasis [Lekkerkerker S et al. Curr Pharm Des. 18(27), 4093-102 (2012)]. Thus, progressive fibrosis is a condition that cannot be considered homeostatic and/or serves no longer as a regenerative process; it might be associated with a disease or may constitute a condition that requires medical treatment.

(82) The present inventors have unexpectedly recognized that fluvoxamine, the potent Sigma-1 receptor (S1R) agonists is useful in the prevention and/or inhibition of fibrotic remodeling of ECM and thereby in progressive fibrotic conditions. Specifically the inventors discovered that fluvoxamine successfully improves the impairment of renal function associated with renal fibrosis (confirmed by the improvement in conventional, gold-standard, clinical parameters e.g. GFR, creatinine, serum urea nitrogen etc). Furthermore they demonstrated that S1R agonists have an anti-fibroproliferative potential also in various tissues.

(83) As explained below it is understood herein that accumulation of myofibroblasts and/or overproduction and deposition of ECM components may be indicative of and/or associated with progressive fibrosis associated with pathological conditions and which may occur in several disorders. Treatment of progressive fibrosis, however, can be differentiated from the treatment of the disease which is accompanied by said progressive fibrosis and may even be independent therefrom.

(84) Thus, according to the invention in a given condition or in a given subject/patient the treatment may be directed to prevention, control, reversal or inhibition of fibrotic remodeling of ECM preferably including accumulation of myofibroblast and/or excessive production and deposition of ECM components, e.g. fibrillar components thereof, including collagen, preferably type I and III collagen or fibronectin or excessive production and/or rearrangement of actin filaments in the fibrotic cells. Thereby the present invention may lead to the amelioration of the patient's condition regarding the underlying or causative disease e.g. as listed herein.

(85) The invention provides compounds and compositions for use in the prevention or treatment of progressive fibrosis, in particular in the prevention, control, reversal or inhibition of progressive fibrosis. Once fibrotic processes are inhibited or prevented, this may allow the regenerative mechanisms of the organism to take place. Thereby a fibrotic condition may even be reversed.

(86) The Sigma Receptor

(87) The Sigma receptor is as a ligand-regulated molecular chaperon in the endoplasmic reticulum. Sigma receptors consist of two subtypes, Sigma-1 and Sigma-2 (S1R and S2R) receptors (alternative names: sigma non-opioid intracellular receptor, AAG8, ALS16, Aging-associated gene 8 protein, OPRS1, SIG-1R). The S1R was cloned in 1996 and its molecular conformation was then explored. S1R and S2R receptors have no close homology to any other mammalian proteins. The human 223-amino acid protein S1R is localized in various tissues including the brain, intestine, liver, spleen, lung, kidney, skeletal muscle, adrenal glands, genital tract, skin and eye [Hanner M et al. Proc Natl Acad Sci USA. 93(15). 8072-8077 (1996)]. S1R can be found in a large number on the endoplasmic reticulum, in particular on the mitochondria-associated ER membrane where they proposed to function as receptor chaperones However outside the central nervous system the function and regulation of the S1R is almost unknown.

(88) S1R has been suggested to take part in a number of diseases of the central nervous system. The primary therapeutic targets of agonists include schizophrenia, major depression, obsessive-compulsive disorder (OCD), and Alzheimer's disease and major depressive disorder [Ishikawa M et al. Journal of Receptor, Ligand and Channel Research 3, 25-3 (2010)]. Information is scarce about the potential role and use of S1R agonists outside the central nervous system. Furthermore, the study of clinical potential of S1R agonists is in its very beginning.

(89) It is contemplated that in principle any S1R receptor agonists might be applicable in the present invention. Preferred are S1R agonists which are selective over S2R. Also preferred are S1R agonists which have a strong affinity to S1R receptor and which have less side-effects.

(90) A compound is selective for S1R over S2R if it has a higher affinity for S1R than S2R, preferably 5 times higher or 20 times higher or 50 times higher or at least 10.sup.2 higher, at least 10.sup.3 higher or at least 10.sup.4 higher.

(91) S1R agonists belong to various structural groups of compounds. In the present invention compounds as defined in the brief description of the invention are preferred.

(92) In the experimental part illustrative experiments are shown with three S1R agonist compounds: fluvoxamine, SA-4503 (cumetasine) and PRE-84. Each compound has different structures and each of them has been surprisingly found to be active in controlling progressive fibrosis. Fluvoxamine was found to be successful even in preventing, inhibiting and reversing progressive fibrosis in the kidney and in the lung. As it has been shown, other S1R agonists, e.g. SA-4503 (cumetasine) and PRE-84 have the same effect in vivo. Fluvoxamine is particularly preferred.

(93) In a further set of experiment the same three S1R agonist compounds, fluvoxamine, SA-4503 (cumetasine) and PRE-84 were used to inhibit progressive fibrosis in an in vitro model of progressive fibrosis of the eye HTM-5 cell wherein progressive fibrosis has been artificially elicited by PDGF. Excessive fibrotic proliferation of cells, expression of matrix components indicating fibrosis, as well as active overproduction and rearrangement of F-actin filaments were reversed by different Sigma-1 receptor agonists. HTM cells can be obtained e.g. from DELL APPLICATIONS INC, San Diego, Calif., USA.

(94) In Table A below a number of S1R receptor agonist are listed which are contemplated for use according to the invention.

(95) TABLE-US-00001 TABLE A Name Formula IUPAC name fluvoxamine 2-{[(E)-{5-Methoxy-1-[4-(trifluoro- methyl)phenyl]pentylidene}amino]oxy}- ethanamine fluoxetine 0 N-methyl-3-phenyl-3-[4- (trifluoromethyl)phenoxy]propan-1-amine sertraline (1S,4S)-4-(3,4-dichlorophenyl)-N- methyl-1,2,3,4-tetrahydronaphthalen-1- amine SA 4503 (cutamesine) 1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-(3- phenylpropyl)piperazine BD1031 (8aR)-2-[2-(3,4- Dichlorophenyl)ethyl]octahydropyrrolo[1,2- a]pyrazine BD1052 N-[2-(3,4-dichlorophenyl)ethyl]-N-(2- pyrrolidin-1-ylethyl)prop-2-en-1-amine 4-IBP N-(N-Benzylpiperidin-4-yl)-4- iodobenzamide PRE-084 2-morpholin-4-ylethyl 1- phenylcyclohexane-1-carboxylate Pentoxyverine (rINN) or carbetapentane 2-[2-(diethylamino)ethoxy]ethyl 1- phenylcyclopentanecarboxylate ()-PPCC oxalate (S*,R*)-2-[(4-Hydroxy-4-phenyl-1- piperidinyl)methyl]-1-(4-methylphenyl)- cyclopropanecarboxylic acid methyl ester haloperidol metabolite II (reduced haloperidol) 4-[4-(4-Chlorophenyl)-4-hydroxy-1- piperidyl]-1-(4-fluorophenyl)-butan-1-ol ANAVEX2-73 Tetrahidro- N,N-dimetil- 2,2-difenil-3- furnmetnamin HCl 0 Tetrahydro-N,N-dimethyl-2,2-diphenyl- 3-furanmethanamine hydrochloride RC-33 1-[1-(4-biphenyl)-1-methyl- propyl]piperidine

(96) S1R agonists for use in the present invention can be prepared according to methods known for a skilled person or are commercially available like fluvoxamine, SA-4503, PRE-084, 4-IBP, ANAVEX2-73, etc.

(97) For example, fluvoxamine maleate can be prepared as described in U.S. Pat. No. 4,085,225 and in U.S. Pat. No. 6,433,225 B1.

(98) EP2353598A1 discloses synthesis of sigma-receptor ligands including cumetasine and related compounds.

(99) PRE-084 is a high affinity, sigma-receptor agonist, selective for the S1R subtype (Kis=2.2 and 13,091 nM for 1 and 2 receptors, respectively). It is a potent ligand of the S1R (IC50=44 nM) without appreciable affinity for PCP receptors (IC50>100,000 nM) and its availability is described e.g. [Griesmaier E et al. Experimental Neurology 237(2), 388 395 (2012)]. Rossi, Daniela et al. describe the synthesis of sigma-receptor ligands based on arylalkenylaminic scaffold, among others RC-33, see Table A [Rossi D et al. Bioorganic & Medicinal Chemistry 19(21), 6210-6224 (2011)].

(100) It is known for a large number of compounds that they are S1R agonist. To test binding affinity and measure dissociation constant can be done by usual methods in protein and bioorganic chemistry.

(101) For example Xu, Rong et al. disclose the effect of ether modifications to SA-4503 on binding affinity and selectivity for sigma receptors and monoamine transporters and methods to measure these parameters [Rong Xu et al. Bioorganic & Medicinal Chemistry 23(1), 222-230 (2015)].

(102) Furthermore, Rossi, Daniela et al. (see above) selected and identified a potent and selective S1R agonist among a number of compounds and described related methods. Moreover, the authors have developed a three dimensional S1R pharmacophore model using active compounds only to derive this model. The model included two hydrophobes and a positive nitrogen as relevant features and it was able to discriminate between molecules with and without affinity toward 1 receptor subtype. Thus, it is well within the skills of a person skilled in the art to prepare and select compounds according to the invention.

(103) As also shown in the Examples below, it is well within the skills of a person skilled in the art to test whether a potential S1R agonist is actually an agonist.

(104) A usual method to test whether an S1R agonist acts on the S1R is to use a specific antagonist, as a control. Such a well-accepted specific antagonist is NE-100 which is a potent and selective S1R antagonist (K.sub.i=0.86 nM) that displays >55-fold selectivity over S2R and >6000-fold selectivity over D.sub.1, D.sub.2, 5-HT.sub.1A, 5-HT.sub.2 and PCP receptors (4-Methoxy-3-(2-phenylethoxy)-N,N-dipropylbenzeneethanamine hydrochloride). NE-100 exhibits reversible binding (K.sub.d=1.2 nM) [Okuyama S et al. CNS Drug Rev. 2(2), 226-237 (1999), Berardi F et al. Bioorg. Med. Chem. 9(5), 1325-35 (2001)].

(105) Below a few diseases are listed as examples that often or even necessarily associated with progressive fibrosis and a few options to treat progressive fibrosis are mentioned. It is to be understood, however, that these examples though may be preferred, are merely illustrative. As progressive fibrosis may occur in a number of disorders other embodiments of the invention may be reduced into practice without departing from the concept and scope of the present invention. Typically these disease conditions are accompanied by abnormal proliferation of myofibroblasts and/or excessive production of ECM components. Such diseases in which pathogenic progressive fibrosis may be evident or imminent may be acute or chronic. Preferably progressive fibrosis is prevented or treated in chronic diseases or chronic fibroproliferative diseases.

(106) Progressive Fibrosis in the Kidney

(107) A number of diseases such as metabolic, anatomical, mechanical abnormalities, infections and toxic agents or autoimmune diseases can result in loss of kidney function. The most frequent examples of kidney related diseases in which progressive fibrosis are part of the syndrome are: diabetic nephropathy, hypertensive nephropathy, different types of glomerulonephritis, and certain tubulointerstitial disorders. Patients with diabetes and hypertension are at greatest risk and have a higher rate of renal problems than the normal population. Diabetic nephropathy accounts for 25-30% of new patients commencing renal replacement therapy worldwide. Antibiotics, analgesic drugs (aspirin, ibuprofen, acetaminophen etc.), chemotherapic agents, different drugs and various infections have all been also identified as progressive fibrosis inducing agents.

(108) The different forms of renal diseases mimic a sustained injury leading to an excessive accumulation of ECM that may occur in virtually all type of chronic kidney failure. In diabetic nephropathy progressive fibrosis arises through activation of renal myofibroblasts to secrete certain proteins of the connective tissue, most commonly collagen types I, III, and IV and fibronectin and thereby remodel the ECM. Compounds used in diabetic nephropathy include renin-angiotensin-aldosterone system (RAAS) blockers, primarily ACE-inhibitors and ARBs, none of which directly aims at the overproliferation of ECM. A review of medication dosing in patients with chronic kidney disease is provided by Zuber K et al. the principles of which, and references cited therein, may be used as guidance for setting the dose of S1R agonists [Zuber K et al. JAAPA. 26(10), 19-25 (2013)].

(109) Early phase diagnosis is preferred in treatment of the progressive fibrosis of the kidney. Present treatments focus on preventing and improving the symptoms and the progression of the disease itself. Oral administration in this case is preferred. Parenteral intravenous, intramuscular, intracutan or subcutan administration would be also an option, or direct infusion to target the kidney is also possible.

(110) Progressive Fibrosis in the Lung

(111) Infections, long-term exposure to pollutants or toxins (most commonly smoking), allergy, certain medications (e.g. chemotherapics), gastroesophageal reflux, autoimmun diseases (e.g. SLE) are all risk factors or potential causes of lung diseases characterized by inflammation and resulting in abnormal tissue repair. Scar forming and the thickening of the walls of the lungs lead to oxygen shortage and diseases identified under the generic term pulmonary fibrosis.

(112) To the present knowledge scarring that occurs in progressive pulmonary fibrosis cannot be reversed, and no current treatment has proved effective in halting the progression of the disease. Some treatments including corticosteroids or immunsuppressive therapy may improve symptoms temporarily but their efficacy regarding fibrotic conditions is rather questionable with serious side effects.

(113) Administration of the composition of the invention is preferably started at an early phase of the onset of disease. Inhalation represents a preferred option through systemic administration, if possible. Means for this type of administration, like powder inhalers, vaporizers, nebulizers, devices like oxygen mask, nasal cannula and metered dose inhalers are well-known in the art.

(114) Progressive Fibrosis in the Gastrointestinal System

(115) In chronic intestinal inflammatory conditions, inflammation is accompanied by a response where progressive fibrosis is an inevitable or very common component. In Crohn-disease, inflammation is typically transmural and so is the ensuing fibrostenotic response, whereas, in ulcerative colitis, inflammation and a progressive fibrotic response are virtually limited to the mucosal layer. Transmural inflammation and progressive fibrosis typically frequently result in symptomatic stenosis or stricture. Stellate cells are found not exclusively in liver, but also in the pancreas and human intestinal mucosa. Infiltrating immune cells and intestinal stellate cells release different cytokines and growth factors, such as TGF, which contribute to the remodeling of the ECM. Some cells of non-mesenchymal origin also undergo a process of transdifferentiation into mesenchymal cells to become efficient ECM-producing cells.

(116) Although intestinal fibrosis is increasingly recognized as a problem, there is no accepted medication in the art to treat or hinder organ fibrosis of the GI system.

(117) Preferably administration is carried out through the digestive tract. Examples of oral formulations include solid forms like pills, tablets, capsules, pastilles etc. Liquid forms include syrups, emulsions, suspensions, hydrogels, encapsulated forms, preferably in an extended release form.

(118) Progressive Fibrosis in the Liver

(119) A common symptom of fibroproliferative diseases of the liver (e.g. cirrhosis steatohepatis, infectious hepatitis, biliary diseases, storage diseases like hemochromatosis or Wilson's disease) may be the accumulation of excess connective tissue in the liver accompanying hepatocellular damage. Abnormal degradation of the ECM may also contribute to the progressive fibrosis of the liver. During the progression of fibrosis, activated stellate cells (or liver-specific pericytes) show features of smooth muscle-like cells, characterized by expression of a number of contractile filaments including -SMA and myosin. As fibrosis advances, the activated stellate cells progressively impede portal blood flow mediated by pathways that allow interaction with the ECM. The end stage of chronic liver disease, without liver transplantation, frequently leads to death.

(120) Early phase diagnosis is preferred also in treatment of the progressive fibrosis of the liver. Oral administration of compounds of the invention in this case is preferred. Parenteral intravenous, intramuscular, intracutan or subcutan) administration would be also an option.

(121) Progressive Fibrosis in the Organs of the Urogenital System

(122) The urogenital system can also be affected by diseases associated with progressive fibrosis when they are exposed to various infections (e.g. chlamydia, candida or herpes). Women are at risk of injuries to the organs of the reproductive system also during pregnancy, delivery or miscarriages. Irradiation associated fibrosis of the vagina or prostate could be a consequence of the anti-tumor treatment of the urogenital organs (e.g. ovarian or prostate cancer) Endometriosis is a severe progressive fibrotic disorder causing constant pain and infertility. Penile fibrosis is a possible cause of impotency in men.

(123) Oral administration of compounds of the invention in this case is preferred and parenteral administration, like injection or infusion is also possible. Topical e.g. transmucosal administration may be possible if this may provide a better targeting of the drug. Topical formulae may include in this group of disorders ointment, vaginal or rectal suppositories, and rings etc, intrauterine devices.

(124) Progressive Fibrosis in the Skin

(125) Defective wound healing consists of two categories: in the case of chronic wounds (e.g. ulcerative lesions) the healing process is delayed or blocked, while in excessive wound healing (e.g. hypertrophic scars, keloids), the repair process is hyperactivated. Excessive wound healing occurs when ECM synthesis remains high, resulting in overproduction of collagen and other ECM components. This condition may arise from a failure of myofibroblasts to undergo apoptosis and results in hypertrophic scarring, leaving permanent and undesirable marks on the skin. In dermal keloids, the overproduction of collagen type I or type III extend beyond the boundaries of the original injury.

(126) Most often progressive fibrosis related dermatological diseases or conditions are as follows: keloids of various origin (e.g. acne, piercing, chicken-pox), ulcus (e.g. diabetes derived), and various infectious diseases e.g. acne vulgaris, acne inversa). Treatment focuses on improving the symptoms and preventing the progression of fibrosis. Topical administration of the compound of the invention is preferred when treating a skin related symptom. Preventive treatment is contemplated when the patient is at risk of defective wound healing.

(127) In dermal applications topical formulation of the medicament is preferred, among others ointments, topical creams and gels, dermal and transdermal patches and films, hydrogels, creams, lotions and sprays.

(128) Progressive Fibrosis in the Eye

(129) Most diseases that cause loss of vision are related to abnormal wound healing and angiogenesis. While among the most frequent causes are tissue ischemia or inflammation, it is usually, among others, the concomitant fibrosis that results in mechanical disruption of the visual axis [Friedlander, Martin. (2007) 117(3): 576-586].

(130) Fibrosis, as a local response to injuries includes infiltration by inflammatory cells, neovascularization, altered vascular permeability, proliferation of fibroblasts and fibroblast-like cells, modification of the ECM, and, ultimately, some sort of resolution of the damaged tissue. Progressive fibrosis of the eye included fibrosis of the anterior segment of the eye which is not part of the central nervous system and posterior segments i.e. those which are part of the CNS, like retina wherein progressive fibrosis may be called gliosis. Preferably, in the present invention, fibrosis is present in the anterior segments. However, posterior segments i.e. those which are part of the CNS, like retina, have similar processes and in a broader aspect the invention covers those tissues as well.

(131) In preferred embodiments the fibroproliferarive disease of the eye may be retinal fibrosis wherein a dysregulated accumulation of extracellular matrix (ECM) protein occurs. This can occur as the result of an aberrant wound-healing response, as in the case of post-surgical complications of proliferative vitreoretinopathy (PVR). Fibrosis has a role in other retinal diseases including diabetic retinopathy (DR) and age-related macular degeneration (AMD) as well, wherein neovascularization, a typical hallmark of late stage DR just as AMD, involves aberrant ECM assembly [Miller Charles G et al. Minireview: (2016) Fibronectin in retinal disease Experimental Biology and Medicine 242(1), 1-7]

(132) A quite frequent eye disease, glaucoma, is characterized by neuropathy and loss of retinal ganglion cells, cupping and atrophy of the optic nerve head, and associated visual field loss. Primary open-angle (POAG) glaucoma, is a usual form wherein free aqueous humor (AH) outflow occurs, which due to clogging of trabecular meshwork (TM) channels, follows an abnormal route [Zhavoronkov, Alex et al. Pro-fibrotic pathway activation in trabecular meshwork and lamina cribrosa is the main driving force of glaucoma Cell Cycle. 2016; 15(12): 1643-1652]. It is well known that fibrosis plays a role in the glaucoma progression and a number of therapeutic approaches have been studied in an attempt to combat fibrosis [see e.g. Hill, Lisa J, Neural Regen Res. 2016 Jun.; 11(6): 922-923.], however, mechanisms of fibrosis have been largely unknown.

(133) The Trabecular Meshwork

(134) The trabecular meshwork is an important regulator of intraocular pressure. The TM cells (TMC) display characteristic features of several different cell types, like those of endothelia, fibroblasts, smooth muscle and macrophages, owing to the multiple roles and two distinct environments where they operate to maintain intraocular pressure homeostasis [Stamer W. Daniel et al. The many faces of the trabecular meshwork cell Exp Eye Res. 2017 158 112-123.]. TMC make layers of beams, part of a fibrous basement membrane containing ECM cells. Damage and dysfunction of HTMC have clinical significance. For instance, physical changes to the TM include increased fibrosis, fibronectin accumulation, and expression of ECM cross-linking enzymes. This cytoskeletal reorganization and cell loss causes the TM to become rigid and stiff which may play role in several diseases, like glaucoma-related dysfunctions. Thus, fibrosis in TMC are good model for ocular fibrosis.

(135) Concepts of Diagnosis

(136) While it is known that many diseases are associated with progressive fibrosis the present invention is useful to prevent or inhibit the formation of excessive amount of ECM in different tissues and organs. While at present diagnosis of progressive fibrosis has its difficulties, diagnosis of such a condition is possible and advisable.

(137) Inevitably, microscopic examination of tissue biopsies is one of the most reliable methods of diagnosing fibrotic tissue. Detection of the proliferation of mesangial cells (mesangial matrix expansion, (see e.g. Examples 8) and/or myofibroblasts, (e.g. as in Example 11, 13, 15) is clearly a possibility. Measurement of markers of progressive fibrosis like increased presence of Masson's trichrome (e.g. in Example 7, 12, 14) or Sirius red e.g. Example 10 positivity, increased expression of -SMA (e.g. in Example 11, 13, 15) or determination of the amount of fibronectin (e.g. in Example 9) in the tissue is a further option (Example 5).

(138) Several morphometry techniques are used to assess progressive interstitial fibrosis, including morphometry of slides stained with Masson's trichrome or Sirius Red which are specific for collagen types I and III under polarized light and immunohistochemistry method [Farris A B, United States and Canadian Academy of Pathology Annual Meeting (2012)]. The method is, however, invasive and inconvenient to the patient and quite often needs anesthesia. A skilled pathologist is needed for the assessment and the whole evaluation procedure is rather slow to use as routine clinical application. Moreover, invasive methods, while applicable in case of need, have their own risk [Dez J, Circ J. 72, A:A8-12 (2008)].

(139) Thus, from the aspect of patient well-being and compliance non-invasive physical methods are preferred. There are certain functional non-invasive markers that are used as gold-standard values in the estimation of organ function (including kidney: glomerular filtration rate (GFR) and serum creatinine and urea nitrogen, proteinuria [KDIGO, 2013]; lung: spyrometry etc, liver: fibroscan. Pulmonary fibrosis can be diagnosed based on the Guidelines provided by the American Thoracic Society [Raghu et al. Am J Respir Crit Care Med 183, 788-824 (2011)]. Progressive liver fibrosis can be diagnosed by serum markers (hepatic myofibroblast specific single chain antibody C1-3 was conjugate and imaging techniques that are in a research-phase yet [Hill S, Thesis (2012)].

(140) These markers can predict the deterioration of the organ function, but they are not always specific enough for the progressive fibrotic process. Furthermore at present all these markers are expensive and slow to perform, therefore the techniques are expected to improve in the future and new markers will be probably also discovered.

(141) As to biomarkers of the fibrosis in the eye the research is underway. Cynthia Yu-Wai-Man et al. [Cynthia Yu-Wai-Man Personalized Medicine in Ocular Fibrosis: Myth or Future Biomarkers. Adv Wound Care (New Rochelle). 2016 Sep. 1; 5(9): 390-402.] list a number of new biomarkers which are under validation. However, such biomarkers can be used in the present invention to diagnose risk of scarring and to decide on as well as to carry out the antifibrotic treatment regimen to each individual patient.

(142) However, progressive fibrosis in the eye, albeit in a later stage than diagnosis by biomarkers, can be established by the comprehensive ocular examination including the measurement of ocular pressure as well.

(143) As to diagnosis of glaucoma, a fibroproliferative eye disease the treatment of which is preferred according to the invention, preferably the following factors should be examined:

(144) the inner eye pressure (preferably by tonometry),

(145) the examination, eg. shape and color of the optic nerve (preferably by ophthalmoscopy or dilated eye exam or computer measurement),

(146) the complete field of vision (preferably by perimetry or visual field test),

(147) the angle in the eye where the iris meets the cornea (preferably by gonioscopy),

(148) the thickness of the cornea (preferably by pachymetry).

(149) By this method e.g. open or closed angle glaucoma, and preferably glaucoma of the anterior or posterior eye tissues can be diagnosed.

(150) Below the invention is illustrated through specific examples and exemplary embodiments which, however, do not limit the scope of the invention.

EXAMPLES

(151) Methods

(152) Compounds

(153) fluvoxamine (fluvoxamine maleate, Sigma Aldrich, St. Louis, Mo., USA), PRE-084 (2-morpholin-4-dylethyl 1-phenylcyclohexane-1-carboxylate Sigma Aldrich, St. Louis, Mo., USA), SA4503 (1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine; Tocris Bioscience, Bristol, UK); NE100 (N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)-phenyl]-ethylamine monohydrochloride, Tocris Bioscience, Bristol, UK)

(154) Cell Lines

(155) NRK49F rat kidney interstitial fibroblast cell lines (American Type Culture Collection, Manassas, Va., USA) were cultured in Dulbecco's modified Eagle's medium (Gibco, Life Technologies, Carlsbad, Calif., USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Carlsbad, Calif., USA) and 1% Antibiotic-Antimycotic Solution (Sigma-Aldrich Co., St. Louis, Mo., USA) at 37 C. and 5% CO.sub.2.

(156) Cell Viability and Proliferation Assay

(157) To test the possible cytotoxic effect of the said compounds cell viability was determined in 96-well plate by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay after 24-hours treatment with the said S1R compounds (Roche Diagnostics, Mannheim, Germany). Cell viability was also assessed by trypan blue exclusion. Cells were detached with trypsin-EDTA and re-suspended in medium diluted 1:1 with trypan blue solution (Sigma Aldrich, Budapest, Hungary). Live cells from triplicate wells were counted in a Burker chamber.

(158) To investigate the effect of S1R agonists (fluvoxamine, PRE084, SA4503) on PDGFB induced proliferation renal fibroblasts cells were starved in 0.01% FBS for 24 hours then trypsinized and seeded in 6-well plates at a density of 510.sup.5 cells/well. After plating, cells were treated with human recombinant rPDGFBB; (10 ng/mL, R&D Systems, Minneapolis, Minn., USA). A group of cells was treated with rPDGFBB and fluvoxamine (1, 5 and 10 M/L; Cell Signaling Technology Inc., Danvers, Mass., USA). Control cells were treated with solvents (4 mM HCl, Sigma-Aldrich Co., St. Louis, Mo., USA) alone. Subsequently cells were incubated for 24 hours at 37 C. then cell proliferation assay (MTT) was performed.

(159) PicroSyrius Red Stain to Measure Collagen Production

(160) To investigate the deposition of fibrillar collagen Sirius Red staining was performed. 48 hours after the treatment with TGF- and said compounds the NRK-49F cells were incubated for 10 minutes with Kahle fixative solution. 0.1% Sirius Red (Direct Red 80, Sigma-Aldrich) in 1.2% picric acid was added for each well and plates were incubated for 30 minutes at RT. The unconnected dye molecules were washed with distilled water. The bound Sirius Red dye was eluated with 0.1 M NaOH solution, absorbance was recorded at 540 nm in a Hidex Chameleon Microplate Reader (Triathler, Plate Chameleion, 300SL Lablogic Systems, Inc., Brandon, Fla., USA) used MikroWin program. Vehicle treated cells served as controls.

(161) Collagen I-III PCR

(162) Total RNA was isolated from NRK49F cells by RNeasy Micro RNA isolations kit (Qiagen GmbH, Hilden, Germany). 100 ng RNA was reverse-transcribed using SuperScript III RNase H (Gibco, Life Technologies, Carlsbad, Calif., USA) to generate first-strand cDNA. The mRNA expressions of collagen I, collagen III and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by real-time RT-PCR using Light Cycler 480 SYBR Green 1 Master on a Light Cycler system (Roche Diagnostics, Mannheim, Germany). The reaction mix contained 10 mol/l of each PCR primers (Table 1; Invitrogen, Life Technologies, Carlsbad, Calif., USA), 10 l of Light Cycler 480 SYBR Green 1 Master enzyme mix (Roche Diagnostics, Mannheim, Germany) and 1 l of cDNA sample. The conditions of the PCRs were as follows: 1 cycle at 95 C. for 5 minutes, followed by 60 cycles under the appropriate PCR conditions. Quantification was performed with the second-derivative method by monitoring the cycle number at which the fluorescent sign could be distinguished from the background. Results were analyzed with Light Cycler 480 software version 1.5.0.39 (Roche Diagnostics, Mannheim, Germany). The mRNA expression of each gene was determined by comparison with GAPDH as internal control from the same sample.

(163) TABLE-US-00002 TABLE1 Nucleotidesequenceofspecificprimerpairsappliedfortherealtime detectionoftheexaminedgenesandconditionsofthePCRreactions. PCR Gene Primersequences conditions Rat F:5-AGCTCAGGGGCGAAGGCAACAGTC-3 (SEQID 95 C.-5sec collagenI NO:1) 59 C.-7sec R:5-CAGGCGGGAGGTCTTGGT-3 (SEQIDNO:2) 72 C.-7sec Rat F:5-AGGCGGTGCGGGTGCTGAT-3 (SEQIDNO:3) 95 C.-5sec collagenIII R:5-GGGCCAGGGGGACCAATAGGA-3 (SEQIDNO: 59 C.-7sec 4) 72 C.-7sec RatGAPDH F:5-GTCACGGCATGGACTGTG-3 (SEQIDNO:5) 95 C.-5sec R:5-CACCACCATGGAGAAGGCTG-3 60 C.-5sec (SEQIDNO:6) 72 C.-10sec

(164) In Vivo Models of Fibrosis

(165) Animals

(166) The institutional committee on animal welfare approved all experiments. Experiments were performed on Male Wistar rats weighing 20515 g (Toxi-Coop Toxicological Research Center, Dunakeszi, Hungary) or 7-8 week old male C57BL/6 mice (WT; Charles River Laboratories, Sulzfeld, Germany). Animals were housed in a temperature-controlled (221 C.) room with alternating light and dark cycles and had free access to standard rat chow and water.

(167) During surgical procedures or at animal harvest general anesthesia was performed by an i.p. injection of ketamine (75 mg/bwkg) and xylazine (10 mg/bwkg). (Richter Ltd., Budapest, Hungary).

(168) Rat Model of Streptozotocin (STZ) Induced Diabetic Nephropathy

(169) All substances were purchased from Sigma-Aldrich Ltd. (Budapest, Hungary). Diabetes was induced in male Wistar rats by 65 mg/bwkg streptozotocin (STZ) i.v. (dissolved in 0.1 M citrate buffer; pH 4.5). Animals were considered diabetic if blood glucose concentrations increased to 15 mmol/L within 72 h after STZ injection and remained elevated. Animals were randomly divided into groups (n=10-12/group) and received by per os (i) fluvoxamine (20 mg/bwkg/day) for 7 weeks; (ii) fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes, (iii) fluvoxamine (2 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes; or (iv) vehicle (isotonic saline). Additional groups were also treated per os with NE100, a specific inhibitor (antagonist) of S1R (v) fluvoxamine+NE100 (20 mg/bwkg/day+1 mg/bwkg/day) for two weeks, (vi) fluvoxamine+NE100 (2 mg/bwkg/day+1 mg/bwkg/day) for two weeks. Non-diabetic age-matched control animals were injected with citrate buffer and sacrificed after 7 weeks (n=8-10/group).

(170) Before and during and at the end of treatment period rats were placed into metabolic cages to collect 24-hour urinary samples. After 2 weeks of treatment all rats were anesthesized, blood and urinary samples were collected and the kidneys were removed, weighed and a section fixed in formalin (4%, pH=7.4) for histology and the remained immediately snap-frozen for further investigations.

(171) Mice Model of Unilateral Ureteral Obstruction (UUO) Induced Renal Fibrosis

(172) After general anesthesia animals were placed on a thermo controlled table to maintain rectal temperature at 371 C.). After standard midline laparotomy the bowel was gently displaced from the abdomen and covered with sterile saline soaked sterile gauze. Left ureter was isolated by blunt dissection and completely ligated using fine suture material (6/0 Safil, B. Braun Aesculap, Panama, USA). The bowel was then laid back and the muscle and skin were closed with 4-0 nylon sutures. Mice were treated with fluvoxamine (20 mg/bwkg/day, i.p.) or fluvoxamine+NE100 (1 mg/bwkg/day, i.p.). Left kidneys of the mice were surgically removed on the 7th day (n=6) after the onset of UUO. As surgical controls, animals (n=6) underwent identical surgical procedure without occlusion of left ureter. Kidney segments were immediately used for molecular biological measurements or frozen in liquid nitrogen and fixed in formalin (4%, pH=7.4).

(173) Rat Model of Bleomycin Induced Pulmonary Fibrosis

(174) For the induction of pulmonary fibrosis male Wistar rats were anesthetized. Bleomycin (5 mg/bwkg in a 300 l solution of isotonic saline) or 300 l isotonic saline was administered into the trachea using a 30G needle.

(175) Animals were randomly divided into 4 groups of 6 rats each as follows (i)control group was sham operated, received isotonic saline per os daily for 3 weeks, (ii) vehicle treated group: received isotonic saline per os daily for 3 weeks after the induction of lung fibrosis, (iii) fluvoxamine treated group: received fluvoxamine (20 mg/bwkg/day per os for 3 weeks after the induction of lung fibrosis), (iv) fluvoxamine+NE100 treated group: received fluvoxamine (20 mg/bwkg/day; per os) and 1 mg/bwkg/day NE100 (i.p. for 3 weeks after the induction of lung fibrosis.

(176) For the induction of pulmonary fibrosis rats were anesthetized and bleomycin (5 mg/bwkg in a 300 l solution of isotonic saline) or 300 l isotonic saline was administered intratracheally using a 30G needle. Animals were sacrificed 21 days after the induction of pulmonary fibrosis.

(177) Measurement of Metabolic and Renal Parameters

(178) Serum metabolic (glucose, fructoseamine, total and HDL-cholesterol, triglycerides) and renal functional parameters from rat serum (sodium, potassium, creatinine, BUN, GFR and proteinuria) were determined with commercially available kits on a Hitachi 912 photometric chemistry analyzer. Random urine and 24-hour urine samples were also measured. Urinary protein to creatinine ratio was also calculated.

(179) Histological Analysis

(180) PAS Staining

(181) Kidney was fixed in 10% formalin, paraffin embedded, 5 m wide sections were taken and stained with periodic acid-Schiff (PAS) for determination of glomerular matrix expansion, vascular hyalinosis and tubulointerstitial lesions. Briefly, glomerular hypertrophy was determined by measuring the glomerular tuft area of 50 glomerular cross-sections excluding incomplete glomeruli along the sample edge. Hyaline was determined by assessment of PAS-positive and nucleus-free areas within the arterioles. Arteriolar hyalinosis was defined by the average of hyalinized quarters of arterioles. The presence of Armanni-Ebstein lesions was also evaluated. The analysis was performed on a double blinded fashion with computer-assisted morphometry using AxioVision 4.8 software on a Zeiss Axiolmager A1 light-microscope.

(182) Fibronectin Staining

(183) Heat-induced epitope retrieval was performed by boiling the paraffine-embedded tissue sections in citrate buffer (pH 6, HISTOLS, Citrate Buffer, Histopathology Ltd). Slides were peroxidase blocked (HISTOLS Peroxidase Blocking, Histopathology Ltd), and non-specific attachments were inhibited with protein solution (HISTOLS BBPS, Histopathology Ltd). Sections were incubated with policlonal antibody against fibronectin (1:500, Abcam, USA) and peroxidase labelled anti-rabbit antibody (HISTOLS-R, Detection System, Histopathology Ltd). Fibronectin was visualised with HISTOLS-Resistant AEC Chromogen/Substrate System, (Histopathology Ltd.), counterstained with haematoxylin and eosin and mounted with permanent mounting medium.

(184) Masson's Trichrome Staining

(185) To investigate the amount of collagen fibers the formalin-fixed and paraffin embedded tissue samples were dewaxed and cut into 4-10 m slices. Slides were immersed in Weigert's hematoxylin (Sigma-Aldrich Co., St. Louis, Mo., USA) then were stained serially with acid fuschin, phosphomolybdic acid and methyl blue. The color was fixed in 1% acetic acid. Then the slides were dehydrated using increasingly higher concentration of alcohol, fixed in toluene, mounted in Permount (Fisher Scientific Inc., Waltham, Mass., USA.) and air-dried overnight before observation and photography. The nuclei of the cells appear as blue-black, the collagen fibers stained blue, the cytoplasm is red.

(186) The stained sections were viewed and photographed with Pannoramic 250 Flash and Pannoramic Viewer 1.15.2 (3D HISTECH Ltd. Budapest, Hungary) Adobe Photoshop 13.0 and Scion Image for Windows software were used for the analysis. The blue staining of fibrotic tissue was marked using the color-recognizer option of Adobe Photoshop software. The number of blue stained pixels (i.e. the area of the fibrotic tissue) was divided by the number of pixels in the whole section, thus giving the ratio of fibrotic tissue to all tissue. Finally these ratios were statistically analyzed in all of the treatment groups.

(187) Protein Isolation and Western Blotting

(188) Tissue samples were lysed in buffer containing leupeptin, aprotinin, Triton X-100, Tris-HCl, Ethylene glycol-bis (2-aminoethylether),N,N,N,N-tetraacetic-acid, NaF, Phenylmethylsulphonylfluoride and Na-orthovanadate (each substance were purchased from Sigma-Aldrich Co., St. Louis, Mo., USA) and centrifuged to pellet nuclei and large cellular fragments. Protein concentration of the supernatants was determined by Bradford assay (Bio-Rad Laboratories, Hercules, Calif., USA). Ten micrograms were separated by 10% SDS-PAGE at 120 V (40 mA, 90 min) (Penguin Dual-Gel Water Cooled Systems, Owl, N.H., USA). Pre-stained protein mixture (BenchMark, Gibco/BRL, Eggenstein, Germany) was used as marker of molecular mass. The separated proteins were transferred into nitrocellulose membrane (GE Haelthcare, Little Chalfont, UK) at 70 V (220 mA, 90 min) (MiniTank electroblotter, Owl, N.H., USA). Non-specific binding sites were blocked in 5% non-fat dry milk containing blot solution. Membranes were incubated with monoclonal antibody specific to mouse -SMA (Sigma-Aldrich Co., St. Louis, Mo., USA) diluted to 1:1000. Blots were washed and incubated (30 min, room temperature) with peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich Co.) diluted to 1:10000. Equal protein loading to the gel was confirmed by staining with a goat polyclonal IgG antibody raised against the carboxy (C-11) terminus of the -actin (Santa Cruz Biotechnology Inc.). Immunoreactive bands were visualized using enhanced chemiluminescence Western blotting detection protocol (AP Biotech, Buckinghamshire, UK). Bands were analyzed with Quantity One software version 4.6.9. (Bio-Rad). Ponceau staining was used as a loading control and an internal control was used as well.

(189) Fluorescent Immunohistochemistry

(190) Frozen kidney sections were embedded in Shandon cryomatrix (Thermo Fisher Scientific) and cut to 5-7 m slides with a cryostat. Samples were incubated for one hour with the specific mouse -SMA (1:2000, Sigma-Aldrich Co., St. Louis, Mo., USA) or S1R (1:100, Sigma-Aldrich Co., St. Louis, Mo., USA) antibody. After repeated washing slides were incubated with goat anti-mouse Alexa Fluor 488 conjugate and counterstained with Hoechst 33342 (Sigma-Aldrich Ltd.) to visualize nuclei. Appropriate controls were performed omitting the primary antibody to assure the specificity and to avoid autofluorescence. Sections were analyzed with a Zeiss LSM 510 Meta confocal laser scanning microscope with objectives of 20 and 63 magnification.

(191) Statistical Analysis

(192) Data were analyzed using GraphPad Prism software (GraphPad Software Inc., La Jolla, Calif., USA). After testing the normality with Kolmogorov-Smirnov test, numerical datasets from all experiments were analyzed using the Mann-Whitney U-test for two group's comparison and Kruskal-Wallis test when there were 3 or more groups. P values less than 0.05 were considered to indicate statistically significant differences. Values for all measurements were expressed as mean+SEM.

(193) Experiments with Human Trabecular Meshwork-5 (HTM-5) Cells

(194) Cell Cultures

(195) Human trabecular meshwork-5 (HTM-5) were cultured in DMEM containing 25 mM glucose (Thermo Fisher Scientific, Waltham, Mass., USA) supplemented with 10% FBS, 1% penicillin/streptomycin and 1% L-glutamine (Sigma-Aldrich St. Luis, Mo., USA). Cells were incubated at 37 C. in a humidified atmosphere of 5% CO.sub.2 and 95% air.

(196) Methyl-Thiazoletetrazolium (MTT) Cell Proliferation Assay

(197) HTM-5 cells were plated in serum free fresh medium in 96 well plates at a density of 40 000 cells/well and treated with 20 ng/mL of platelet-derived growth factor (PDGF; R&D Systems, Minneapolis, Minn., USA), 10-15 M fluvoxamine (PDGF+FLU; Sigma-Aldrich, St. Luis, Mo., USA), 5-15 M SA-4503 (PDGF+SA; Tocris Bioscience, Bristol, UK), and with 5-15 M PRE-087 (PDGF+PRE; Sigma-Aldrich, St. Luis, Mo., USA) for 24 h. Control cells were treated with vehicle (HCl) alone (n=6 wells/group). After the treatment period cells were incubated with methyl-thiazoletetrazolium (MTT) for 4 h followed by solubilisation in DMSO-ethanol (1:1). The formation of water-insoluble formazan was determined by measuring optical density at 570 nm in a Plate CHAMELEON V Fluorometer-Luminometer-Photom reader (Hidex, Turku, Finland).

(198) Immunocytochemistry

(199) Morphological changes/cytoskeleton rearrangements of HTM-5 cells caused by PDGF was visualised by immunostaining of F-actin. Cells were plated at a density of 200 000 cells/well on 0.1% gelatine coated tissue culture chambers in serum-free media. Cells were treated with PDGF (20 ng/mL) or FLU (10 M) for 24 h, then cells were washed with PBS, fixed in 4% formalin, washed again, permeabilised with Triton X-100 (Sigma-Aldrich, St. Luis, Mo., USA) and blocked with 5% BSA (Sigma-Aldrich, St. Luis, Mo., USA). Repeated washes with PBS were followed by staining with phalloidin-Alexa-Fluor 546 (1:40; Invitrogen, Carlsbad, Calif., USA) for F-actin and with Hoechst (Sigma-Aldrich, St. Luis, Mo., USA) for DNA. After PBS washes, coverslips were mounted onto slides using ProLong Anti-Fade (Life Technologies, Waltham, Mass., USA). Images were obtained at 10 and 60 magnification on Nikon Ti2 fluorescent microscope.

(200) Reverse Transcription Polymerase Chain Reaction (RT-PCR)

(201) Total RNA was extracted using the RT050 Total RNA isolation Mini Kit (Geneaid Biotech, New Taipei City, Taiwan). The quality and quantity of isolated RNA was measured on a NanoDrop ND-1000 spectrophotometer (Baylor College of Medicine, Houston, Tex., USA). One hundred fifty nanograms each of FN1, COL1A1 and MMP2 mRNA from HTM-5 cells were reverse-transcribed using a First Strand cDNA Synthesis Kit for RT-PCR (Thermo Fisher Scientific, Waltham, Mass., USA). The expression of the mRNA was determined in triplicate with 1 L cDNA samples obtained by qPCR using 10 L SYBR Green I Master enzyme mix (Roche Diagnostics, Mannheim, Germany) and 10 pmol L-1 of each specific primer (Invitrogen, Carlsbad, Calif., USA), following sequences designed by Lasergene PrimerSelect software version 7.1.0 (DNASTAR, Medison, Wis., USA) based on nucleotide sequences from National Center for Biotechnology Information's nucleotide database. Results were analysed by LightCycler 480 software version 1.5.0 (Roche Diagnostics, Indianapolis, Ind., USA). The mRNA expression of interest was normalised against mRNA expression of 18S ribosomal RNA (RN18S) from the same samples as internal control.

(202) Statistical Analysis

(203) Statistical analyses were performed using GraphPad Prism software version 6.01 (GraphPad Software Inc., San Diego, Calif., USA). To test if the values were from a Gaussian distribution, a Kolmogorov-Smirnov normality test was performed. Data were analysed by one-way ANOVA followed by Bonferroni's multiple-comparison post hoc test for all parametrical comparisons, or in the case of non-parametric data, by Kruskal-Wallis ANOVA on ranks. Significance was set a priori at P<0.05, corrected for multiple comparisons.

Example 1S1R is Expressed in Various In Vitro, In Vivo and Human Samples

(204) Fluorescent immunohistochemistry confirmed the presence of SIR in various models (see also the chapter Fluorescent immunohistochemistry). In vitro in myofibroblasts (1A) SIR was localized in the whole cytoplasm with a predominant enrichment in the endoplasmic reticulum. In in vivo samples SIR showed a perinuclear staining pattern of proximal tubules, however it was also visible in the cytoplasm (1B). Immunhistochemistry of the total kidney of diabetic rats (1C) revealed that SIR is not expressed in renal glomeruli. In renal biopsies of patients diagnosed with obstructive uropathy (1D) SIR staining and patchy co-localization with -smooth muscle actin (SMA) indicates that SIR is expressed in the myofibroblasts also in humans.

Example 2S1R Agonist Compounds (Fluvoxamine, SA-4503, PRE-084) are not Cytotoxic in Myomyofibroblasts

(205) None of the selective SIR agonists (fluvoxamine (FIG. 2A), SA-4503 (FIG. 2B) or PRE-084 (FIG. 2C)) inhibited cell viability of NRK49F cells, which confirms that applied concentrations of the said SIR compounds are not cytotoxic in myofibroblasts in the commonly used doses (1-10 M) and therefore they can be administered in in vitro studies (see also the chapter In vitro experiments on myofibroblastsMTT assay).

Example 3S1R Agonist Compounds (Fluvoxamine, SA-4503, PRE-084) Decreases PDGF Induced Cell-Proliferation

(206) PDGF treatment of myofibroblasts for 24 hours resulted in significantly increased cell proliferation compared to controls (FIG. 3; see also the chapter In vitro experiments on myofibroblastsMTT assay). Pretreatment with different concentrations of the said SIR agonists (fluvoxamine (FIG. 3A), SA-4503 (FIG. 3B) or PRE-084 (FIG. 3C)) significantly decreased PDGF-induced myofibroblast proliferation. Co-incubation with the S1R antagonist NE-100 (3 M) suspended the effect of fluvoxamine (10 M), which suggests that the anti-proliferative effect is SIR mediated.

Example 4S1R Agonist Compound Fluvoxamine Minimizes TGF-Induced Collagen 1 and Collagen 3 Production of Myofibroblasts in a Time-Dependent Manner

(207) NRK49F myofibroblast cells were treated with 50 nM TGF to induce collagen production. (FIG. 4; the model used is described in the chapter In vitro experiments on myofibroblastsRT-PCR). 48 hours of treatment resulted in a significant production of ECM components collagen-1 (FIG. 4A), and collagen-3 (FIG. 4B). Compared to TGF treated cells fluvoxamine treatment remarkably diminished mRNA expression of the said collagens already as early as by 24 hours. By 48 hours collagen production of fluvoxamine treated cells returned to the level of normal controls.

Example 5Sigma-1 Receptor (S1R) Agonist Compounds (Fluvoxamine, SA-4503, PRE-084) Inhibit TGF-Induced Extracellular Matrix (ECM) Production

(208) 24 hours of TGF induction led to significant ECM production of NRK49F myofibroblasts compared to controls (FIG. 5; the model used is described in the chapter In vitro experiments on myofibroblastsSirius Red staining). All the applied concentrations (even the smallest 1 M) of the said S1R agonist compounds (fluvoxamine (FIG. 5A), SA-4503 (FIG. 5B) or PRE-084 (FIG. 5C)) significantly inhibited TGF-induced ECM production.

Example 6S1R Agonist Fluvoxamine Improves Diabetes Induced Impairment in Renal Function

(209) Renal parameters of control, diabetic and treated diabetic rats (Table 2-3) were measured. The model used is described in the chapter Rat model of streptozotocin induced diabetic nephropathy. Diabetes induced severe renal impairment with increased serum creatinine and blood urea nitrogen values. Fractional sodium excretion (FeNa) was increased, significant albuminuria was present and the glomerular filtration rate (GFR) was decreased, all indicating the development of diabetic nephropathy. Fluvoxamine treatment, specifically the long-term (7-weeks) treatment remarkably improved renal function, prevented GFR decline. This beneficial effect was diminished by co-administration of the specific S1R antagonist NE-100 (Table 3), which confirms that any non-specific effect on a receptor other than S1R could be excluded. These data prove that S1R agonists are renoprotective and the treatments improve those gold standard markers of renal function that are used also in human clinical routine for the assessment of kidney failure.

(210) TABLE-US-00003 TABLE 2 Renal parameters of control, diabetic and diabetic rats treated with the S1R agonist fluvoxamine Control Diabetes (D) D7FLU D + FLU D + FLU2 Blood Glucose (mmol/L) 17.3 0.95 46.6 2.85* 50.31 3.7.sup. 36.6 2.62.sup. 26.45 3.11.sup. Fructosamine (mol/L) 152 11.0 254 8.52* 276 11.2.sup. 252 18.5.sup. 242 12.8.sup. Blood Urea Nitrogen (mmol/L) 7.06 0.19 26.6 2.42* 17.3 1.49.sup. 17.3 2.30.sup. 18.8 1.68.sup. Blood Creatinine (mol/L) 22.0 0.93 42.0 2.39* 27.0 2.24.sup. 34.5 2.74.sup. 31.8 2.94.sup. GFR (mL/min/100 g) 12.8 0.57 3.15 0.20* 6.77 1.15.sup. 3.73 0.49.sup. 4.73 0.69.sup. FeNa (%) 0.22 0.02 3.12 0.75* 0.40 0.03.sup. 0.90 0.23.sup. 0.62 0.12.sup. Urinary albumin excretion (mg/mL) 3.25 2.39 42.5 6.38* 20.8 9.51.sup. 21.5 4.99.sup. 24.3 5.77.sup.

(211) Table 2 shows renal function parameters of Streptozotocin-(65 mg/bwkg iv.) induced type 1 diabetic rats treated per os with (D): vehicle (isotonic saline); (D7FLU): fluvoxamine (20 mg/bwkg/day) for 7 weeks or (D+FLU): fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes or (D+FLU2): fluvoxamine (2 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes. GFR-glomerular filtration rate, FeNa: fractional sodium excretion. *p0.05 vs. Control; p0.05 vs. Diabetes (n=8-10 group, MeanSEM).

(212) TABLE-US-00004 TABLE 3 Renal parameters of diabetic rats and diabetic rats treated with either only S1R agonist fluvoxamine, or with the S1R agonist fluvoxamine + antagonist NE-100. D + FLU + Diabetes (D) D + FLU NE-100 D + FLU2 D + FLU2 + NE-100 Blood Glucose (mmol/L) 46.6 2.85 36.6 2.62.sup. 48.5 2.40.sup.$ 26.45 3.11.sup. .sup.40.8 3.00.sup.# Fructosamine (mol/L) 254 8.52 252 18.5.sup. 267 7.66.sup. 242 12.8.sup. 264 11.1 Blood Urea Nitrogen (mmol/L) 26.6 2.42 17.3 2.30.sup. 24.3 2.27.sup.$ 18.8 1.68.sup. 22.3 1.43 Blood Creatinine (mol/L) 42.0 2.39 34.5 2.74.sup. 37.0 4.39.sup. 31.8 2.94.sup. 40.0 3.72 GFR (mL/min/100 g) 3.15 0.20 3.73 0.49.sup. 3.25 0.25.sup. 4.73 0.69.sup. 4.06 0.58 FeNa (%) 3.12 0.75 0.90 0.23.sup. 1.33 0.39.sup. 0.62 0.12.sup. 0.96 0.12 Urinary albumin excretion (mg/mL) 42.5 6.38 21.5 4.99.sup. 73.3 15.8.sup.$ 24.3 5.77.sup. .sup.42.8 7.92.sup.#

(213) Table 3 shows renal function parameters of Streptozotocin-(65 mg/bwkg iv.) induced type 1 diabetic rats treated per os with (D): vehicle (isotonic saline); (D+FLU): fluvoxamine (20 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes,) or (D+FLU2): fluvoxamine (2 mg/bwkg/day) for 2 weeks from the 5.sup.th week of diabetes. Additional groups were also treated per os with NE-100, a specific antagonist of S1R; (D+FLU+NE-100): fluvoxamine+NE-100 (20 mg/bwkg/day+1 mg/bwkg/day) for two weeks or D+FLU2+NE-100):fluvoxamine+NE-100 (2 mg/bwkg/day+1 mg/bwkg/day) for two weeks from the 5.sup.th week of diabetes. GFR-glomerular filtration rate, FeNa: fractional sodium excretion. p<0.05 vs. Diabetes; $p0.05 vs. D+FLU; # p0.05 vs.D+FLU2; (n=8-10 group, MeanSEM).

Example 7Sigma-1 Receptor (S1R) Agonist Compound Fluvoxamine Diminishes Diabetes Induced Renal Interstitial Fibrosis

(214) The rat model used was the same as in Example 6. To evaluate the fibrotic lesion of diabetic kidney, paraffin embedded tissue sections of rat kidneys were stained with Masson's trichrome reagent. Diabetes induced development of tubulointerstitial fibrosis (FIG. 6B) is marked by the light blue or light grey regions. Fluvoxamine treatment ameliorated the diabetes induced tubulointerstitial fibrosis (FIGS. 6C, 6D and 6F). Specifically the long-term (7-weeks) treatment restored almost the normal renal structure (FIG. 6C). The co-administration of the S1R specific antagonist NE-100 inhibited the protective effect of fluvoxamine in rats treated with 20 mg fluvoxamine (FIG. 6E). The results are summarized on the column diagram on FIG. 6H.

Example 8Sigma-1 Receptor (S1R) Agonist Compound Fluvoxamine Compound Treatment Decreases Diabetes Induced Mesangial Matrix Expansion in the Kidney of Diabetic Rats

(215) The rat model used was the same as in Example 6. Paraffin embedded tissue sections of rat kidneys were stained with PAS. The increase of PAS positive (dark purple or dark grey) area showed a significantly more robust mesangial matrix expansion in diabetic animals compared to controls (FIG. 7B). Similarly to tubulointerstitial fibrosis the extent of mesangial matrix expansion was remarkably diminished by all doses of fluvoxamine (FIGS. 7C, 7D and 7F). Long-term (7-weeks) treatment was the most effective in preventing renal tissue damage (FIG. 7C). The co-administration of the S1R specific antagonist NE-100 prohibited the renoprotection of fluvoxamine (FIGS. 7E and 7G) suggesting a directly S1R mediated effect. The results are summarized on the column diagram on FIG. 7H.

Example 9Sigma-1 Receptor (S1R) Agonist Compound Fluvoxamine Treatment Decreases Diabetes Induced Fibronectin Accumulation in the Kidney of Diabetic Rats

(216) The development of fibrosis in diabetic rats (see the chapter Rat model of streptozotocin (STZ) induced diabetic nephropathy) was confirmed by fibronectin staining as well (FIG. 8). In fluvoxamine treated rats the fibrotic lesion (brown area or darker-medium grey area) is smaller (FIG. 8C) than in diabetic rats (FIG. 8B), and again the S1R antagonist

(217) NE-100 suspended this beneficial effect (FIG. 8D). The results are summarized on the column diagram of FIG. 8E.

Example 10Sigma-1 Receptor (S1R) Agonist Compound Fluvoxamine Decreases Diabetes Induced Extracellular Matrix (ECM) Production in the Kidney of Diabetic Rats

(218) The rat model used was the same as in Example 6. ECM components in the kidney tissue sections were determined by Sirius Red staining (FIG. 9). Diabetes induced excessive ECM accumulation (as seen in FIG. 9B), was significantly reduced by the long term 7-weeks fluvoxamine treatment (FIG. 9C).

Example 11Sigma-1 Receptor (S1R) Agonist Compound Fluvoxamine Treatment Decreases

Diabetes Induced Alpha Smooth Muscle Actin (SMA) Protein Level in the Kidney of Diabetic Rats

(219) Diabetes induces proliferation and ECM production of myofibroblasts in the kidney, which can be investigated also by the measurement of the protein level of SMA, a typical marker of myofibroblast. As seen in FIG. 10 SMA increased by 300% in diabetic rats compared to controls. Fluvoxamine treatment, (predominantly the dose of 20 mg) reduced SMA protein level by the half. The beneficial effect of fluvoxamine was suspended by the co-administration of the S1R specific antagonist NE-100.

Example 12Sigma-1 Receptor (S1R) Agonist Compound Fluvoxamine Treatment Minimizes Tubulointerstitial Fibrosis in the Kidney after Unilateral Ureteral Obstruction (UUO)

(220) To confirm the anti-fibroproliferative effect in other models of progressive fibrosis, fluvoxamine was administered to mice with unilateral ureter obstruction (UUO) that is the gold-standard animal model of fibrosis (FIG. 11). The model used is described in the chapter Mice model of unilateral ureteral obstruction (UUO) induced renal fibrosis. A serious tubulointerstitial fibrosis was induced by the ureter obstruction and fluvoxamine treatment decreased tubulointerstitial fibrosis (FIG. 11B-11C). Similar to diabetic rats, in mice NE-100, the specific S1R antagonist, suspended the beneficial effect of fluvoxamine (FIG. 11D). The results are summarized on the column diagram on FIG. 11E.

Example 13Sigma-1 Receptor (S1R) Agonist Compound Fluvoxamine Treatment Minimizes

Alpha Smooth Muscle Actin (SMA) Production in the Kidney after Unilateral Ureteral Obstruction (UUO)

(221) The mice model used was the same as in Example 12. One week after the induction of UUO the protein amount of SMA was six times higher in UUO mice than in controls (FIG. 12). One week fluvoxamine treatment successfully decreased UUO-induced SMA production in mice, which suggest a significant anti-fibroproliferative effect of fluvoxamine treatment even in the long-term.

Example 14Sigma-1 Receptor (S1R) Agonist Compound Fluvoxamine Treatment Ameliorates Interstitial Fibrosis of the Lung in a Rat Model of Bleomycin-Induced Lung Fibrosis

(222) To prove the beneficial anti-fibroproliferative effect of fluvoxamine also in other organs, fluvoxamine was tested in the progressive fibrosis of the lung in the rat model of bleomycin-induced lung fibrosis described in chapter Rat model of bleomycin-induced pulmonary fibrosis, (FIG. 13). While fibrotic lesions of the lung (marked by light blue or continuous medium grey area FIG. 13B) significantly increased after bleomycin treatment compared to controls; fluvoxamine prevented almost totally the fibrotic effect of bleomycin (FIG. 13C). S1R antagonist NE-100 suspended the effect of fluvoxamine (FIG. 13D). The results are summarized on the column diagram on FIG. 13E.

Example 15Sigma-1 Receptor (SIR) Agonist Compound Fluvoxamine Treatment Diminishes SMA Production in a Rat Model of Bleomycin-Induced Lung Fibrosis

(223) The rat model used was the same as in Example 14. Three weeks after the intrathecal injection of bleomycin SMA protein level was significantly increased in the lung compared to controls (FIG. 14). Fluvoxamine treatment successfully reduced bleomycin-induced SMA production nearly to the level of controls. Similar to previous results the S1R antagonist NE-100 suspended the beneficial effect of fluvoxamine, which underlines the S1R-mediated antiproliferative effect of fluvoxamine also in other organs, e.g. in the lung.

Example 16Sigma-1 Receptor (S1R) Agonists Suspend Platelet-Derived Growth Factor (PDGF)-Induced Cell Proliferation in Human Trabecular Meshwork-5 (HTM-5) Cells

(224) Human trabecular meshwork-5 (HTM-5) cells were cultured and MTT cell proliferation assay was carried out as described in Materials and Methods. Briefly, the HTM-5 cells were plated in 96 well plates at a density of 40 000 cells/well and treated with a pro-fibrotic substance, PDGF, in the absence or presence of Sigma-1 receptor agonists fluvoxamine (FLU), SA-4503 and PRE-087 for 24 h. Control cells were treated with vehicle (HCl) alone. Thus, in this eye fibrosis model the inventors studied whether Sigma-1 receptor agonists of very different chemical structure are able to reduce artificially elicited progressive fibrosis.

(225) After the treatment period cells were incubated with methyl-thiazoletetrazolium (MTT) for 4 h followed by solubilisation in DMSO-ethanol (1:1) to measure the level of fibrotic proliferation. The formation of water-insoluble formazan was determined by measuring optical density at 570 nm.

(226) The results are shown in FIG. 15.

(227) It can be seen that in a dose-dependent manner each of the three Sigma-1 receptor (SIR) agonists has reduced fibrotic proliferation to the level or even below the level of control cells.

(228) Statistical analysis was carried out as described in the Materials and Methods throughout the Examples.

Example 17SIR Receptor Agonist Fluvoxamine Reduces Fibrotic Markers in HTM-5 Cells

(229) The aim of this experiment was to measure the mRNA expression level of fibrotic markers, i.e. cellular matrix elements fibronectin (FN1), collagenlal (COL1A1) and matrix metalloproteinase 2 (MMP2) in the HTM-5 eye fibrosis model and the effect of S1R agonists effect on progressive fibrosis characterized by these markers.

(230) Human trabecular meshwork-5 (HTM-5) cells were cultured and reverse transcription polymerase chain reaction (RT-PCR) was carried out as described in the Materials and Methods. Briefly, total RNA was extracted, qualitatively and quantitatively characterized and subjected to reverse transcription. The expression levels were measured by real-time quantitative PCR (qPCR) by SYBR green detection. Primer sequences were based on nucleotide sequences from National Center for Biotechnology Information's nucleotide database. The mRNA expression of interest was normalised against mRNA expression of 18S ribosomal RNA (RN18S) from the same samples as internal control.

(231) FIG. 16 shows that fluvoxamine inhibit the expression of each fibrosis marker tested in a dose dependent manner.

Example 18. Effect of PDGF and S1R Agonist FLU on Actin Cytoskeleton Rearrangement in HTM-5 Cells

(232) In this example of the eye fibrosis model of human trabecular meshwork-5 (HTM-5) cells, treated with PDGF, F-actin were immunostained to visualize morphological changes/cytoskeleton rearrangements of HTM-5 cells caused by PDGF.

(233) Cells were cultured and plated in serum-free media as described in the Materials and Methods. Cells were treated with PDGF or FLU or both, washed, fixed and permealized and blocked. This preparation and repeated washes with PBS were followed by staining with phalloidin-Alexa-Fluor 546 for F-actin and with Hoechst dye for DNA to show nuclei. Images were made as described in Materials and methods in various magnifications and are shown in FIG. 17 (magnification: 1000) and FIG. 18 (magnification: 6000) The results clearly show that in the present eye fibrosis model fibrotic rearrangement and active production of actin filaments (see FIGS. 17B and 18B), a hallmark of progressive fibrosis, are blocked by S1R agonist fluvoxamine (see FIGS. 17C and 18C) to a level similar that of the control (see FIGS. 17A and 18A),

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