Protected succinate for use in the treatment of fibrotic and interstitial lung disease

Abstract

The present invention relates to a cell membrane permeable protected succinate according to formula (II) for use in the treatment of a disease selected from a fibrotic disease and an interstitial lung disease. Moreover, the present invention relates to a composition comprising the cell membrane permeable protected succinate for use in the treatment of a disease selected from a fibrotic disease and an interstitial lung disease and to a method of treating a disease selected from a fibrotic disease and an interstitial lung disease in a human subject.

Description

Our ref. : M 12200WO / ADW. KWOPROTECTED SUCCINATE FOR USE IN THE TREATMENT OF FIBROTIC AND INTERSTITIALLUNG DISEASETECHNICAL FIELD

[0001] The present invention relates to a cell membrane permeable protected succinate according to formula (II) for use in the treatment of a disease selected from a fibrotic disease and an interstitial lung disease. Moreover, the present invention relates to a composition comprising the cell membrane permeable protected succinate for use in the treatment of a disease selected from a fibrotic disease and an interstitial lung disease and to a method of treating a disease selected from a fibrotic disease and an interstitial lung disease in a human subject.BACKGROUND OF THE INVENTION

[0002] It is estimated that about 5-40 in 100,000 people worldwide suffer from idiopathic pulmonary fibrosis, and many more from other fibrotic diseases and other interstitial lung diseases.

[0003] Presently, only very limited treatment options exist for diseases associated with increasing fibrosis of the parenchyma of various organs, with pulmonary fibrosis presenting an especially stark example thereof. Previous pharmacological therapy has been marked by numerous setbacks; the most impressive of which was the failure of glucocorticoids, which had previously been used as the standard therapy for pulmonary fibrosis, in clinical trials; proving them ineffective in practice. Numerous candidate treatments, such as sildenafil, interferon gamma, ziritaxestat, and simtuzumab, have also proven ineffective.

[0004] Only two substances, nintedanib and pirfenidone, have been shown to have any treatment effect. However, these substances can only slow the progression of the disease, but not stop it completely. No beneficial effect on mortality has yet been proven conclusively (Xu H et al., Ann Am Thorac Soc. 2024 Oct;21 (10):1407-1415; Kyle AR, Dempsey TM. An Eternal Dilemma: Exposing the Impact of Immortal Time Bias on Antifibrotic Effectiveness. Ann Am Thorac Soc. 2024 Oct;21 (10):1383-1384). The average cost of treatment per patient per month with nintedanib or pirfenidone amounts to about 1500 Euros, presenting a significant financial strain on patients. In addition, nintedanib and pirfenidone often lead to gastrointestinal and other side effects that may make it necessary to discontinue therapy.

[0005] If the loss of functioning lung parenchyma becomes too great in a given patient with pulmonary fibrosis, a lung transplant is ultimately necessary. This, too, however is often not a valid treatment option, since on the one hand, there are not enough donor organs available and, on the other hand, not every patient is eligible for such a transplant (e.g. due to comorbidities).

[0006] A number of cell membrane permeable protected succinates were identified in a screen in WO2014053857A1 for the purpose of enhancing mitochondrial function in mitochondrial diseases (but not for the treatment of fibrotic disease or interstitial lung disease). These cell membrane permeable protected succinates increase mitochondrial respiration in rotenone-inhibited intact cells compared to mitochondrial respiration in the rotenone-inhibited cell prior to addition of the cell membrane permeable protected succinate.ADW:KWO

[0007] For these reasons, there is a great and continued need for further additional therapeutic strategies that have fewer side effects, can more efficiently slow or even stop disease progress, and / or are cheaper than available treatments.SUMMARY OF THE INVENTION

[0008] The inventors surprisingly found that cell membrane permeable protected succinate compounds, such as e.g., diethylsuccinate (DES), can treat fibrotic changes, e.g. by reducing profibrotic processes such as collagen secretion and expression of smooth muscle cell actin, can increase apoptosis in fibroblasts derived from pulmonary fibrosis patients, and can improve lung function and weight in an animal model of pulmonary fibrosis.

[0009] DES is a toxicologically harmless product commonly used as a flavoring agent in food and as a fragrance in perfumes. Despite its widespread use, no occurrences of allergies or other adverse effects have been recorded. This is of particular importance, as pulmonary fibrosis and other fibrotic diseases and interstitial lung diseases typically require long-term therapy. DES and other cell membrane permeable protected succinate compounds can be chemically synthesized in a cost-effective and straightforward manner.

[0010] It was therefore an object of the present invention to provide protected succinate compounds for use in the treatment of a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, which are cell membrane permeable.

[0011] Thus, in a first aspect, the present invention relates to a cell membrane permeable protected succinate for use in the treatment of a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, preferably a fibrotic interstitial lung disease, wherein the cell membrane permeable succinate comprises a succinate core of formula (I):Formula (I).

[0012] The cell membrane permeable protected succinate for use as defined herein has formula (II):Formula (II) wherein R1is H, a pharmaceutically acceptable salt, an alkyl group or a group of formula (III) and R2is independently an alkyl group, or a group according to formula (III) where formula (III) is:Formula (III) wherein R3is H, C C3alkyl, or is linked together with R5by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring;R4is H;R5is OCORa, OCOORb, OCONRcRd, CONRcRdor is linked to R3by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; where Rais CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms.

[0013] In a preferred embodiment of the first aspect, the cell membrane permeable protected succinate is diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate.

[0014] In an embodiment of the first aspect, the cell permeable protected succinate of any embodiment of the first aspect increases mitochondrial respiration in a rotenone-inhibited intact cell compared to mitochondrial respiration in the rotenone-inhibited cell prior to addition of the cell permeable protected succinate, preferably wherein mitochondrial respiration is measured by a high resolution extracellular metabolic flux analyzer.

[0015] In a second aspect, the present invention relates to a composition comprising the cell membrane permeable protected succinate for use as defined herein for use in the treatment of a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, preferably a fibrotic interstitial lung disease.

[0016] In a preferred embodiment of the first or second aspect, the fibrotic disease is selected from the group consisting of pulmonary fibrosis regardless of underlying aetiology, renal fibrosis, hepatic fibrosis, systemic sclerosis, and pulmonary collagenosis.

[0017] In one such embodiment of the first or second aspect, the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, diffuse pulmonary fibrosis, and chronic pulmonary fibrosis, preferably the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis.

[0018] In an embodiment of the first or second aspect, the interstitial lung disease is selected from the group consisting of alveolar proteinosis, pulmonary alveolar microlithiasis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-Rich syndrome, idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component, preferably wherein the interstitial lung disease is selected from the group consisting of idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component.

[0019] In a third aspect the present invention relates to a method of treating a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, preferably a fibrotic interstitial lung disease in a human subject, the method comprising administering to the subject an effective amount of a cell membrane permeable protected succinate or a composition comprising the same and a pharmaceutically acceptable carrier, wherein the cell membrane permeable succinate comprises a succinate core of formula (I):Formula (I).

[0020] In said method the cell membrane permeable protected succinate has formula (ii):Formula (II) wherein R1is H, a pharmaceutically acceptable salt, an alkyl group or a group of formula (III) and R2is independently an alkyl group, or a group according to formula (III) where formula (III) is:Formula (III) wherein R3is H, C C3alkyl, or is linked together with R5by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring;R4is H;R5is OCORa, OCOORb, OCONRcRd, CONRcRdor is linked to R3by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; where Rais CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms.

[0021] In a preferred embodiment of said method, the cell membrane permeable protected succinate is diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate.

[0022] In any embodiment of the third aspect, the cell permeable protected succinate increases mitochondrial respiration in a rotenone-inhibited intact cell compared to mitochondrial respiration in the rotenone-inhibited cell prior to addition of the cell permeable protected succinate, preferably wherein mitochondrial respiration is measured by a high resolution extracellular metabolic flux analyzer.

[0023] In any embodiment of the third aspect, the fibrotic disease is selected from the group consisting of pulmonary fibrosis, renal fibrosis, hepatic fibrosis, systemic sclerosis, and pulmonary collagenosis.

[0024] In one such embodiment of the third aspect, the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, diffuse pulmonary fibrosis, and chronic pulmonary fibrosis, preferably wherein the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis.

[0025] In an embodiment of the third aspect, the interstitial respiratory disease is selected from the group consisting of alveolar proteinosis, pulmonary alveolar microlithiasis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-Rich syndrome, idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component, preferably wherein the interstitial lung disease is selected from the group consisting of idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1. Diethylsuccinate (DES) reduces the expression of fibrosis-associated markers in fibroblasts derived from interstitial pulmonary fibrosis (IPF) patients. IPF patient derived fibroblasts were treated with 0 (VEH), 1 , 3 or 10mM DES. After treatment for 72 hours, cells were lysed for western blot analysis of aSMA or RNA isolation for CTHRC1 qPCR, and the cell culture medium supernatants were collected for ELISA. (A) Western blot analysis for aSMA. (B) ELISA for Coll A1 and (C) fibronectin. (D) qPCR for CTHRC1 . One-way ANOVA for repeated measurements (A-C) and paired t-test (D) were used to assess statistical significance. *=p<0.05, **=p<0.01 . VEH- OmM DES / vehicle.

[0027] Figure 2. DES induces apoptosis in IPF patient-derived fibroblasts in vitro. IPF patient derived fibroblasts were treated with 0 (VEH), 1 , 3 or 10mM DES. After treatment for 72 hours, apoptosis was evaluated by colorimetric measurement of caspase 3 / 7 activity (A) and TUNEL staining (B), FAS-L- Fas ligand (positive control). One-way ANOVA for repeated measures was used to assess statistical significance. *=p<0.05, **=p<0.01.

[0028] Figure 3. DES improves fibrosis in the murine bleomycin model of pulmonary fibrosis.C57 / BI6 J mice were intra-tracheally instilled with bleomycin or saline on day 0, followed by intraperitoneal administration of DES from day 11 until day 21 post bleomycin administration. On day 11 lung function parameters were assessed in mice using the Flexivent system (Scireq, Montreal, CA). (A) Schematic representation of the bleomycin model of pulmonary fibrosis in mice used to evaluate the potential anti-fibrotic role of DES in vivo. Lung function parameters including pressure-volume curves (B), static compliance (C), inspiratory capacity (D) and forced vital capacity (FVC) (E) were assessed. Hydroxyproline assay was performed on the lung homogenates of the respective groups of mice treated with bleomycin (F). Masson-trichrome staining was performed on mouse lung tissue sections obtained (G). The stained sections were then scored using the Modified Ashcroft score method (H). One way ANOVA was used to assess statistical significance. *=p<0,05, **=p<0,01 ,*** =p<0,001 , ****=p<0,0001 . VEH VEH- Vehicle + vehicle, VEH DES- Vehicle + 85mg / kg DES, BLEO VEH- Bleomycin + VEH, BLEO DES- Bleomycin + 85mg / kg DES, BLEO LOW DES- Bleomycin + 25mg / kg DES.

[0029] Figure 4. DES does not inhibit succinate dehydrogenase activity. Lung tissue of mice receiving vehicle or diethylsuccinate for 10 days (once daily, 85mg / kg) was homogenized in ddH2O. Using a commercial kit for measuring SDH activity, lysates were incubated for 25 minutes according to the manufacturer’s instructions and absorbance was measured. Vehicle: vehicle treated mice, DES: 85 mg / kg DES, vehicle + added DES: diethylsuccinate at 10 mM was added exogenously immediately prior to the start of the assay. (A) SDH activity as calculated per the manufacturer’s instructions, (B) absorbance traces.

[0030] Figure 5. Schematic figure of screening assay for identifying cell membrane permeable protected succinates. The schematic shows the protocol for evaluating novel protected succinates for cell membrane permeability. In the assay, mitochondrial function in intact cells is repressed with the respiratory complex I inhibitor rotenone. Candidate compounds are compared with the endogenous substrate succinate, which is not cell membrane permeable, before and after permeabilization of the plasma membrane to evaluate bioenergetic enhancement or inhibition.

[0031] Figure 6. DES alters the transcriptome of IPF patient derived fibroblasts. (A) Schematic representation of experimental setup for RNA sequencing analysis of IPF patient fibroblasts treated with or without DES (B) Heatmap of RNA sequencing data from IPF patient-derived fibroblasts treated with orwithout DES. (C) Top genes upregulated and downregulated by DES treatment of IPF fibroblasts. n=12, data represented as mean ± SD. DES- Diethyl succinate, IPF- Idiopathic Pulmonary Fibrosis, COL1A1- Collagen 1 A1 , CTHRC1- Collagen Triple Helix Repeat Containing 1 , DEPP1- Decidual Protein Induced by Progesterone, CEMIP- Cell Migration Inducing and Hyaluronan Binding Protein, COL1A2- Collagen 1A2, PLIN2- Perilipin 2, PPARy- Peroxisome Proliferation Activated Receptor gamma, AKR1C1- Aldo- Keto Reductase family 1 member C1 .

[0032] Figure 7. DES induces myogenic to lipogenic transdifferentiation in IPF fibroblasts. (A) Schematic representation of the experimental set up to investigate DES as a mediator of myogenic to lipogenic transition in IPF fibroblasts. (B) Western blotting analysis of PLIN2 expression in IPF fibroblasts that recieved vehicle control or DES. Vinculin was used as a housekeeping control. (C) Quantification of B. (D) ELISA for procollagen 1a1 in cell culture supernatants from IPF patient fibroblasts treated with or without DES. (E) Representative immunofluorescent images for LIPID TOX green staining of of IPF patient fibroblasts treated with or without DES. (F) Quantification of E. n=6, data represented as mean ± SD. Statistical analysis was performed using paired t-tests. DES- Diethyl Succinate, IPF- Idiopathic Pulmonary Fibrosis.

[0033] Figure 8. Dimethylsuccinate (DMS) also reduces the expression of fibrosis-associated markers in fibroblasts derived from interstitial pulmonary fibrosis (IPF) patients. IPF patient derived fibroblasts were treated with 0 (VEH) or 10mM DMS. After treatment for 72 hours, cells were lysed for western blot analysis of aSMA, and the cell culture medium supernatants were collected for ELISA. (A) Western blot analysis for aSMA. (B) ELISA for Coll A1 . Student’s t-test was used to assess statistical significance. VEH- OmM DMS / vehicle.DETAILED DESCRIPTION OF THE INVENTION

[0034] As has been set out above, the present invention concerns in one aspect a cell membrane permeable protected succinate for use in the treatment of a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, preferably a fibrotic interstitial lung disease, wherein the cell membrane permeable succinate comprises a succinate core of formula (I):Formula (I)

[0035] The cell membrane permeable protected succinate for use as defined herein has formula (II):Formula (II) wherein R1is H, a pharmaceutically acceptable salt, an alkyl group or a group of formula (III) and R2is independently an alkyl group, or a group according to formula (III) where formula (III) is:Formula (III) wherein R3is H, C C3alkyl, or is linked together with R5by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; R4is H;R5is OCORa, OCOORb, OCONRcRd, CONRcRdor is linked to R3by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; where Rais CH3, CH2CH3, CH(CH3)2, C(CH3)3 or cycloalkyl;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3 or cycloalkyl;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms. Specific, non-limiting embodiments of Formula II are specified below in the section “Cell membrane permeable protected succinates”. In a particularly preferred embodiment of the first aspect, the cell membrane permeable protected succinate is diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate. In a particularly preferred embodiment of the first aspect, the cell membrane permeable protected succinate is diethyl succinate (DES).

[0036] In an embodiment of the first aspect, the cell permeable protected succinate of any embodiment of the first aspect increases mitochondrial respiration in a rotenone-inhibited intact cell compared to mitochondrial respiration in the rotenone-inhibited cell prior to addition of the cell permeable protected succinate, preferably wherein mitochondrial respiration is measured by a high resolution extracellular metabolic flux analyzer. In a preferred embodiment, diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate increases mitochondrial respiration in a rotenone-inhibited intact cell compared to mitochondrial respiration in the rotenone-inhibited cell prior to addition of the cell permeable protected succinate, preferably wherein mitochondrial respiration is measured by a high resolution extracellular metabolic flux analyzer. In a particularly preferred embodiment, diethyl succinate (DES) increases mitochondrial respiration in a rotenone-inhibited intact cell compared to mitochondrial respiration in the rotenone- inhibited cell prior to addition of the cell permeable protected succinate, preferably wherein mitochondrial respiration is measured by a high resolution extracellular metabolic flux analyzer.

[0037] In a second aspect, the present invention relates to a composition comprising the cell membrane permeable protected succinate for use as defined herein for use in the treatment of a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, preferably a fibrotic interstitial lung disease. In a preferred embodiment, the composition for use in the treatment of a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, preferably a fibrotic interstitial lung disease comprises diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate. In a particularly preferred embodiment, the composition for use in the treatment of a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, preferably a fibrotic interstitial lung disease comprises diethyl succinate (DES).

[0038] In a preferred embodiment of the first or second aspect, the fibrotic disease is selected from the group consisting of pulmonary fibrosis regardless of underlying aetiology, renal fibrosis, hepatic fibrosis, systemic sclerosis, and pulmonary collagenosis. In one such embodiment of the first or second aspect,the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, diffuse pulmonary fibrosis, and chronic pulmonary fibrosis, preferably the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis.

[0039] In a preferred embodiment, diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate or a composition comprising diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, b is- (1 -acetoxy- ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate is for use in in the treatment of a disease selected from the group consisting of pulmonary fibrosis regardless of underlying aetiology, renal fibrosis, hepatic fibrosis, systemic sclerosis, and pulmonary collagenosis. In another preferred embodiment, diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate or a composition comprising diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate is for use in in the treatment of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, diffuse pulmonary fibrosis, and chronic pulmonary fibrosis, preferably the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis.

[0040] In a particularly preferred embodiment, diethyl succinate (DES) or a composition comprising diethyl succinate (DES) is for use in in the treatment of a disease selected from the group consisting of pulmonary fibrosis regardless of underlying aetiology, renal fibrosis, hepatic fibrosis, systemic sclerosis, and pulmonary collagenosis. In another particularly preferred embodiment, diethyl succinate (DES) or a composition comprising diethyl succinate (DES) is for use in in the treatment of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, diffuse pulmonary fibrosis, and chronic pulmonary fibrosis, preferably the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis.

[0041] In an embodiment of the first or second aspect, the interstitial lung disease is selected from the group consisting of alveolar proteinosis, pulmonary alveolar microlithiasis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-Rich syndrome, idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component, preferably wherein the interstitial lung disease is selected from the group consisting of idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component.

[0042] In a preferred embodiment, diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate or a composition comprising diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, b is- (1 -acetoxy- ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate is for use in in the treatment of a disease selected from the group consisting of alveolar proteinosis, pulmonary alveolar microlithiasis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-Rich syndrome, idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component. In another preferred embodiment, diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1- acetoxyethyl acetoxymethyl succinate or a composition comprising diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate is for use in in the treatment of a disease selected from the group consisting of idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component.

[0043] In a particularly preferred embodiment, diethyl succinate (DES) or a composition comprising diethyl succinate (DES) is for use in in the treatment of a disease selected from the group consisting of alveolar proteinosis, pulmonary alveolar microlithiasis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-Rich syndrome, idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component. In another preferred embodiment, diethyl succinate (DES) or a composition comprising diethyl succinate (DES) is for use in in the treatment of a disease selected from the group consisting of idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component.

[0044] In a third aspect the present invention relates to a method of treating a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, preferably a fibrotic interstitial lung disease in a human subject, the method comprising administering to the subject an effective amount of a cell membrane permeable protected succinate or a composition comprising the same and a pharmaceutically acceptable carrier, wherein the cell membrane permeable succinate comprises a succinate core of formula (I):Formula (I)

[0045] In said method, the cell membrane permeable protected succinate has formula (II):Formula (II) wherein R1is H, a pharmaceutically acceptable salt, an alkyl group or a group of formula (III) and R2is independently an alkyl group, or a group according to formula (III) where formula (III) is:Formula (III) wherein R3is H, C C3alkyl, or is linked together with R5by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring;R4is H;R5is OCORa, OCOORb, OCONRcRd, CONRcRdor is linked to R3by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; where Rais CH3, CH2CH3, CH(CH3)2, C(CH3)3 or cycloalkyl;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3 or cycloalkyl;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms. Specific, non-limiting embodiments of Formula II are specified below in the section “Cell membrane permeable protected succinates”. In a particularly preferred embodiment of the third aspect, the cell membrane permeable protected succinate is diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethylsuccinate. In a particularly preferred embodiment of the third aspect, the cell membrane permeable protected succinate is diethyl succinate (DES). Specific, non-limiting embodiments of Formula II are specified below in the section “Cell membrane permeable protected succinates”. In a particularly preferred embodiment of the third aspect, the cell membrane permeable protected succinate is diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1- acetoxyethyl acetoxymethyl succinate. In a particularly preferred embodiment of the third aspect, the cell membrane permeable protected succinate is diethyl succinate (DES).

[0046] In an embodiment of the third aspect, the cell permeable protected succinate of any embodiment of the third aspect increases mitochondrial respiration in a rotenone-inhibited intact cell compared to mitochondrial respiration in the rotenone-inhibited cell prior to addition of the cell permeable protected succinate, preferably wherein mitochondrial respiration is measured by a high resolution extracellular metabolic flux analyzer. In a preferred embodiment, diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate increases mitochondrial respiration in a rotenone-inhibited intact cell compared to mitochondrial respiration in the rotenone-inhibited cell prior to addition of the cell permeable protected succinate, preferably wherein mitochondrial respiration is measured by a high resolution extracellular metabolic flux analyzer. In a particularly preferred embodiment, diethyl succinate (DES) increases mitochondrial respiration in a rotenone-inhibited intact cell compared to mitochondrial respiration in the rotenone- inhibited cell priorto addition of the cell permeable protected succinate, preferably wherein mitochondrial respiration is measured by a high resolution extracellular metabolic flux analyzer.

[0047] In any embodiment of the third aspect, the fibrotic disease is selected from the group consisting of pulmonary fibrosis regardless of underlying aetiology, renal fibrosis, hepatic fibrosis, systemic sclerosis, and pulmonary collagenosis. In one such embodiment of the third aspect, the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, diffuse pulmonary fibrosis, and chronic pulmonary fibrosis, preferably the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis.

[0048] In a preferred embodiment, the method is a method of treating a disease selected from the group consisting of pulmonary fibrosis regardless of underlying aetiology, renal fibrosis, hepatic fibrosis, systemic sclerosis, and pulmonary collagenosis, and the cell membrane permeable protected succinate is diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate. In another preferred embodiment, the method is a method of treating a disease selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, diffuse pulmonary fibrosis, and chronic pulmonary fibrosis, preferably the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, and the cell membrane permeable protected succinate is diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate.

[0049] In a particularly preferred embodiment, the method is a method of treating a disease selected from the group consisting of pulmonary fibrosis regardless of underlying aetiology, renal fibrosis, hepatic fibrosis, systemic sclerosis, and pulmonary collagenosis, and the cell membrane permeable protected succinate is diethyl succinate (DES). In another preferred embodiment, the method is a method of treating a disease selected from the group consisting of idiopathic pulmonary fibrosis, progressivepulmonary fibrosis, diffuse pulmonary fibrosis, and chronic pulmonary fibrosis, preferably the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, and the cell membrane permeable protected succinate is diethyl succinate (DES).

[0050] In an embodiment of the third aspect, the interstitial respiratory disease is selected from the group consisting of alveolar proteinosis, pulmonary alveolar microlithiasis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-Rich syndrome, idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component, preferably wherein the interstitial lung disease is selected from the group consisting of idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component.

[0051] In a preferred embodiment, the method is a method of treating a disease selected from the group consisting of alveolar proteinosis, pulmonary alveolar microlithiasis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-Rich syndrome, idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component, preferably wherein the interstitial lung disease is selected from the group consisting of idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component, and the cell membrane permeable protected succinate is diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate.

[0052] In a particularly preferred embodiment, the method is a method of treating a disease selected from the group consisting of alveolar proteinosis, pulmonary alveolar microlithiasis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-Rich syndrome, idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component, preferably wherein the interstitial lung disease is selected from the group consisting of idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component, and the cell membrane permeable protected succinate is diethyl succinate (DES).Cell membrane permeable protected succinates

[0053] In the present invention, the cell membrane permeable protected succinate comprises a succinate core of formula (I):Formula (I)

[0054] The cell membrane permeable protected succinate has formula (II):Formula (II) wherein R1is H, a pharmaceutically acceptable salt, an alkyl group or a group of formula (III) and R2is independently an alkyl group, or a group according to formula (III) where formula (III) is:Formula (III) wherein R3is H, C C3alkyl, or is linked together with R5by a group of formula COO(CR R )O to form aring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring;R4is H;R5is OCORa, OCOORb, OCONRcRd, CONRcRdor is linked to R3by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; where Rais CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms.

[0055] It is to be understood that in the following preferred embodiments regarding the cell membrane permeable protected succinate comprising a succinate core of formula (I)Formula (I), and having the formula (II)Formula (II) are defined hereinafter, which are relevant for all aspects of the present invention, including where compositions and / or methods are concerned.

[0056] With regard to the succinate core of formula (I) it is to be understood that the curled lines in Formula (I) refer to the attachment of the succinate core to the protection groups. The protection groups of the cell membrane permeable protected succinate comprising the succinate core of formula (I) include all possible and chemical feasible protection groups, which are available for protecting the COOH moieties of the succinate core.

[0057] The protection groups of the succinate core of formula (I) are further defined by the substituents R1and R2in the cell membrane permeable succinate having the formula (II)Formula (II).

[0058] The cell membrane permeable protected succinate has the formula (II)Formula (II) wherein R1is H, a pharmaceutically acceptable salt, an alkyl group or a group of formula (III) and R2is independently an alkyl group, or a group according to formula (III) where formula (III) is:Formula (III) wherein R3is H, C C3alkyl, or is linked together with R5by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring;R4is H;R5is OCORa, OCOORb, OCONRcRd, CONRcRdor is linked to R3by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; where Rais CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms.

[0059] In the following preferred embodiments the substituent R1is defined. It is to be understood that the embodiments are preferred on their own as well as in combination with the remaining embodiments of the present invention. Further, it is to be understood that the below embodiments are particularly preferred with regard to the cell membrane permeable protected succinate according to the present invention, in particular the cell membrane permeable protected succinate according to formula (II).

[0060] In the cell membrane permeable protected succinate according to formula (II)Formula (II)R1is H, a pharmaceutically acceptable salt, an alkyl group or a group of formula (III).

[0061] In a preferred embodiment of the present invention of the cell membrane permeable protected succinate, in particular the cell membrane permeable protected succinate according to formula (II)Formula (II),R1is H or a pharmaceutically acceptable salt.

[0062] In this regard it is to be understood that in case R1is H, the cell membrane permeable protected succinate according to the present invention does not have a protection group on one side of the succinate moiety, where R1is defined. In other words, the cell membrane permeable succinate moiety according to the present invention has a COOH group on one side of the succinate moiety.

[0063] Further, it is to be understood that in case R1is a pharmaceutically acceptable salt, the cell membrane permeable succinate according to the present invention is present in the salt form on the side of the succinate moiety where R1is defined. In other words, the COOH group on the side of the succinate moiety where R1is defined may be present in deprotonated form to form a pharmaceutically acceptable salt as further defined above or below.

[0064] In another preferred embodiment of the present invention of the cell membrane permeable protected succinate, in particular the cell membrane permeable protected succinate according to formula (II)Formula (II),R1is an alkyl group or a group of formula (III).

[0065] In a more preferred embodiment of the present inventionR1is methyl, ethyl or propyl, preferablyR1is methyl or ethyl.In other words,R1is CH3or CH2CH3.

[0066] In another more preferred embodiment of the present inventionR1is a group of formula (III)Formula (III) wherein R3is H, C C3alkyl, or is linked together with R5by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring;R4is H;R5is OCORa, OCOORb, OCONRcRd, CONRcRdor is linked to R3by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; where Rais CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms.

[0067] In connection with the above embodiments it is to be understood that the substituents R2, R3, R4, R5, Ra, Rb, Rc, Rd are as defined above or further below.

[0068] In certain embodiments of the cell membrane permeable protected succinate R1can be methyl or ethyl.

[0069] In further certain embodiments of the cell membrane permeable protected succinate R1can be methyl.

[0070] In the following preferred embodiments the substituent R2is defined. It is to be understood that the embodiments are preferred on their own as well as in combination with the remaining embodiments of the present invention. Further, it is to be understood that the below embodiments are particularly preferred with regard to the cell membrane permeable protected succinate according to the present invention, in particular the cell membrane permeable protected succinate according to formula (II).

[0071] In the cell membrane permeable protected succinate according to formula (II)Formula (II)R2is independently an alkyl group, or a group according to formula (III).

[0072] In a preferred embodiment of the present invention of the cell membrane permeable protected succinate according to formula (II)Formula (II)R2is an alkyl group, preferablyR2is methyl or ethyl.In other words,R2is CH3or CH2CH3.

[0073] In another preferred embodiment of the present invention of the cell membrane permeable protected succinate according to formula (II)Formula (II)R2is a group according to formula (III) where formula (III) isFormula (III) wherein R3is H, C C3alkyl, or is linked together with R5by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring;R4is H;R5is OCORa, OCOORb, OCONRcRd, CONRcRdor is linked to R3by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; where Rais CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms.

[0074] In connection with the above embodiments it is to be understood that the substituents R1, R3, R4, R5Ra, Rb, Rcand Rdare as defined above or further below.

[0075] In the following preferred embodiments the substituents R3, R4and R5are defined. It is to be understood that the embodiments are preferred on their own as well as in combination with the remaining embodiments of the present invention. Further, it is to be understood that the below embodiments are particularly preferred with regard to the cell membrane permeable protected succinate according to thepresent invention, in particular the cell membrane permeable protected succinate according to formula(II).

[0076] As outlined in detail above with regard to the substituents R1and R2each of the substituents R1and R2in the compound of formula (II) can be independently of each other a group according to formula(III) where formula (III) isFormula (III) wherein R3is H, C C3alkyl, or is linked together with R5by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring;R4is H;R5is OCORa, OCOORb, OCONRcRd, CONRcRdor is linked to R3by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; where Rais CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms.

[0077] In certain embodiments of the cell membrane permeable protected succinate R5can be an alkyl ester providing the alkyl ester does not contain a further succinate (OCOCH2CH2COO) moiety.

[0078] In a preferred embodiment of the compound of formula (II), in particular with regard to the group of formula (III) where formula (III) isR5is OCORa, OCOORb, OCONRcRd, CONRcRd.

[0079] In a more preferred embodiment of the compound of formula (II), in particular with regard to the group of formula (III) where formula (III) isFormula (III)R3is H, CH3or CH2CH3;R4is H; andR5is OCORa, or OCOORb.

[0080] In certain embodiments of the cell membrane permeable protected succinate R3can be methyl or ethyl.

[0081] In other certain embodiments of the cell membrane permeable protected succinate R3can be H.

[0082] In an even more preferred embodiment of the compound of formula (II), in particular with regard to the group of formula (III) where formula (III) isFormula (III)R3is H or CH3;R4is H; andR5is OCORa.

[0083] In connection with the above embodiments, it is to be understood that the substituents Ra, Rband Rcare as defined above or further below.

[0084] In a particularly preferred embodiment of the compound of formula (II), in particular with regard to the group of formula (III) where formula (III) isFormula (III)R3is H or CH3;R4is H; andR5is OCORa; whereinRais CH3, CH2CH3, CH(CH3)2, C(CH3)3, more preferably Rais CH3.

[0085] In certain embodiments of the cell membrane permeable protected succinate R5can be OCORawhere R-i is CH3, CH2CH3, CH(CH3)2.

[0086] In other embodiments of the cell membrane permeable protected succinate, the cell membrane permeable protected succinate has the formula (II)Formula (II) wherein R1is H or an alkyl group or a group of formula (III) and R2is independently an alkyl group or a group according to formula (III) where formulaFormula (III) wherein R3and R5are linked together to form a ring and the ring comprises a moiety of formula (IV) where formula (IV) isFormula (IV) wherein R4is H and R’ and R” are independently H, C C3alkyl or are linked together to form a ring.

[0087] In other embodiments of the cell membrane permeable protected succinate, the compound may be a compound according to formula (Iva), where formula (Iva) isFormula (IVa).

[0088] In other embodiments of the cell membrane permeable protected succinate, R3and R5can be linked together to form a ring. The ring may be an all carbon ring or may contain additional heteroatoms. The R3-R5 ring may contain one or more OCR’R”O linkages where R’ and R” are independently H, C C3alkyl or are linked together to form a ring.

[0089] In further embodiments of the cell membrane permeable protected succinate R5can be an alkyl ester providing the alkyl ester does not contain a further succinate (O2CCH2CH2CO2) moiety. R5can be an alkyl ester with the exception of butyl ester. R5can be OCORawhere Rais CH3, CH2CH3, CH(CH3)2or C(CH3)3.

[0090] In one embodiment of the present invention, exemplary compounds of the present invention may be of the following formulawhereinR3is H or C C3alkyl;R4is H;Rais CH3, CH2CH3, CH(CH3)2or C(CH3)3; andR1is H, or a pharmaceutically acceptable salt group of formula (III) where formula (III) isFormula (III) whereinR3is H, or C C3alkyl;R4is H; and whereinR5is selected from OCORa, OCOORb, OCONRcRd, or CONRcRd, whereinRais CH3, CH2CH3, CH(CH3)2, C(CH3)3;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms.

[0091] In other embodiments the cell membrane permeable protected succinate can be a compound of formula (II)Formula (II)wherein R1is H or an alkyl group or a group of formula (III) and R2is independently an alkyl group or a group according to formula (III)R5is OCOORb, whereinRbis CH3, CH2CH3, CH(CH3)2, C(CH3)3, preferably CH3, CH2CH3

[0092] In further embodiments the cell membrane permeable protected succinate can be a compound of the following formulawhereinR3is H or C C3alkyl;R4is H; andRbis CH3, CH2CH3, CH(CH3)2, or C(CH3)3, preferably CH3, CH2CH3;R1is H or a pharmaceutically acceptable salt group of formula (III) where formula (III) isFormula (III) whereinR3is H, or C C3alkyl;R4is H, and whereinR5is selected from OCORa, OCOORb, OCONRcRd, or CONRcRdwhereinRais CH3, CH2CH3, CH(CH3)2, or C(CH3)3;Rbis CH3, CH2CH3, CH(CH3)2, or C(CH3)3;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms.

[0093] In other embodiments the cell membrane permeable protected succinate can be a compound of formula (II)Formula (II) wherein R1is H or an alkyl group or a group of formula (III) and R2is independently an alkyl group or a group according to formula (III) where formula (III) isFormula (III) whereinR3is linked together with R5by a group of formula COO(CR’R”)O to form a ring, wherein R’ and R” are independently H, C C3alkyl or are linked together to form a ring;R4is H; andR5is linked to R3by a group of formula COO(CR’R”)O to form a ring, wherein R’ and R” are independently H, C C3alkyl or are linked together to form a ring.

[0094] In further embodiments the cell membrane permeable protected succinate can be a compound of formula (II)Formula (II) wherein R1is H or an alkyl group or a group of formula (III) and R2is independently an alkyl group or a group according to formula (III) where formulaFormula (III) wherein alkyl;R5is selected from OCONRcRdor CONRcRdwhereinRcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms. In such cases each Rcand Rdcan be methyl or ethyl.

[0095] In other embodiments the cell membrane permeable protected succinate can be a compound of the following formulawhereinR3is H or C C3alkyl;R4is H; andRcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms; andR1is H or a pharmaceutically acceptable salt group of formula (III) where formula (III) isFormula (III) whereinR3is H or C C3alkyl;R4is H; andR5is selected from OCORa, OCOORb, OCONRcRdor CONRcRdwhereinRais CH3, CH2CH3, CH(CH3)2, or C(CH3)3;Rbis CH3, CH2CH3, CH(CH3)2, or C(CH3)3;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms. In such cases each Rcand Rdcan be methyl or ethyl.

[0096] In other embodiments the cell membrane permeable protected succinate can be a compound of the following formulaRcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms; andR1is H or a pharmaceutically acceptable salt group of formula (III) where formula (III) isFormula (III) whereinR3is H or C C3alkyl;R4is H; andR5is selected from OCORa, OCOORb, OCONRcRdor CONRcRdwhereinRais CH3, CH2CH3, CH(CH3)2, or C(CH3)3;Rbis CH3, CH2CH3, CH(CH3)2, or C(CH3)3;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms. In such cases each Rcand Rdcan be methyl or ethyl.

[0097] In other embodiments of the cell membrane permeable protected succinate one or both ends of the succinate compound can be protected with a moiety selected fromwhere R3is H, methyl or ethyl andRais CH3, CH2CH3, CH(CH3)2, or C(CH3)3.

[0098] With regard to the above embodiment it is to be understood that R1can be H or a pharmaceutically acceptable salt or alkyl or a group of formula (III) where formula (III) isR5is selected from OCORa, OCOORb, OCONRcRdor CONRcRdwhereinRais CH3, CH2CH3, CH(CH3)2, or C(CH3)3;Rbis CH3, CH2CH3, CH(CH3)2, or C(CH3)3;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms. In such cases each Rcand Rdcan be methyl or ethyl.

[0099] In further preferred embodiments of the cell membrane permeable succinate compound one of the ends of the succinate compound is protected with a moiety selected fromwhere R3is H, methyl or ethyl andRcand Rdare independently H, CH3or CH2CH3.

[0100] In a particularly preferred embodiment of the present invention, the cell membrane permeable protected succinate is diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 - acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate.

[0101] With regard to the above embodiment, it is to be understood that the cell membrane permeable protected succinate is further defined by the following chemical structures:diethyl succinate dimethyl succinate bis(acetoxymethyl) succinatebis-(1 -acetoxy-ethyl) succinate 1 -acetoxyethyl acetoxymethyl succinate

[0102] In an even more particularly preferred embodiment of the present invention, the cell membrane permeable protected succinate is diethyl succinate (DES) having the following structure:diethyl succinate

[0103] A number of cell membrane permeable protected succinates were identified in a screen in WO2014053857A1 for the purpose of enhancing mitochondrial function in mitochondrial diseases (but not for the treatment of fibrotic disease or interstitial lung disease). These cell membrane permeable protected succinates increase mitochondrial respiration in rotenone-inhibited intact cells compared to mitochondrial respiration in the rotenone-inhibited cell prior to addition of the cell membrane permeable protected succinate. They are thus useful in the inventive methods of treatment described herein.

[0104] The term “protected succinate” indicates that, upon removal of the protection groups attached to the succinate core according to Formula (I), succinate is released.

[0105] The term “compound(s) of / according to the present invention” or “compound(s) according to formula” comprises the compound(s) as defined herein as well as pharmaceutical salts thereof.

[0106] If the compounds of the present invention comprise chiral centres, all optical isomers of the compounds are included by the present invention, whether in the form of racemates or resolved enantiomers.

[0107] Also included herein are any solvates of the compounds of the present invention and their salts. The term “solvates” as used herein refers to solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the present invention of molecules of a non-toxic pharmaceutically acceptable solvent. Examples of solvents include but are not limited to water, alcohols and dimethyl sulfoxide. The solvates can be stoichiometric or non-stoichiometric. Examples of solvates are hydrates, e.g. hemihydrates, monohydrates and dihydrates.

[0108] The term “pharmaceutically acceptable salt(s)” as used herein includes acid addition salt and based addition salts of the cell membrane permeable protected succinate. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of the protected succinate with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of the solvent or medium using standard techniques. Salts of the protected succinate of the present invention can be also prepared by exchanging a counter-ion of the protected succinate in the form of a salt with another counter-ion, for example using a suitable exchange resin. Examples of pharmaceutically acceptable salts of the protected succinate of the present invention include but are not limited to acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or potassium and calcium or organic bases such as ammonium, ethanolamine, N,N-dialkylethanolamines or morpholine salts. Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulfonic acids (e.g. benzenesulfonic, naphthalene- 2-sulfonic, naphthalene-1 ,5-disulfonic and p- toluenesulfonic), ascorbic (e.g. L- ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1 S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1 ,2-disulfonic, ethanesulfonic, 2- hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a- oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L-malic), malonic,(±)-DL-mandelic, metaphosphoric, methanesulfonic, 1 -hydroxys- naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

[0109] The organic moieties mentioned in the above definitions of the variables are collective terms for individual listings of the individual group members. The prefix Cn-Cmindicates in each case the possible number of carbon atoms in the groups.

[0110] The term “succinate” as used herein refers to the salts or esters of succinic acid.

[0111] The term “alkyl or alkyl group” as used in the present invention denotes in each case a straight chain or branched alkyl group having usually from 1 to 4 carbon atoms, preferably from 1 to 3 carbon atoms. Preferred alkyl groups include but are not limited to methyl, ethyl, n-propyl or iso-propyl.

[0112] The term “cycloalkyl” as used herein denotes in each case a monocyclic cycloaliphatic radical having usually from 3 to 10 or 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl.

[0113] The cell membrane permeable protected succinates for use according to the present invention may be either commercially available or may be prepared in different ways by known chemical synthesis. For the preparation by chemical synthesis, it is referred for example to WO2014053857A1 , which describes such synthesis in detail.Diseases

[0114] The cell membrane permeable protected succinates of any of the embodiments described herein, and in particular those described in the section “Cell membrane permeable protected succinates” above, can be used in methods of treatment of a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, preferably a fibrotic interstitial lung disease. The following definitions apply to all other embodiments described herein.

[0115] The term “fibrotic disease” as used herein refers to diseases characterized by fibrosis, i.e. by the thickening and / or scarring of normal parenchymal tissue to form permanent scar tissue comprised of fibroblasts and extracellular matrix. Fibrotic diseases can affect many tissues of the body, for example, but not limited to, lung tissue, liver tissue, kidney tissue, and heart tissue. Examples of fibrotic disease include, but are not limited to, pulmonary fibrosis regardless of underlying aetiology, idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, diffuse pulmonary fibrosis, chronic pulmonary fibrosis, renal fibrosis, hepatic fibrosis, and systemic sclerosis.

[0116] The term “pulmonary fibrosis regardless of underlying aetiology” as used herein refers to all disorders that lead to an aberrant deposition of extracellular matrix or aberrant accumulation of fibrotic tissue in the lung. This can be diagnosed by HR-CT, biopsy or other suitable diagnostic modalities (e.g. MRT). It is sometime referred to as fibrotic or fibrosing interstitial lung disease (F-ILD).

[0117] The term “idiopathic pulmonary fibrosis” as used herein refers to idiopathic pulmonary fibrosis as defined in ICD-10: J84.1-.

[0118] The term “progressive pulmonary fibrosis” as used herein refers to silicotic fibrosis (massive) of lung as defined in ICD-10: J.62.-, bauxite fibrosis (of lung) as defined in ICD-10: J63.1 , and graphite fibrosis (of lung) as defined in ICD-10: J63.3. The term “progressive pulmonary fibrosis” as used herein also refers to an interstitial lung disease other than idiopathic pulmonary fibrosis that shows radiologicalsigns of fibrosis and evidence of progression over time, as described in Rajan SK et al., Eur Respir J. 2023 Mar 30;61 (3):2103187, to which we explicitly refer.

[0119] The term “diffuse pulmonary fibrosis” as used herein refers to diffuse pulmonary fibrosis as defined in ICD-10: J84.1-.

[0120] The term “chronic pulmonary fibrosis” as used herein refers to chronic pulmonary fibrosis due to inhalation of chemicals, gases, fumes, or vapors as defined in ICD-10: J68.4 and to chronic pulmonary fibrosis following radiation as defined in J70.1.

[0121] The term “renal fibrosis” as used herein refers to renal fibrosis as specified in ICD-10: N26.9, wherein in this case “ICD-10” refers to the American 2025 ICD-10-CM / PCS with effective date of 1 October 2024 as available at https: / / www.icd10data.com / . The term “renal fibrosis” also refers to excessive proliferation of fibroblasts and deposition of extracellular matrix in the kidney that accompanies or causes a decline in renal function such as glomerular filtration rate.

[0122] The term “hepatic fibrosis” as used herein refers to fibrosis and cirrhosis of the liver as defined in ICD-10: K74.- and includes hepatic fibrosis as specified in ICD-10: K74.0, hepatic sclerosis as specified in ICD-10: K74.1 , hepatic fibrosis with hepatic sclerosis as defined in ICD-10: K74.2, primary biliary cirrhosis as defined in ICD-10: K74.3, secondary biliary cirrhosis as defined in ICD-10: K74.4, biliary cirrhosis, unspecified as defined in ICD-10: K74.5, and other and unspecified cirrhosis of the liver as specified in ICD-10: K74-6.

[0123] The term “systemic sclerosis” as used herein refers to systemic sclerosis as defined in ICD-10: M34.-, and includes progressive systemic sclerosis as defined in ICD-10: M34.0, CR(E)ST syndrome as defined in ICD-10: M34.1 , systemic sclerosis induced by drug and chemical as defined in ICD-10: M34.2, other forms of systemic sclerosis as defined in ICD-10: M34.8, and systemic sclerosis, unspecified as defined in ICD-10: M34-9. The term “scleroderma” is used synonymously with “sclerosis” herein.

[0124] The term “pulmonary collagenosis” as used herein refers to a systemic connective tissue disorder / collagenosis with pulmonary manifestation as specified in ICD-10: M30-36 and ICD-10: J99.1 .

[0125] The term “interstitial lung disease” as used herein refers to interstitial lung disease as defined in ICD-10: J84.-, i.e. to alveolar and parieto-alveolar conditions as defined in ICD-10: J84.0-, other interstitial pulmonary diseases with fibrosis as defined in ICD-10: J84-1-, lymphoid interstitial pneumonia as defined in ICD-10: J84.2-, other specified interstitial pulmonary diseases as specified in ICD-10: J84-8-, and interstitial pulmonary disease, unspecified as defined in ICD-10: J84-9-. Preferably, the interstitial lung diseases is a fibrotic interstitial lung disease.

[0126] The term “fibrotic interstitial lung disease” as used herein refers to interstitial lung diseases with fibrosis as defined in ICD-10: J84.1-.

[0127] The term “alveolar proteinosis” as used herein refers to alveolar proteinosis as defined in ICD-10: J84.0-.

[0128] The term “pulmonary alveolar microlithiasis” as used herein refers to pulmonary alveolar microlithiasis defined in ICD-10: J84.0-.

[0129] The term “fibrosing alveolitis (cryptogenic)” as used herein refers to cryptogenic fibrosing alveolitis as defined in ICD-10: J84.1-.

[0130] The term Hamman-Rich syndrome as used herein refers to Hamman-Rich syndrome as defined in ICD-10: J84.1-.

[0131] The term “idiopathic pulmonary fibrosis” as used herein refers to idiopathic pulmonary fibrosis as defined in ICD-10: J84.1-.

[0132] The term “interstitial pneumonia with a fibrotic component” as used herein

[0133] When reference is made herein to ICD-10, this refers to “ICD-10-GM Version 2025, Systematisches Verzeichnis, Internationale statistische Klassifikation der Krankheiten und verwandter Gesundheitsprobleme, 10. Revision, German Modification, Stand: 13. September 2024“ published by Bundesinstitut fur Arzneimittel und Medizinprodukte (BfArM) im Auftrag des Bundesministeriums fur Gesundheit (BMG) unter Beteiligung der Arbeitsgruppe ICD des Kuratoriums fur Fragen der Klassifikation im Gesundheitswesen (KKG) in Cologne, Germany, in 2024, file name „icd10gm2025syst_referenz_20240913.pdf“ available at https: / / www.bfarm.de / SharedDocs / Downloads / DE / Kodiersysteme / klassifikationen / icd-10- gm / version2025 / icd10gm2025syst- pdf_zip.html?nn=841246&cms_dlConfirm=true&cms_calledFromDoc=841246 unless otherwise indicated.Other Definitions

[0134] In the following, further definitions important for understanding the present invention are given that apply to all embodiments described herein.

[0135] As used herein, the singular forms of “a” and “an” also include the respective plurals unless the context clearly dictates otherwise.

[0136] Further, in the context of the present invention it is to be understood that the term “comprising” is not limiting. For the purposes of the present invention the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. As used herein, if a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group, which preferably consists of these embodiments only.

[0137] The term “mitochondrial respiration” as used herein refers to the metabolic processes occurring in the mitochondria and requiring oxygen to convert the energy stored in nutrients into adenosine triphosphate (ATP). In other words, mitochondrial respiration is ATP production in the mitochondria via oxidative phosphorylation (OXPHOS) complexes, which are present in the inner membrane of mitochondria. These complexes are known as NADH: ubiquinone oxidoreductase (“complex I”), succinate dehydrogenase (“complex II”), ubiquinol-cytochrome c oxidoreductase (“complex III”, or “cytochrome bc1 complex”), cytochrome c oxidase (“complex IV”), and ATP synthase (“complex V”). Electrons are shuttled down a series consisting of complex I, complex II, complex III, and complex IV known as the “respiratory chain”. The oxidation of succinate occurs at Complex II (succinate dehydrogenase complex) and FAD is part of the complex. The respiratory complexes are embedded in the inner membrane of the mitochondrion. Complex IV, at the end of the chain, transfers the electrons to oxygen, which is reduced to water. The energy released as these electrons traverse the complexes is used to generate a proton gradient across the inner membrane of the mitochondrion, which creates an electrochemical potential across the inner membrane. Complex V (which is not directly associated with Complexes I, II, III and IV) uses the energy stored by the electrochemical gradient to convert ADP into ATP.

[0138] The term “rotenone” as used herein refers to an odorless, colorless, crystalline isoflavone of the formula:It occurs naturally in the seeds and stems of several plants, such as the jicama vine, and in the roots of several other members of the Fabaceae Rotenone (a natural extract from plants) is a broad-spectrum insecticide and pesticide. Rotenone inhibits mitochondrial complex I and activates the production of reactive oxygen species (ROS).

[0139] Rotenone can be used to screen for additional cell membrane permeable protected succinate compounds to be used in the inventive methods of treatment. In the screening assay, mitochondrial function in intact cells, e.g. in intact platelets or fibroblasts, is repressed with the respiratory complex I inhibitor rotenone. Such a screening method is disclosed in Ehinger, J. K.et al. Cell-permeable succinate prodrugs bypass mitochondrial complex I deficiency. Nat.Commun. 7:12317 and WO2014053857A, to which it is referred. Briefly, the screen can be performed with intact cells, e.g. with isolated intact platelets in solution or isolated fibroblasts seeded on collagen-coated plates, in a buffer containing 110 mM sucrose, HEPES 20 mM, taurine 20 mM, K-lactobionate 60 mM, MgCI2 3 mM, KH2P04 10 mM, EGTA 0.5 mM, BSA 1 g / l, pH 7.1 . After baseline respiration with endogenous substrates is established, complex I is inhibited with Rotenone at 2 mM. Candidate protected succinates dissolved in DMSO are titrated to the rotenone-inhibited cells in steps to 100 pM, 500 pM and 5 mM final concentration. Subsequently, cell membranes are permeabilized with digitonin (1 mg / 1*106 pit). After stabilized respiration, succinate 10mM was added and after the respiration stabilized the experiment was terminated by addition of the complex III inhibitor Antimycin af final concentration 1 pg / mL and the residual respiration measured. Figure 5 shows the protocol for evaluating novel cell-permeable mitochondrial substrates. Drug candidates are compared with endogenous substrates (succinate) before and after permeabilization of the plasma membrane to evaluate bioenergetic enhancement or inhibition. The ideal compound stimulates respiration in rotenone-inhibited intact cells at low concentration without inhibitory effect on succinate stimulated respiration after permeabilization. That is, if addition of a candidate protected succinate leads to an increase of mitochondrial respiration in an intact cell in which complex I is inhibited by rotenone, then the candidate protected succinate is cell membrane permeable and releases succinate inside the cell, which then can act on complex II. Such a cell membrane permeable protected succinate is suitable for use in the inventive methods of treatment. If no further increase of respiration occurs after permeabilization of the cell membrane with digitonin, then the cell membrane permeable protected succinate is highly cell membrane permeable, and is especially suitable for use in the inventive methods of treatment. All respiration measurements are performed with a high resolution extracellular metabolic flux analyzer.

[0140] The term “intact cell” as used herein refers to a cell with an intact cell membrane, i.e. an unbroken cell membrane that has not been permeabilized. Preferred intact cells are intact platelets and intact fibroblasts.

[0141] The term “high resolution extracellular metabolic flux analyzer” as used herein refers to an instrument which measures and reports the oxygen consumption rate (OCR), proton efflux rate (PER) or extracellular acidification rate (ECAR), as well as ATP production rates of live cells. For cells in monolayers, e.g. fibroblasts, the Seahorse Bioscience XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, NorthBillerica, USA) is particularly suitable. For cells in suspension, such as platelets, the Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) is particularly suitable.

[0142] The term “treating” or “treatment” as used herein refers to any type of a beneficial effect, e.g. amelioration of at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement over baseline, e.g. with regard to severity of a symptom or symptoms (e.g. lung capacity, breathlessness, cough, kidney function (e.g. GFR), liver function (e.g. MELD-score)), overall health (e.g. weight, quality of life). An effective treatment can for example slow or even entirely halt the progression of and possibly reverse fibrosis in a diseased tissue. In current clinical trials of pulmonary fibrosis medications, typical readouts include decrease of forced vital capacity from baseline, mortality, exacerbation rates or sometimes 6 minutes walking distance.

[0143] The term “administering” as used herein refers to delivery of a cell membrane permeable protected succinate of any of the embodiments described herein or a composition comprising the same to a subject such that the cell membrane permeable protected succinate or the composition is taken into the body of the subject. Administration may be parenteral, oral, or occur by injection, via suppository, or by inhalation.

[0144] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages I an “effective amount” as used herein and in vivo half-lives for the cell membrane permeable protected succinates described herein can be made using conventional methodologies or on the basis of in vivo testing using a suitable animal model.

[0145] The term “pharmaceutically acceptable carrier” as used herein can be any substance used for the preparation of pharmaceutical dosage forms, including coating materials, film-forming materials, fillers, disintegrating agents, release-modifying materials, carrier materials, diluents, binding agents and other adjuvants. Typical pharmaceutically acceptable excipients include substances like sucrose, mannitol, sorbitol, starch and starch derivatives, lactose, and lubricating agents such as magnesium stearate, disintegrants and buffering agents. The term “carrier” denotes pharmaceutically acceptable organic or inorganic carrier substances with which the active ingredient is combined to facilitate the application.Suitable pharmaceutically acceptable carriers include, for instance, water, salt solutions, alcohols, oils, preferably vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, surfactants, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone and the like. The compositions can be sterilized and if desired, mixed with auxiliary agents, like lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and / or aromatic substances and the like which do not deleteriously react with the active compound.

[0146] If liquid dosage forms are considered for the present invention, these can include pharmaceutically acceptable emulsions, solutions, suspensions and syrups containing inert diluentscommonly used in the art such as water. These dosage forms may contain e.g. micro-crystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer and sweeteners / flavouring agents.

[0147] For parenteral application, particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Pharmaceutical formulations for parenteral administration are particularly preferred and include aqueous solutions in water-soluble form. Additionally, suspensions may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0148] Particularly preferred dosage forms are injectable preparations of a composition for use of the present invention. Thus, sterile injectable aqueous or oleaginous suspensions can for example be formulated according to the known art using suitable dispersing agents, wetting agents and / or suspending agents. A sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be used are water and iso-tonic sodium chloride solution. Sterile oils are also conventionally used as solvent or sus-pending medium.

[0149] Suppositories for rectal administration of a composition of the present invention can be prepared by e.g. mixing the compound with a suitable non-irritating excipient such as cocoa butter, synthetic triglycerides and polyethylene glycols which are solid at room temperature but liquid at rectal temperature such that they will melt in the rectum and release the active agent from said suppositories.

[0150] For administration by inhalation, the composition comprising a compound according to the present invention may be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., di-chlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the com-pound and a suitable powder base such as lactose or starch.

[0151] Oral dosage forms may be liquid or solid and include e.g. tablets, troches, pills, capsules, powders, effervescent formulations, dragees and granules. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and pro-cessing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tab-lets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. The oral dosage forms may be formulated to ensure an immediate release of the active agent or a sustained release of the active agent.Examples

[0152] The following Examples serve to further describe and exemplify embodiments of the present invention, but do not limit the scope of the invention.Example 1 - Diethylsuccinate (DES) reduces the expression of fibrosis-associated markers and induces apoptosis in fibroblasts derived from IPF patients in vitro

[0153] Idiopathic pulmonary fibrosis (IPF) patient derived fibroblasts cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Prior to treatment cells were serum- starved. Cells were then treated with 0 (VEH), 1 , 3 or 10mM DES. After treatment for 72 hours, cells were lysed in RIPA buffer supplemented with Halt protease inhibitor cocktail. 5pg of protein was resolved on a SDS-PAGE followed by transfer of the resolved proteins to a on a polyvinylidene fluoride membrane using the iBIot dry transfer system (Thermo Fisher Scientific); thereafter the membrane was blocked (5% non-fat dry milk) for 1 hour and incubated with primary antibodies overnight. Secondary HRP-conjugated antibodies followed by HRP- detection substrate (Bio-Rad) was used to visualize chemiluminescence by ChemiDoc Touch Imaging system (Bio-Rad). After stripping with respective buffer (Restore PLUS, Thermo Fisher Scientific), membranes were re-probed with antibodies for respective loading controls The western blot analysis was performed to assess the changes associated with aSMA (alpha-smooth muscle actin). Additionally, the cell culture medium supernatants were collected and used for ELISA for Coll A1 and fibronectin using the respective ELISA kits (R&D biosystems) as per the manufacturer’s protocols. One-way ANOVA for repeated measures was used to assess statistical significance. The results are shown in Figure 1 and demonstrate that aSMA, Coll A1 , and Fibronectin, ECM-proteins, are all reduced upon treatment of the fibroblasts with DES. Additional investigations showed a decrease of CTHRC1 , a marker of a subpopulation of highly pro-fibrotic fibroblasts. These results show that DES leads to a decrease of factors contributing to tissue fibrosis and might change fibroblast fate towards a less fibrotic phenotype. Therefore, as accumulation of these ECM components is a hallmark of fibrosis, decreasing their synthesis represents a beneficial therapeutic strategy.

[0154] IPF patient derived fibroblasts were cultured and treated with 0 (VEH), 1 , 3 or 10mM DES for 72 hours, and apoptosis was evaluated by colorimetric measurement of caspase 3 / 7 activity using the Caspase-Gio® 3 / 7 Assay System (Promega corporation) as per manufacturer’s instructions and compared to the positive control Fas ligand (FAS-L). Apoptosis was also measured using the TUNEL staining. After respective treatments of IPF patient fibroblasts with with 0 (VEH), 1 , 3 or 10mM DES for 72 hours, cells were fixed in 4% parafornaldehyde for 20 min at RT and subsequently washed with PBS. Cells were permeabilized for 2 minutes on ice using PBS containing 0,1% Triton X, and 0,1% sodium citrate. Afterward cells were incubated with TUNEL reaction mix for 1 h at 37°C in a humidified chamber in the dark, washed with PBS and mounted using DAPI containing mounting medium. One-way ANOVA for repeated measures was used to assess statistical significance. The results are shown in Figure 2 and demonstrate that treatment with DES induced apoptosis. As impaired fibroblast apoptosis is believed to contribute to the accumulation of fibrotic tissue in fibrotic disease, increasing apoptosis would represent an attractive mode of action and could potentially contribute to a decrease in fibrotic tissue.Example 2 - DES improves fibrosis in the murine bleomycin model of pulmonary fibrosis

[0155] C57 / BI6 J mice were intra-tracheally instilled with bleomycin or saline on day 0, followed by intraperitoneal administration of DES from day 11 until day 21 post bleomycin administration. In brief, mice were anesthetized with isoflurane, and bleomycin or vehicle (saline solution) was instilled intratracheally with a Hamilton syringe at a dose of 1 .75 U / kg. On day 21 post bleomycin administration, lung function parameters were assessed in mice using the Flexivent system (Scireq, Montreal, CA) to evaluate the potential anti-fibrotic role of DES in vivo. Lung function parameters including pressure-volume curves, static compliance, inspiratory capacity, and forced vital capacity (FVC) were assessed. Lungs were weighed, right upper lobes were excised, fixed in Histofix and paraffin-embedded for histologic assessment, while for all other measurements, the remaining lung was snap frozen and subsequently pulverized in liquid nitrogen. Hydroxyproline assay was performed on the lung homogenates from all treatmemt groups. 10mg of lung tissue were dissolved in 100pL of water. The samples were incubated with equal volumes of 12N HCI for 3 hours. 10pL of each sample was transferred to a 96-well plate. A hydroxyproline standard curve was generated by adding 0, 0.2, 0.4, 0.6, 0.8 and 1 pg in a series of wells. The samples were then treated with a 100pL of Chloramine T for 60 minutes at room temperature. This was followed by treatment of the samples with 10OpL of 4-(Dimethylamino) benzaldehyde (DMAB) was added to each well and incubated at 60°C for 90 minutes. The absorbance was recorded at 560nm on a microplate reader. Hydroxyproline content per lung was then calculated using the following formula: (B / V*D) *total lung weight where B is the hydroxproline concentration as derived from the standard curve (pg), V is the sample volume in each well (pL) and D is the sample dilution factor Masson-trichrome staining was performed on mouse lung tissue sections obtained. The stained sections were then scored using the Modified Ashcroft score method method to histologically assess collagen deposition in the lung tissue. One way ANOVA was used to assess statistical significance. The results are shown in Figure 3 and demonstrate that treatment with DES improved fibrosis in mice. This is especially important as the bleomycin model with delayed initiation of treatment (i.e. after day 10) is considered the gold standard for preclinical testing of potential antifibribotic compounds (Jenkins et al., Am J Respir Cell Mol Biol. 2017 May;56(5):667-679). Importantly, no decrease in weight with DES treatment or other gross morphologic or behavioral changes as compared to vehicle were observed, therefore showing no indication of toxicity in this model.Example 3 - DES does not affect activity of succinate dehydrogenase (SDH)

[0156] Lung tissue of mice receiving vehicle or diethylsuccinate for 10 days (once daily, 85mg / kg) was homogenized in ddH2O. Using a commercial kit for measuring SDH activity (Succinate Dehydrogenase Assay Kit | MAK197-1 KT), lysates were incubated for 25 minutes according to the manufacturer’s instructions and absorbance was measured. Vehicle: vehicle treated mice, DES: 85 mg / kg DES, vehicle + added DES: diethylsuccinate at 10 mM was added exogenously immediately prior to the start of the assay. (A) SDH activity as calculated per the manufacturer’s instructions, (B) absorbance traces.The results are shown in Figure 4 and demonstrate that DES has no impact on SDH activity, i.e. that the mechanism of action of DES does not involve succinate SDH.Example 4 - DES alters the transcriptome of IPF patient derived fibroblasts in vitro

[0157] Idiopathic pulmonary fibrosis (IPF) patient derived fibroblasts cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Prior to treatment cells were serum-starved. Cells were then treated with 0 (VEH) or 10mM DES. After treatment for 72 hours, RNA was isolated in TRIZOL and RNASeq was performed by Azenta. Alignment to the T2T genome was performed using STAR on the medbionode cluster and differential gene expression via edgeR package.The results are shown in Figure 6 and demonstrate that DES alters the transcriptome of fibroblasts in IPF patients. This is especially important as multiple downregulated genes (COL1 A1 , CTHRC1 , DEPP1 , COL1A2, CEMIP) are associated with a profibrotic fibroblast phenotype and multiple upregulated genes (PLIN2, PPARy, AKR1 C1) relate to a pro-resolving or lipofibroblast phenotypeExample 5 - DES induces myogenic to lipoqenic transdifferentiation in IPF fibroblasts in vitro

[0158] Idiopathic pulmonary fibrosis (IPF) patient derived fibroblasts cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Prior to treatment cells were serum- starved. Cells were then treated with 0 (VEH) or 10mM DES. After treatment for 72 hours cells were lysed in RIPA buffer supplemented with Halt protease inhibitor cocktail. 5pg of protein was resolved on a SDS- PAGE followed by transfer of the resolved proteins to a on a polyvinylidene fluoride membrane using the iBIot dry transfer system (Thermo Fisher Scientific); thereafter the membrane was blocked (5% non-fat dry milk) for 1 hour and incubated with primary antibodies overnight. Secondary HRP-conjugated antibodies followed by HRP- detection substrate (Bio-Rad) was used to visualize chemiluminescence by ChemiDoc Touch Imaging system (Bio-Rad). After stripping with respective buffer (Restore PLUS, Thermo Fisher Scientific), membranes were re-probed with antibodies for vinculin. The western blot analysis was performed to assess the changes associated with PLIN2 (perilipin-2). Additionally, the cell culture medium supernatants were collected and used for ELISA for Coll A1 using the ELISA kit (R&D biosystems) as per the manufacturer’s protocol. Chamber slides were used for microscopy and stained with Lipidtoxto label neutral lipids, a hallmark of lipofibroblast accumulation. DES induced upregulation of PLIN2 and lipid accumulation in IPF-patient derived fibroblasts and led to a decrease in collagen secretion.Student’s t-test was used to assess statistical significance. The results are shown in Figure 7and demonstrate that DES induces myogenic to lipogenic transdifferentiation of fibroblasts in IPF patients. This has been previously associated with an antifibrotic phenotype and lipofibroblasts are believed to promote regeneration and contribute to alveolar epithelial cell function and health.Example 6 - Dimethylsuccinate (DMS), another cell permeable succinate also exerts antifibrotic effects. Idiopathic pulmonary fibrosis (IPF) patient derived fibroblasts were cultured in DMEM medium containing 10% fetal bovine serum and 1 % penicillin / streptomycin. Prior to treatment cells were serum-starved. Cells were then treated with 0 (VEH) or 10mM DMS. After treatment for 72 hours, cells were lysed in RIPA buffer supplemented with Halt protease inhibitor cocktail. 5pg of protein was resolved on an SDS-PAGE gel followed by transfer of the resolved proteins to a polyvinylidene fluoride membrane using the iBIot dry transfer system (Thermo Fisher Scientific); thereafter the membrane was blocked (5% non-fat dry milk) for 1 hour and incubated with primary antibodies overnight. Secondary HRP-conjugated antibodies followed by HRP- detection substrate (Bio-Rad) was used to visualize chemiluminescence by ChemiDoc Touch Imaging system (Bio-Rad). After stripping with respective buffer (Restore PLUS, Thermo Fisher Scientific), membranes were re-probed with antibodies for vinculin. The western blot analysis was performed to assess the changes associated with aSMA (alpha-smooth muscle actin). Additionally, thecell culture medium supernatants were collected and used for ELISA for Coll A1 using the ELISA kit (R&D biosystems) as per the manufacturer’s protocol.Student’s t-test was used to assess statistical significance. The results are shown in Figure 8 and demonstrate that aSMA and Col1A1 are reduced upon treatment of the fibroblasts with DMS.

Claims

CLAIMS1 . A cell membrane permeable protected succinate for use in the treatment of a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, preferably a fibrotic interstitial lung disease, wherein the cell membrane permeable succinate comprises a succinate core of formula (I):Formula (I) and has formula (ii):Formula (II) wherein R1is H, a pharmaceutically acceptable salt, an alkyl group or a group of formula (III) and R2is independently an alkyl group, or a group according to formula (III) where formula (III) is:Formula (III) wherein R3is H, C C3alkyl, or is linked together with R5by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring;R4is H;R5is OCORa, OCOORb, OCONRcRd, CONRcRdor is linked to R3by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; where Rais CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms.

2. The cell membrane permeable protected succinate for use according to claim 1 , wherein the cell membrane permeable protected succinate is diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate.

3. The cell membrane permeable protected succinate for use according to claim 1 or claim 2, wherein the cell permeable protected succinate increases mitochondrial respiration in a rotenone-inhibited intact cell compared to mitochondrial respiration in the rotenone-inhibited cell prior to addition of the cell permeable protected succinate, preferably wherein mitochondrial respiration is measured by a high resolution extracellular metabolic flux analyzer.

4. A composition comprising the cell membrane permeable protected succinate for use according to any one of claims 1 -3 for use in the treatment of a disease selected from the group consisting of a fibroticdisease and an interstitial lung disease, preferably a fibrotic interstitial lung disease.

5. The cell membrane permeable protected succinate for use according to any one of claims 1 -3 or the composition for use according to claim 5, wherein the fibrotic disease is selected from the group consisting of pulmonary fibrosis regardless of underlying aetiology, renal fibrosis, hepatic fibrosis, systemic sclerosis, and pulmonary collagenosis6. The cell membrane permeable protected succinate for use or the composition for use according to claim 5, wherein the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, diffuse pulmonary fibrosis, and chronic pulmonary fibrosis, preferably wherein the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis.

7. The cell membrane permeable protected succinate for use according to any one of claims 1 -3, or the composition for use according to claim 5, wherein the interstitial lung disease is selected from the group consisting of alveolar proteinosis, pulmonary alveolar microlithiasis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-Rich syndrome, idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component, preferably wherein the interstitial lung disease is selected from the group consisting of idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component.

8. A method of treating a disease selected from the group consisting of a fibrotic disease and an interstitial lung disease, preferably a fibrotic interstitial lung disease in a human subject, the method comprising administering to the subject an effective amount of a cell membrane permeable protected succinate or a composition comprising the same and a pharmaceutically acceptable carrier, wherein the cell membrane permeable succinate comprises a succinate core of formula (I):Formula (I) and has formula (ii):Formula (II) wherein R1is H, a pharmaceutically acceptable salt, an alkyl group or a group of formula (III) and R2is independently an alkyl group, or a group according to formula (III) where formula (III) is:Formula (III) wherein R3is H, C C3alkyl, or is linked together with R5by a group of formula COO(CR’R")O to form a ring,where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring;R4is H;R5is OCORa, OCOORb, OCONRcRd, CONRcRdor is linked to R3by a group of formula COO(CR’R")O to form a ring, where R’ and R" are independently H, C C3alkyl, or are linked together to form a ring; where Rais CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rbis CH3, CH2CH3, CH(CH3)2, C(CH3)3or cycloalkyl;Rcand Rdare independently H, methyl or ethyl or are linked together to form a ring which may contain one or more further heteroatoms.

9. The method according to claim 8, wherein the cell membrane permeable protected succinate is diethyl succinate (DES), dimethyl succinate, bis(acetoxymethyl) succinate, bis-(1 -acetoxy-ethyl) succinate or 1 -acetoxyethyl acetoxymethyl succinate.

10. The method according to claim 8 or claim 9, wherein the cell permeable protected succinate increases mitochondrial respiration in a rotenone-inhibited intact cell compared to mitochondrial respiration in the rotenone-inhibited cell prior to addition of the cell permeable protected succinate, preferably wherein mitochondrial respiration is measured by a high resolution extracellular metabolic flux analyzer.1 1 . The method according to any one of claims 8-10, wherein the fibrotic disease is selected from the group consisting of pulmonary fibrosis, renal fibrosis, hepatic fibrosis, systemic sclerosis, and pulmonary collagenosis.

12. The method according to claim 11 , wherein the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, diffuse pulmonary fibrosis, and chronic pulmonary fibrosis, preferably wherein the pulmonary fibrosis is selected from the group consisting of idiopathic pulmonary fibrosis, progressive pulmonary fibrosis.

13. The method according to any one of claims 8-10, wherein the interstitial respiratory disease is selected from the group consisting of alveolar proteinosis, pulmonary alveolar microlithiasis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-Rich syndrome, idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component, preferably wherein the interstitial lung disease is selected from the group consisting of idiopathic pulmonary fibrosis, and interstitial pneumonia with a fibrotic component.

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