阅读说明：本技术 用β型糖原合酶激酶3抑制剂治疗特发性肺纤维化 (Treatment of idiopathic pulmonary fibrosis with a beta-type glycogen synthase kinase 3 inhibitor ) 是由 托里·A·塔克 史蒂文·艾德尔 于 2019-05-16 设计创作，主要内容包括：描述了药物组合物和方法,其依赖于糖原合酶激酶3(β型；GSK-3β)抑制剂(最优选9-ING-41)以在数种小鼠模型中抑制体内纤维化肺重塑(包括肌成纤维细胞增殖和分化为纤维化成纤维细胞)。用临床上可用的特异性抑制剂9-ING-41对GSK-3β进行的治疗性靶向减轻了体内纤维化肺重塑,并且提供了通过用9-ING-41进行特异性GSK-3β抑制对人IPF进行治疗的方式。(Pharmaceutical compositions and methods are described that rely on glycogen synthase kinase 3 (beta-type; GSK-3 beta) inhibitors (most preferably 9-ING-41) to inhibit fibrotic lung remodeling (including myofibroblast proliferation and differentiation into fibrotic fibroblasts) in vivo in several mouse models. Therapeutic targeting of GSK-3 β with the clinically available specific inhibitor 9-ING-41 reduces fibrotic lung remodeling in vivo and provides a means of treatment for human IPF through specific GSK-3 β inhibition with 9-ING-41.)

1. A method of treating Idiopathic Pulmonary Fibrosis (IPF) in a mammalian subject comprising administering to the mammalian subject an effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof,

wherein:

x is independently-H, halogen, 5, 6-methylenedioxy, -CN, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-CH₂CH₂CO₂H or-CH₂CH₂CO₂Et；

R¹is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

R²is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl; and is

Y is independently-H, halogen, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-NO₂-CN or-C ═ CH₂。

2. A method of treating Idiopathic Pulmonary Fibrosis (IPF) in a mammalian subject comprising administering to the mammalian subject a pharmaceutical composition comprising:

(i) an effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof,

wherein:

x is independently-H, halogen, 5, 6-methylenedioxy, -CN, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-CH₂CH₂CO₂H or-CH₂CH₂CO₂Et；

R¹is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

R²is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

y is independently-H, halogen, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-NO₂-CN or-C ═ CH₂(ii) a And

(ii) a pharmaceutically acceptable carrier or excipient.

3. The method of claim 1 or claim 2, wherein the compound of formula I is 9-ING-41:

4. the method of any one of claims 1, 2 or 3, wherein the mammalian subject is a human.

5. The method of any one of claims 1 to 4, wherein the administration is by inhalation.

6. A method for inhibiting proliferation and/or differentiation of pulmonary myofibroblasts into Fibrotic Lung (FL) fibroblasts, or reducing FL fibroblast proliferation, in a mammalian subject in need thereof, comprising contacting the myofibroblasts or FL fibroblasts with an antifibrotic effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof,

wherein:

x is independently-H, halogen, 5, 6-methylenedioxy, -CN, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-CH₂CH₂CO₂H or-CH₂CH₂CO₂Et；

R¹is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

R²is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl; and is

Y is independently-H, halogen, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-NO₂-CN or-C ═ CH₂。

7. A method for inhibiting proliferation and/or differentiation of pulmonary myofibroblasts into Fibrotic Lung (FL) fibroblasts, or reducing FL fibroblast proliferation in a mammalian subject in need thereof, comprising contacting the myofibroblasts or FL fibroblasts with a pharmaceutical composition comprising:

(i) an anti-fibrotic effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof,

wherein:

x is independently-H, halogen, 5, 6-methylenedioxy, -CN, -OMe, -OH, -O, an acid, an ester, a salt, a ketoneOBn、-CF₃、-CH₂OH、-CH₂OMe、-CH₂CH₂CO₂H or-CH₂CH₂CO₂Et；

R¹is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

R²is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

y is independently-H, halogen, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-NO₂-CN or-C ═ CH₂(ii) a And

(ii) a pharmaceutically acceptable carrier or excipient.

8. The method of claim 6 or claim 7, wherein the compound of formula I is 9-ING-41:

9. the method of any one of claims 6, 7 or 8, wherein the mammalian subject is a human.

10. The method of any one of claims 6 to 9, wherein said contacting is effected by administering said compound or pharmaceutical composition to said mammalian subject via inhalation.

11. A pharmaceutical composition comprising:

(i) an anti-fibrotic effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof,

wherein:

x independentImmediately is-H, halogen, 5, 6-methylenedioxy, -CN, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-CH₂CH₂CO₂H or-CH₂CH₂CO₂Et；

R¹is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

R²is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

y is independently-H, halogen, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-NO₂-CN or-C ═ CH₂(ii) a And

(ii) a pharmaceutically acceptable carrier or excipient.

12. The pharmaceutical composition of claim 11, wherein the compound of formula I is 9-ING-41:

13. the pharmaceutical composition of any one of claims 11 to 12, formulated for administration by inhalation.

14. Use of the composition of any one of claims 11 to 13 for treating IPF in a mammalian subject, wherein an effective amount of the composition is administered to a mammalian subject suffering from IPF.

15. The use of claim 14, wherein the composition:

(i) (ii) inhibits GSK-3 β;

(ii) inhibiting proliferation and/or differentiation of lung myofibroblasts into FL fibroblasts; and

(ii) reducing the FL fibroblast proliferation.

16. The use according to any one of claims 14 or 15, wherein the composition comprises 9-ING-41 or a pharmaceutically acceptable salt or solvate thereof.

17. The use according to any one of claims 14 to 16, wherein the composition is administered by inhalation.

18. The use according to any one of claims 14 to 17, wherein the mammalian subject is a human.

19. Use of a composition according to any one of claims 11 to 13 for the preparation of a medicament for treating IPF in a subject in need thereof.

20. The use of claim 19, wherein the composition:

(i) (ii) inhibits GSK-3 β;

(ii) inhibiting proliferation and/or differentiation of lung myofibroblasts into FL fibroblasts; and

(ii) reducing the FL fibroblast proliferation.

21. The use of any one of claims 19 or 20, wherein the composition comprises 9-ING-41 or a pharmaceutically acceptable salt or solvate thereof.

22. The use of any one of claims 19 to 21, wherein the composition is administered by inhalation.

23. The use according to any one of claims 19 to 22, wherein the mammalian subject is a human.

Technical Field

The present invention, which is in the fields of biochemistry and medicine, relates to methods and compositions for inhibiting fibrotic pulmonary remodeling (IPF) in vivo and thereby treating Idiopathic Pulmonary Fibrosis (IPF) using inhibitors of the β -type glycogen synthase kinase 3 (form β, GSK-3 β), most preferably 9-ING-41.

Background

Idiopathic Pulmonary Fibrosis (IPF) is a poorly understood progressive and fatal lung disease for which there is little, if any, treatment other than lung transplantation. Currently available treatments for pulmonary fibrosis are known not to be curative and only slow down disease progression. Median Survival Time (MST) is 3 years after diagnosis (and median survival less than 20% 5 years after diagnosis). Most forms of interstitial lung disease and other forms of pulmonary fibrosis are characterized by: fibrotic lesions, progressive distortion of alveolar structure and replacement by fibrotic or scar tissue with excessive extracellular matrix (ECM) deposition, lead to progressive respiratory difficulties and loss of lung function.

GSK-3 β is a serine/threonine kinase and is one of two GSK-3 isoforms (α and β). GSK-3 β can modulate the function of a variety of targets, including transcription factors. GSK-3 β also regulates many signaling pathways, which in turn affect the transcriptional activity of many inflammatory mediators.

Since current treatments only slow down the progression of the disease, there is a need to identify new more effective targets, as well as a need for better therapeutic modalities that can significantly reverse IPF, or reduce mortality and cure IPF.

Disclosure of Invention

The present disclosure relates to the treatment of both pleural fibrosis and pulmonary fibrosis. In some preferred embodiments, these methods are achieved by administering 9-ING-41 that is well tolerated even at high doses. It also limits scarring in mouse models of pleural fibrosis and pulmonary fibrosis as described herein. There is no effective treatment for the treatment of scarring and tissue reorganization associated with pleural fibrosis, and existing methods available for pulmonary fibrosis only slow their progression without cure. 9-ING-41 alleviates fibrotic lung remodeling in vivo, addressing the urgent need for effective treatment of pulmonary fibrosis.

There is currently no effective treatment to reverse pulmonary fibrosis. The present invention addresses this important gap and provides novel compounds and methods for targeting fibrotic lung ("FL") fibroblasts (or myofibroblasts) to effectively treat IPF.

The present invention relates to a method for inhibiting mesenchymal transition (MesoMT) of resident Pleural Mesothelial Cells (PMC) that contribute to myofibroblast expansion in progression to IPF.

The present invention relates to a method of treating Idiopathic Pulmonary Fibrosis (IPF) in a mammalian subject by administering to a mammalian subject in need thereof an effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof,

wherein:

x is independently-H, halogen, 5, 6-methylenedioxy, -CN, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-CH₂CH₂CO₂H or-CH₂CH₂CO₂Et；

R¹is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

R²is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

y is independently-H, halogen, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-NO₂-CN or-C ═ CH₂。

The present invention also relates to a method of treating IPF in a mammalian subject by: administering to a mammalian subject in need thereof a pharmaceutical composition comprising: i) an effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof; and ii) a pharmaceutically acceptable carrier or excipient.

The present invention also relates to a method for treating IPF in a mammalian subject, most preferably a human, suffering from or developing a disease or disorder characterized by pulmonary fibrosis, comprising administering to the subject an effective amount of a pharmaceutical composition as described above. The compound or composition preferably comprises 9-ING-41 or an analogue thereof which has at least 20% of the biological or biochemical activity of 9-ING-41, e.g. in an in vitro or in vivo assay. The most preferred compound is 9-ING-41.

In some preferred embodiments of the method of treating IPF in a mammalian subject, the compound of formula I is 9-ING-41 or a pharmaceutically acceptable salt or solvate thereof.

In some preferred embodiments of the method of treating a PF in a mammalian subject, the mammalian subject is a human.

In some preferred embodiments of the method of treating IPF in a mammalian subject, the administering is by inhalation.

Also provided are methods for inhibiting proliferation and/or differentiation of pulmonary myofibroblasts into Fibrotic Lung (FL) fibroblasts and reducing FL fibroblast proliferation comprising contacting myofibroblasts and FL fibroblasts in a mammalian subject in need thereof with an antifibrotic effective amount of a compound of formula I or a pharmaceutical composition comprising an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier.

In some preferred embodiments of the methods for inhibiting proliferation and/or differentiation of lung myofibroblasts into FL fibroblasts and reducing proliferation of FL fibroblasts, the compound or formula I is preferably 9-ING-41 or a pharmaceutically acceptable salt or solvate thereof.

In some preferred embodiments of the methods for inhibiting proliferation and/or differentiation of lung muscle fibroblasts into FL fibroblasts and reducing proliferation of FL fibroblasts, the mammalian subject is a human.

In some preferred embodiments of the methods for inhibiting proliferation and/or differentiation of lung muscle fibroblasts into FL fibroblasts and reducing proliferation of FL fibroblasts, the contacting is performed by inhalation.

The present invention also provides a pharmaceutical composition comprising: (i) an anti-fibrotic effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, and (ii) a pharmaceutically acceptable carrier or excipient.

In some preferred embodiments, formula I in the pharmaceutical compositions of the present invention is 9-ING-41.

In some preferred embodiments, the pharmaceutical compositions of the present invention are formulated for administration by inhalation.

In another embodiment, the present invention relates to the use of the above-described composition for treating IPF in a mammalian subject, wherein an effective amount of the composition is administered to a mammalian subject suffering from IPF.

In some preferred embodiments, the composition inhibits GSK-3 β, and inhibits lung myofibroblast proliferation and/or differentiation into FL fibroblasts, and reduces said FL fibroblast proliferation.

In some preferred embodiments, the composition comprises an effective amount of 9-ING-41, or a pharmaceutically acceptable salt or solvate thereof.

In some preferred embodiments, the mammalian subject is a human.

In some preferred embodiments, the composition is administered by inhalation.

Another embodiment relates to the use of a composition as above for the preparation of a medicament for the treatment of IPF in a mammalian subject, preferably a human, in need thereof.

In some preferred embodiments, the composition inhibits GSK-3 β activity, and inhibits lung myofibroblast proliferation and/or differentiation into FL fibroblasts, and reduces the FL fibroblast proliferation.

In this use, the compound is preferably 9-ING-41 or a pharmaceutically acceptable salt or solvate thereof. In the above use, the medicament is prepared for administration to a human by inhalation for the treatment of IPF.

Drawings

FIGS. 1A to 1B are graphs showing that inhibition of GSK-3 β with 9-ING-41 attenuates pulmonary fibrosis. Bleomycin sulfate (0.8U/kg) was administered intratracheally to C57Bl/6J mice. After 14 days, mice were treated with DMSO (vehicle) or the GSK-3 β inhibitor 9-ING-41(30mg/kg) by intraperitoneal injection for the next 14 days. At the completion of the 28-day time course, lung compliance (compliance) was determined using Scireq flexible (fig. 1A). Lung reconstructions were also collected by gated CT scanning and used to determine lung volume (fig. 1B).

FIGS. 2A to 2B show lung tissue sections (5 μm) from mice treated with DMSO vehicle (FIG. 2B) and with 9-ING-41 (FIG. 2A). Sections were immunostained to visualize collagen deposition by confocal microscopy (shown in grey). Solid arrows indicate areas of increased collagen-1 deposition. The images are representative of 30 fields/slides/conditions. n-6 mice.

Fig. 3 is a graph showing lung compliance in mice administered intratracheally with TGF-beta adenovirus to induce pulmonary fibrosis. After 7 days, mice received intraperitoneal injections of 9-ING-41(30mg/kg) daily for the next 7 days. At the completion of the 14 day time course, lung compliance was determined. Data are expressed as mean ± SEM. n-6 mice/condition. Indicates statistical significance of p < 0.05 by Mann-Whitney U test (Mann-Whitney U test) compared to control GFP-adenovirus and DMSO treatments. And denotes p < 0.05 compared to GFP adenovirus 9-ING-41 treatment.

Figures 4A to 4B show lung tissue sections from mice in which TGF-beta adenovirus induced pulmonary fibrosis. Mice were treated with DMSO (FIG. 4A) or 9-ING-41 (FIG. 4B) (see description of FIG. 3). Sections were trichrome stained to reveal the lesion and collagen areas. Solid arrows indicate areas of injury and increased collagen deposition. The images are representative of 30 fields/slides/conditions. n-6 mice.

Fig. 5A to 5F show lung tissue sections (5 μm) from vehicle (fig. 5A to 5C) and 9-ING-41 (fig. 5D to 5F) treated mice. Sections were deparaffinized and trichromatic stained to detect changes in lung architecture and collagen deposition (blue staining). Images were acquired at 20X and represent 30 fields/slide/mouse, n ═ 6 animals/treatment. Fibrotic foci are indicated by solid yellow arrows.

Fig. 6A to 6B show lung sections from vehicle (fig. 6B) and 9-ING-41 (fig. 6A) treated mice immunostained for collagen 1 (colagen 1, Col-1) deposition and imaged by confocal microscopy at 40X. Solid arrows indicate areas of collagen deposition within the damaged lung. These data indicate that GSK-3 β inhibition with 9ING41 reduced collagen deposition in fibrotic lung lesions.

Figures 7A to 7B show lung sections from vehicle (figure 7B) and 9-ING-41 (figure 7A) treated mice immunostained for a-SMA, a marker of myofibroblast differentiation. Images were acquired by confocal microscopy at 40X. The filled arrows indicate the regions of collagen alpha-SMA expression. Images are representative of 30 fields/mouse and n-3 mice/treatment.

Detailed Description

9-ING-41 as GSK-3 beta inhibitors

Compounds useful in the methods of the present invention include 9-ING-41, which is described in U.S. Pat. No. 8,207,216(Kozikowski et al), which is incorporated by reference in its entirety.

Also useful in the present invention is a broader class of benzofuran-3-yl- (indol-3-yl) maleimide families that share GSK-3 β inhibitory properties. Such compounds are encompassed by formula I:

wherein:

x is independently-H, halogen, 5, 6-methylenedioxy, -CN, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-CH₂CH₂CO₂H or-CH₂CH₂CO₂Et；

R¹is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

R²is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl;

y is independently-H, halogen, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-NO₂-CN or-C ═ CH₂。

In the compounds of formula I, X is independently-H, halogen, 5, 6-methylenedioxy, -CN, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-CH₂CH₂CO₂H or-CH₂CH₂CO₂Et. The substituents X may be independently present at one or more of the 4, 5, 6 or 7 positions of the indole ring in formula I.

In some embodiments, X is H.

In other embodiments, X is halogen (i.e., -F, -Cl, -Br, -I). In some embodiments, X is 5-F. In other embodiments, X is 5-Br. In other embodiments, X is 5-I.

In some embodiments, X is 5-F, 6-Cl. In other embodiments, X is 5, 7-dibromo.

In some embodiments, X is 5, 6-methylenedioxy.

In some embodiments, X is — CN.

In some embodiments, X is-OMe (i.e., -O-CH)₃)。

In some embodiments, X is-OH.

In some embodiments, X is-OBn (i.e., -O-benzyl).

In some embodiments, X is-CF₃。

In some embodiments, X is-CH₂OH。

In some embodiments, X is-CH₂OMe。

In some embodiments, X is-CH₂CH₂CO₂H。

In some embodiments, X is-CH₂CH₂CO₂Et。

In the compounds of the formula I, R¹Is H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl.

In some embodiments, R¹Is H.

In other embodiments, R¹Is a lower alkyl group. Lower alkyl as used herein refers to a saturated straight or branched chain hydrocarbon group. Some examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, and the like. In some embodiments, R¹is-CH₃(i.e., methyl).

In other embodiments, R¹Is lower alkyl substituted by hydroxy.

In other embodiments, R¹Is by-NH₂Substituted lower alkyl.

In the compounds of the formula I, R²is-H, lower alkyl substituted by hydroxy or by-NH₂Substituted lower alkyl.

In some embodiments, R²Is H.

In other embodiments, R²Is a lower alkyl group. In some embodiments, R²is-CH₃(i.e., methyl).

In other embodiments, R²Is lower alkyl substituted by hydroxy.

In other embodiments, R²Is by-NH₂Substituted lower alkyl.

In the compounds of formula I, Y is independently-H, halogen, -OMe, -OH, -OBn, -CF₃、-CH₂OH、-CH₂OMe、-NO₂-CN or-C ═ CH₂. The substituents Y may be independently present in formula I at one or more of the 4, 5, 6 or 7 positions of the benzofuran ring.

In some embodiments, Y is H.

In other embodiments, Y is halo (i.e., -F, -Cl, -Br, -I). In some embodiments, Y is 5-F. In other embodiments, Y is 5-Br. In other embodiments, Y is 5-I.

In some embodiments, Y is — CN.

In some embodiments, Y is-OMe (i.e., -O-CH)₃). In some embodiments, Y is 7-OCH₃。

In some embodiments, Y is — OH.

In some embodiments, Y is-OBn (i.e., -O-benzyl).

In some embodiments, Y is-CF₃。

In some embodiments, Y is-CH₂And (5) OH. In some embodiments, Y is 6-CH₂OH。

In some embodiments, Y is-CH₂OMe。

In some embodiments, Y is-C ═ CH₂。

In some embodiments, Y is-NO₂。

In some embodiments of the compounds of formula I, X is preferably 5, 6-methylenedioxy (substituent in 9-ING-41),

in some embodiments, R¹Selected from H, lower alkyl substituted by hydroxy, by-NH₂Substituted lower alkyl, and preferably methyl; in some embodiments, R²Selected from H, lowLower alkyl, lower alkyl substituted by hydroxy, -NH₂Substituted lower alkyl, and preferably is H;

in some embodiments, Y is selected from a 5-or 6-halo group, 5-or 6-NO₂-CN and-C ═ CH₂A group; y is preferably 5-fluoro (5-F).

In some embodiments of the compounds of formula I, X is 5, 6-methylenedioxy; r¹is-CH₃；R²Is H, and Y is 5-F. This embodiment is 9-ING-41, which has the structure shown in formula II below.

The above compounds having different substituents in formula I compared to 9-ING-41 are considered to be analogues thereof.

General methods for synthesizing compounds of formula I are known in the art, some of which are described in the' 216 patent (supra).

In vitro testing of compositions

Compounds of the invention are tested for biological activity, e.g., anti-fibrotic activity, their ability to inhibit the enzyme GSK-3 β and/or inhibit proliferation and collagen production fibroblasts/myofibroblasts, or fibrotic lung fibroblasts, using any of the methods or assays described and/or exemplified herein, or other methods or assays known in the art.

In vivo testing of compositions

The ability of a compound to inhibit pulmonary fibrosis in an animal, preferably a mouse treated with BLM or a constitutively active TGF-beta adenoviral vector (see example VI) is one preferred test for assessing the functional/pharmaceutical activity of a compound. Other tests known in the art that measure the same type of activity may also be used.

Methods for preventing or treating lung injury and fibrosis

The compounds and compositions described herein are used in methods of inhibiting the enzyme GSK-3 β, inhibiting the proliferation of collagen-producing fibroblasts/myofibroblasts in vitro or in vivo, and treating pulmonary fibrosis/IPF.

Pharmaceutical and therapeutic compositions and their administration

Compounds that may be used in the pharmaceutical compositions of the present invention include the compounds described above, preferably 9-ING-41, and analogs thereof, as well as pharmaceutically acceptable salts or solvates of such compounds. "pharmaceutically acceptable salt" refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Sample acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid; and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like.

Sample base addition salts include those derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides, such as, for example, tetramethylammonium hydroxide. Chemical modification of a pharmaceutical compound (i.e., drug) into a salt is a technique well known to pharmaceutical chemists to achieve improved physical and chemical stability, hygroscopicity, flowability, and solubility of compounds. See, e.g., H.Ansel et al, Pharmaceutical Dosage Forms and Drug Delivery Systems (6)^thEd.1995), for example, at pages 196 and 1456-1457.

As mentioned above, the compounds of the invention have the ability to inhibit the enzyme GSK-3 β and are useful in the treatment of pulmonary fibrosis.

The compounds of the present invention and pharmaceutically acceptable salts or solvates thereof may be incorporated into suitable dosage forms such as capsules, impregnated wafers, tablets or preferably injectable formulations. Solid or liquid pharmaceutically acceptable carriers can be used. "pharmaceutically acceptable" such as pharmaceutically acceptable carriers, excipients, and the like, means pharmacologically acceptable and substantially non-toxic to a subject to which a particular compound is administered.

Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, saline, water, dextrose, glycerol, and the like. Similarly, the carrier or diluent may include any extended release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. When a liquid carrier is used, the formulation may be in the form of: syrups, elixirs, emulsions, soft gelatin capsules, sterile injectable liquid (e.g., solutions), such as ampoules (ampoules), or aqueous or non-aqueous liquid suspensions. An overview of such pharmaceutical compositions can be found, for example, in Gennaro, AR, Remington: the Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishers; version 21, 2005 (or latest version).

Pharmaceutical formulations are prepared according to conventional techniques of pharmaceutical chemistry involving, for example, the following steps: mixing, granulating and compressing (as needed for tablet form), or mixing, filling and dissolving the ingredients (as the case may be) to give the desired product for oral, parenteral, topical, transdermal, intravaginal, intracapsular, intranasal, intrabronchial, intracranial, intraocular, intraaural and rectal administration. The pharmaceutical compositions may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like.

The invention may be used to treat any of a number of animal genera and species, and is equally applicable in the practice of human or veterinary medicine. Accordingly, the pharmaceutical compositions are useful for treating domesticated and commercial animals, including birds, and more preferably mammals, and most preferably humans.

The compositions of the present invention may be administered to a subject by any suitable route of administration, including orally, by inhalation, subcutaneously, intramuscularly, intravenously, transdermally, vaginally, rectally, or any combination thereof.

In some embodiments, the compositions of the present invention are administered orally, by inhalation, subcutaneously, intramuscularly, or intravenously.

In some embodiments, the compositions of the present invention are administered orally.

In other embodiments, the compositions of the present invention are administered orally.

In other embodiments, the compositions of the present invention are administered by inhalation.

In other embodiments, the compositions of the present invention are administered subcutaneously.

In other embodiments, the compositions of the present invention are administered intramuscularly.

In other embodiments, the compositions of the present invention are administered intravenously.

The term "systemic administration" refers to administration of a therapeutic compound in a manner, such as intravenous (i.v.) injection or infusion, that results in the compound being introduced into the circulatory system of the subject or otherwise allows the compound to diffuse systemically. By "regional" administration is meant administration into a specific and somewhat more restricted anatomical space, such as instillation or inhalation into the lungs (the preferred route), or intrapleural, intraperitoneal, intrathecal, subdural administration, or administration to a specific organ. Other examples include: intranasally, which is a route corresponding to instillation or inhalation into the lungs; intrabronchial, in ear or in eye, etc. The term "topical administration" refers to the administration of a composition or drug into a confined or restricted anatomical space, such as subcutaneous (s.c.) injection, intramuscular (i.m.). The skilled person will appreciate that local or regional administration also typically results in entry of the composition into the circulatory system, and therefore s.c. or i.m. is also a route of systemic administration. The instillable, injectable or infusible formulations may be prepared in conventional forms such as solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to injection or infusion, or as emulsions. While the preferred regional route of administration is into the lungs, the pharmaceutical composition may be administered systemically or topically or transdermally, either separately or simultaneously with instillation or inhalation into the lungs.

Other pharmaceutically acceptable carriers for use in the compositions of the invention are liposomes, in which the active polypeptide comprised in the pharmaceutical composition is dispersed or otherwise present in bodies consisting of aqueous concentric layers adhered to a lipid layer. The active compounds are preferably present in the aqueous and lipid layers, internally or externally, or in any case in heterogeneous systems, commonly known as liposomal suspensions. The hydrophobic or lipid layer typically, but not exclusively, comprises phospholipids (such as lecithin and sphingomyelin), steroids (such as cholesterol), more or less ionic surface active substances (such as dicetyl phosphate, stearylamine or phosphatidic acid) and/or other substances of a hydrophobic nature. Other suitable embodiments of the liposomal formulation of the present invention will be understood by those skilled in the art.

The therapeutic dose administered is a therapeutically effective amount as known or readily determinable by one of skill in the art. The dosage will also depend on the age, health and weight of the recipient, the nature of concurrent therapy (if any), the frequency of treatment, and the nature of the desired effect.

Method of treatment

The methods of the invention are useful for treating pulmonary fibrosis (also known as IPF) in a subject in need thereof. The term "treating" is broadly defined to include at least the following: inhibiting, alleviating, ameliorating, preventing, reducing the incidence of, or recurrence of a disease or disorder being treated or prevented, including the frequency and/or time of recurrence or the severity of symptoms thereof. This may occur as a result of any of inhibiting epithelial cell death, inhibiting fibroblast proliferation, other biological or biochemical mechanisms disclosed herein as being associated with or leading to IPF.

Benzofuran-3-yl- (indol-3-yl) maleimide GSK-3 β inhibitors (i.e. compounds of formula I), most preferably 9-ING-41 or a pharmaceutically acceptable salt or solvate thereof, are preferably administered as a pharmaceutical composition as described above.

The dose of the compound preferably comprises a pharmaceutical dosage unit comprising an effective amount of 9-ING-41. Dosage unit form refers to physically discrete units suitable as unitary dosages for mammalian subjects; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the invention are indicated by and directly depend on the following: (a) the unique characteristics of the active substance and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the art of compounding such active compounds with respect to the treatment and sensitivity of individual subjects.

By "effective amount" is meant an amount sufficient to achieve a measurable decrease in regional or steady state concentration of any relevant parameter that causes disease in vivo.

By "antifibrotic effective amount" is meant an amount sufficient to inhibit or reduce proliferation and/or differentiation of pulmonary myofibroblasts into Fibrotic Lung (FL) fibroblasts, or sufficient and reduced proliferation of FL fibroblasts.

The amount of active compound administered depends on which compound (e.g., 9-ING-41) is selected, the exact disease or disorder, the route of administration, the health and weight of the recipient, the presence of other concurrent treatments, if any, the frequency of treatment, the nature of the desired effect, and the judgment of the skilled practitioner.

A preferred single dose for once daily administration of a compound of from about 0.2mg/kg to about 250mg/kg (e.g., 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245 or 250 mg/kg), from about 0.2mg/kg to about 5mg/kg, from about 0.5mg/kg to about 5mg/kg (e.g., 0.2, 1, 5, 3, 3.5, 4, 5, 6, or preferably from about 10mg/kg to about 50mg/kg, for example by inhalation. Such doses may be administered daily for any period of time from about 3 days to one or more weeks. Chronic administration is also possible, but as is well known in the art, downward adjustment of the dosage may be required. However, the foregoing ranges are suggestive, as the number of variables in an individual treatment regimen is large, and considerable deviations from these preferred values are expected.

For continuous administration, e.g., by a pump system (e.g., osmotic pump used in some of the experiments described below), the total dose for a time course of about 1 to 2 weeks is preferably 1mg/kg to 1g/kg (e.g., 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 780, 790, 800, 840, 870, 830, 850, 890, 880, 890, 860, 800, 860, 160, 180, 150, 160, 180, 150, 180, 900. 910, 920, 930, 940, 950, 960, 970, 980, 990 or 1000mg/kg), preferably 20 to 300mg/kg, more preferably 50 to 200 mg/kg. Following such a continuous dosing regimen, the total concentration of active compound is preferably from about 0.5 to about 50 μ M (e.g., 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μ M), preferably from about 1 to about 10 μ M.

An effective concentration of an active compound for inhibiting GSK-3 β or preventing proliferation of myofibroblasts and fibrotic lung fibroblasts in vitro is from about 0.5 μ M to about 100 μ M (e.g., 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 μ M), more preferably from about 2 μ M to about 20 μ M. Effective concentrations, dosages, and optimal dosage ranges can be determined in vitro using the methods described herein.

In the methods of the invention, the compound or composition may be administered to the subject by any suitable route of administration, including orally, by inhalation, subcutaneously, intramuscularly, intravenously, transdermally, vaginally, rectally, or any combination thereof.

In some embodiments, the compound or composition is administered orally, by inhalation, subcutaneously, intramuscularly, or intravenously.

In some embodiments, the compound or composition is administered orally.

In other embodiments, the compound or composition is administered orally.

In other embodiments, the compound or composition is administered by inhalation.

In other embodiments, the compound or composition is administered subcutaneously.

In other embodiments, the compound or composition is administered intramuscularly.

In other embodiments, the compound or composition is administered intravenously.

Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless otherwise specified. The examples are included to illustrate some preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example I

Materials and methods and two pulmonary fibrosis models

All Animal studies were approved by the Institutional Animal Care and Use Committee of the University of Texas, Taylor Health Science Center at Tyler. C57BL/6 mice (10 to 12 weeks old, about 20g (from Jackson Laboratory, Bar Harbor ME)) were first anesthetized by Intraperitoneal (IP) injection with xylazine/ketamine. Lung injury was then triggered by some modification according to (Sisson TH et al, Am J Pathol 2015, 185: 969-. Bleomycin sulfate (Teva, 0.8 units/kg) was administered intratracheally via an endotracheal tube. Animals were monitored daily for signs of respiratory distress, significant weight loss, or moribund status. The control group received physiological saline under the same conditions.

Treatment was started 14 days after injury initiation. For the GSK-3 β inhibitor study, treatment with 30mg/kg of 9-ING-41 (given by actual Therapeutics, Ft. worth, TX) or vehicle control dimethyl sulfoxide/DMSO in 40 μ l volumes was administered daily by IP injection for up to 14 days.

At the completion of the 28 day time course, the mice were evaluated for changes in lung function (including resilence and compliance) by flexive (Tucker T et al, Am J Respir Cell Mol Biol 2019 (doi: 10.1165/rcmb.2018-0121OC. electronic edition before printing), Kamata H et al, Sci Rep 2017, 7: 4556, Boren J et al, supra, Tucker TA et al, 2014, supra, Tucker TA et al, Clin Transl Med 5: 17, 2016). Briefly, mice were anesthetized with a chloraminone/xylazine mixture. Anesthetized mice were intubated by inserting a sterile # 20 intravenous cannula through the vocal cords into the trachea and maintained under anesthesia during the pulmonary function test using isoflurane. Measurements were performed using the flexiVent system (SCIREQ, Tempe AZ). The lung compliance was determined using the "snapshot perturbation method" according to the manufacturer's instructions. On day 28, mice were euthanized and lungs were harvested in one piece (en bloc). Lung morphometry was determined by trichrome staining of 5 μm lung tissue sections as described in the references cited above. All tissue sections were first deparaffinized and antigen-repaired using citrate buffer for 20 minutes at 95 ℃. Tissue analytes, collagen deposition and localization were first assessed by trichrome staining. Immunofluorescence was used to visualize α -SMA (MAB1420, R & D) and then confocal microscopy was used to visualize immunofluorescence and co-localization of the markers. Images were acquired from the field of view at 40x using an LSM 510 Meta confocal system (Carl Zeiss) in 0.4 μm z axis increments as described.

In a second model, pulmonary fibrosis was induced by intratracheal instillation of a constitutively active TGF-beta adenoviral vector (Ad-TGF-beta) carrying the C223S/C225S mutation, as reported by (Mackinnon AC et al, Am J Respir Crit Care Med 2012, 185: 537-46) with some modifications. Briefly, 3 × 10⁸pfu of Ad-TGF-. beta.or eGFP adenovirus control vector (Ad-eGFP) was administered intratracheally in a volume of 40. mu.l. Mice were then monitored daily untilUntil the 14 day time course was completed. For the GSK-3 β inhibition study, mice received 9-ING-41 treatment daily 7 days after adenoviral vector administration. At the end of the time course, pulmonary function tests and CT scans were performed as described (Tucker T et al, 2019, supra; Kamata H et al, 2017, supra; Boren J et al, 2017, supra; Tucker TA et al, 2016, supra).

All statistical analyses of animal studies were performed using the mann-whitney U test (student's t test for in vitro studies). P-values less than 0.05 were considered significant.

Example II

9-ING-41 ameliorates bleomycin-mediated lung function and volume reduction

As shown in FIGS. 1A-1B, inhibition of GSK-3 β with 9-ING-41 attenuated pulmonary fibrosis. Bleomycin sulphate (0.8U/kg) was administered intratracheally to C57Bl/6J mice and after 14 days, treated with DMSO (vehicle) or the GSK-3 β inhibitor 9-ING-41(30 mg/kg). The drug was delivered by intraperitoneal (ip) injection in a volume of 40 μ l for up to 14 days. At the completion of the 28 day course, lung compliance was determined using Scireq flexive. Lung reconstructions were also collected by gated CT scanning. These reproductions are then used to determine lung volume. Treatment with 9-ING-41 significantly improved the reduction in lung compliance (p ═ 0.04) and volume (p ═ 0.026).

Example III

9-ING-41 treatment blocked bleomycin-induced collagen deposition in pulmonary fibrosis: three colors

Lung tissue sections (5 μm) were prepared from vehicle and treated with 9-ING-41. The sections were deparaffinized and trichromatic stained to detect changes in lung structure and collagen deposition (trichromatic images not shown). Images were acquired at 20X and represent 30 fields/slide/mouse, n ═ 6 animals/treatment.

Fibrotic foci consistent with bleomycin-induced pulmonary fibrosis were found throughout the injured lung. The matrix deposition area is easily and uniformly present throughout the lung. Mice treated with 9-ING-41 also showed areas of injury; however, these areas are fewer in number and smaller than the areas found in vehicle-treated animals. Furthermore, collagen deposition within fibrotic foci was overall reduced in 9-ING-41 treated mice (FIG. 2A) compared to DMSO-treated controls (FIG. 2B). See also fig. 5A to 5F.

Example IV

9-ING-41 treatment blocks collagen deposition in pulmonary fibrosis: collagen imaging

Lung sections from vehicle and 9-ING-41 treated mice were immunostained for α -SMA, a marker of myofibroblast differentiation. Images were acquired by confocal microscopy at 40X (fig. 6A and 6B). 9-ING-41 treatment reduced alpha-SMA expression in 9-ING-41 treated mice when compared to vehicle-treated control (DMSO). The filled arrows indicate the regions of collagen alpha-SMA expression. Images are representative of 30 fields/mouse and n-3 mice/treatment.

Lung sections from vehicle and 9-ING-41 treated mice were immunostained for collagen 1 deposition and imaged by confocal microscopy at 40X. 9-ING-41 reduced collagen 1 deposition in bleomycin-damaged lungs compared to vehicle (DMSO) -treated controls. These results indicate that inhibition of GSK-3 β with 9-ING-41 reduces collagen deposition in fibrotic lung lesions.

Example V

9-ING-41 shows a marker for decreasing MesoMT: alpha-SMA

Lung sections from vehicle and 9-ING-41 treated mice were immunostained for α -SMA, a marker of myofibroblast differentiation. Images were acquired by confocal microscopy at 40X (fig. 7A and 7B). The filled arrows indicate the regions of collagen alpha-SMA expression. Images are representative of 30 fields/mouse and n-3 mice/treatment. 9-ING-41 treatment reduced alpha-SMA expression in drug-treated mice when compared to vehicle-treated controls.

Example VI

9-ING-41 ameliorating TGF-beta-adenovirus inductionInjury of lung

Pulmonary fibrosis was induced by intratracheal instillation of a constitutively active TGF-beta adenoviral vector (Ad-TGF-beta) carrying the C223S/C225S mutation, as previously reported (Mackinnon AC et al, Am J Respir Crit Care Med 2012, 185: 537-46) with some modifications. Briefly, 3 × 10⁸Ad-TGF-beta or eGFP adenovirus control vector (Ad-eGFP) per plaque forming unit (pfu) was administered intratracheally in a volume of 40. mu.l. Mice were then monitored daily until the 14 day time course was completed. For the GSK-3 β inhibition study, mice received 9-ING-41 treatment daily 7 days after administration of the adenoviral vector. At the end of the time course, pulmonary function tests and CT scans were performed as described (Tucker T et al, 2019, supra; Kamata H et al, 2017, supra; Boren J et al, 2017, supra; Tucker TA et al, 2016, supra).

The results are shown in FIG. 3. Mice treated with 9-ING-41 showed significantly improved lung compliance compared to each vehicle-treated or pseudovehicle-treated control. FIGS. 4A/4B show a reduction in the area of injury and increased collagen deposition in 9-ING-41 treated mice compared to DMSO-treated controls.

The IPF is characterized by: increased myofibroblast presence and subsequent increased deposition of extracellular matrix (ECM) proteins such as collagen and fibronectin. In the IPF model of the invention, bleomycin sulphate and TGF-beta adenovirus are introduced via an endotracheal tube, which is a method to produce robust and consistent lesions (thus requiring fewer animals to produce statistically significant results).

Bleomycin and TGF-beta-adenovirus mediated fibrosis is characterized by: fibrotic foci exist that contain collagen and other ECM matrix proteins that promote scarring of the lung, loss of normal lung structure, and impaired lung function. In addition, these fibrotic foci also contain increased numbers of myofibroblasts, which are considered to be a major source of increased ECM deposition.

To evaluate the efficacy of the preferred GSK-3 β inhibitory compound 9-ING-41, IPF intervention was performed at a time point where changes in lung structure and function could be detected. Based on the preliminary study, 14 day time points were selected for bleomycin damage studies and 7 days were selected for TGF-beta adenovirus studies. Upon completion of the time course (28 days and 14 days, respectively), 9-ING-41 treated mice in both models showed not only improved lung function but also increased lung capacity.

Based on the histopathological analysis discussed and exemplified above, the inventors concluded that: the reduced number of fibrotic foci combined with reduced scarring contributes to the restoration of lung function due to 9-ING-41 treatment. Confocal microscopy analysis further showed that fibrotic foci were found in mice treated with 9-ING-41 in fewer numbers, smaller and contained significantly less collagen than in vehicle-treated control animals. These similar findings in two independent models of pulmonary fibrosis suggest that 9-ING-41 treatment significantly improved IPF outcome. Furthermore, this effect is believed to be caused, at least in part, by a diminished fibroblast-myofibroblast differentiation. The present results indicate that GSK-3 β inhibition with 9-ING-41 represents a novel treatment for IPF.

These were the earliest studies indicating that the GSK-3 β signaling pathway is critical for inducing myofibroblast differentiation. These studies also indicate that therapeutic targeting of GSK-3 β attenuates the progression of pulmonary fibrosis; it is also contemplated that the effect occurs in a human subject. Since therapeutic targeting of GSK-3 β alleviates fibrotic lung remodeling in vivo, this work provides a basis for targeting the GSK-3 β signaling pathway to control fibroblast-myofibroblast differentiation and pulmonary fibrosis fate. The significantly improved compliance and capacity produced by 9-ING-41 in particular is also an important indicator of its effectiveness in treating IPF in mouse models, and this is expected to occur in human IPF patients as well.

The references cited above are all incorporated herein by reference, whether specifically incorporated or not.

Having now fully described this invention, it will be understood by those skilled in the art that the same may be performed within a wide variety of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

The present disclosure also encompasses the following aspects:

an anti-fibrotic pharmaceutical composition formulated for injection or pulmonary instillation comprising:

(i) a benzofuran-3-yl- (indol-3-yl) maleimide compound that inhibits the enzyme glycogen synthase kinase 3 type beta (GSK-3 β), which has at least 20% of the biological or biochemical activity of 9-ING-41 in an in vitro or in vivo assay;

(ii) a pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition of aspect 2. aspect 1, wherein the compound is 9-ING-41 or an analog of 9-ING-41 that has at least 20% of the biological or biochemical activity of 9-ING-41 in an in vitro or in vivo assay, the 9-ING-41 having the formula:

the pharmaceutical composition of aspect 3. aspect 2, wherein the compound is 9-ING-41.

Aspect 4. the pharmaceutical composition of any one of aspects 1 to 3, formulated for pulmonary instillation.

Aspect 5. a method for inhibiting proliferation and/or differentiation of pulmonary myofibroblasts into Fibrotic Lung (FL) fibroblasts and reducing proliferation of said FL fibroblasts, comprising providing to said myofibroblasts and FL fibroblasts an effective GSK-3 β inhibitory amount of a compound or composition of any one of aspects 1 to 4.

The method of aspect 6. aspect 5, wherein the compound is 9-ING-41 or the analog thereof.

The method of aspect 7. aspect 6, wherein the compound is 9-ING-41.

The method of aspect 8. aspect 7, wherein the composition is formulated for pulmonary instillation.

The method of aspect 9. aspect 5 to 8, wherein the providing is performed in vivo.

The method of aspect 10. aspect 9, wherein the providing is by pulmonary instillation.

The method of aspect 11, aspect 9 or 10, wherein the providing is to a human.

Aspect 12 a method for treating a mammalian subject suffering from or experiencing a disease or disorder characterized by Idiopathic Pulmonary Fibrosis (IPF), comprising administering to the subject an effective amount of the pharmaceutical composition of any one of aspects 1 to 4.

The method of aspect 13. aspect 12, wherein the compound or composition comprises 9-ING-41 or an analog thereof having at least 20% of the biological or biochemical activity of 9-ING-41 in an in vitro or in vivo assay.

The method of aspect 14, aspect 13, wherein the compound is 9-ING-41.

The method of any one of aspects 15, 12 to 14, wherein the subject is a human.

Aspect 16. the use of a composition of any of aspects 1 to 4 for treating IPF in a mammalian subject, wherein the compound inhibits GSK-3 β, and inhibits lung myofibroblast proliferation and/or differentiation into FL fibroblasts, and reduces said FL fibroblast proliferation; and administering an effective amount of the composition to a subject having IPF.

The use of aspect 17. according to aspect 16, wherein the compound is 9-ING-41 or the analog thereof.

The use according to aspect 17, wherein the compound is 9-ING-41.

The use according to any one of aspects 16 to 18, wherein the composition is administered by pulmonary instillation.

Aspect 20 use of a composition as described in aspects 1 to 4 for the preparation of a medicament for treating IPF in a subject in need thereof, said compound inhibiting GSK3 β activity, and inhibiting lung myofibroblast proliferation and/or differentiation into FL fibroblasts, and reducing said FL fibroblast proliferation.

The use of aspect 21. according to aspect 20, wherein the compound is 9-ING-41 or an analogue thereof which has at least 20% of the biological or biochemical activity of 9-ING-41 in an in vitro or in vivo assay.

The use according to aspect 21, wherein the compound is 9-ING-41.

Aspect 23. the use according to any one of aspects 20 to 22 for the preparation of a medicament for pulmonary instillation in the treatment of IPF.

Aspect 24 the use according to any one of aspects 20 to 24, wherein the subject is a human.