Antifibrotics antagonists of the human TLR4 receptor

Abstract

The present invention relates to compounds for use in the treatment of fibrotic diseases, pharmaceutical compositions comprising said compounds, and methods of treating a fibrotic disease comprising the administration of said compounds.

Description

[0001] - 1 - SIB BW1373R

[0002] ANTIFIBROTICS ANTAGONISTS OF THE HUMAN TLR4 RECEPTOR ABSTRACT

[0003] The present invention relates to compounds for use in the treatment of fibrotic diseases, pharmaceutical compositions comprising said compounds, and methods of treating a fibrotic disease comprising the administration of said compounds.

[0004] STATE OF THE ART

[0005] Fibrosis represents a major global health problem, being a disease that can affect virtually any organ in the human body and contributing significantly to global mortality. Epidemiological studies indicate that fibrosis is responsible for up to 45% of all deaths in industrialized countries, making it a major cause of mortality. Fibrotic diseases include, but are not limited to, idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, and renal fibrosis. These conditions involve a progressive accumulation of abnormal connective tissue, which impairs the function of the affected organ.

[0006] Despite substantial advances in our understanding of the pathophysiology of fibrosis, a significant gap remains between the identification of putative antifibrotic targets and the translation of this knowledge into effective treatments in humans. Current therapeutic options are limited and often fail to halt disease progression, highlighting the urgent need to develop new therapeutic strategies. The complexity of fibrosis lies in its multifactorial nature, involving multiple cellular signaling pathways, inflammatory mediators, and cellmatrix interactions.

[0007] One of the most important molecular events associated with inflammation and fibrosis is the activation of the innate immune receptor Toll-like Receptor 4 (TLR4). TLR4 is a critical component of the innate immune system, which recognizes and responds to pathogens and tissue damage by activating an intracellular signaling cascade. This cascade leads to the production of pro-inflammatory cytokines and other effector molecules that may contribute to chronic inflammation and fibrogenesis.

[0008] Recent studies have shown that TLR4 activation is closely related to fibrosis progression in various experimental models. In particular, it has been observed that TLR4 upregulation is associated with increased fibroblast activity and extracellular matrix deposition, which are hallmarks of fibrosis. Furthermore, TLR4 blockade in preclinical models showed a significant reduction in fibrotic markers, suggesting that TLR4 could be a promising therapeutic target for the management of fibrotic diseases.

[0009] Currently, there are several strategies under development to modulate TLR4 activity. These include the use of specific small molecule antagonists and monoclonal antibodies. However, translating these findings into clinical therapies remains a challenge, mainlySIB BW1373R

[0010] due to the complexity of the immune system and the potential side effects resulting from the inhibition of TLR4, which plays a crucial role in the defense against infections.

[0011] In conclusion, while the understanding of the molecular mechanisms underlying fibrosis has advanced significantly, the effective translation of this knowledge into clinical treatments remains incomplete. Continued research into the role of TLR4 in fibrosis and the development of novel therapeutic strategies aimed at modulating its activity are essential to fill this gap and improve treatment options for patients affected by fibrotic diseases.

[0012] SUMMARY OF THE INVENTION

[0013] The Authors of the present invention have found that compounds of formula I:

[0014] <

[0015]

[0016] Formula I

[0017] wherein R1is a saturated Cn or C13 saturated alkyl group, and wherein R2is a C11 or C13 saturated alkyl group,

[0018] synthetic molecules developed by the same inventors and validated in vitro and in vivo as potent TLR4 antagonists, show surprising antifibrotic properties, particularly on human embryonic fibroblasts MRC5 cells.

[0019] These compounds dose-dependently inhibit the production of the main fibrosis markers in human MRC5 cells, which are a universally accepted and validated model to study fibrosis. The fibrotic phenotype and biochemical markers are clearly reduced by the administration of the compounds both before and after the induction of fibrosis.

[0020] The tests have been carried out by monitoring the main fibrotic markers such as ACTA2 (aSMA), COL1A1 (collagen I), FN1 (fibronectin), which in fibrotic conditions are produced in high quantities by the cells leading to an intracellular (aSMA) and extracellular (Collagen I and fibronectin) accumulation with consequent pathological progression of fibrosis and in the same cells, the variation in production of the inflammatory marker IL-6 was also evaluated. In all experiments, the compound TAK242, a known TLR4 antagonist, was used as a positive control. A significant inhibition of the production of all fibrotic markers is observed when cells treated with FP7 and FP12 before or after the fibrotic stimulus, at a concentration of 5 pM, compared to untreated cells (green line). The positive control TAK-242 is also active in inhibiting fibrotic markers in both pre- and- 3 - SIB BW1373R

[0021] post-treatment conditions. Moreover, in Figure 9 the effect of TGF-pi was tested on mouse fibroblasts (murine) instead of human MRC5 as in Fig. 2, by measuring the production of aSMA (encoded by the ACTA2 gene) by RT-qPCR. The activity of molecules FP7 and FP12 is not high in inhibiting the production of aSMA, but the behavior of TAK242 is very different and even increases its production, indicating a probable toxicity. In Figure 10the effect of the administration of the molecules was tested on human colon fibroblast cells CCD-18Co. Also in this case a toxic effect of TAK242 is clearly observed, with a marked decrease in the expression level of aSMA and collagen I in normal fibroblasts (i.e. not TGFb-activated). On the contrary, FP7 and FP12 molecules do not show this effect while retaining anti-fibrotic properties on the TGFb-treated fibroblasts.

[0022] These experiments clearly demonstrate the effectiveness of FP7 and FP12 in inhibiting the production of fibrotic markers, and at the same time show a different behavior of the FP7 and FP12 molecules and of TAK242, a TLR4 antagonist known in literature and in clinical development for a series of inflammatory diseases. The FP7 and FP12 molecules show a surprising effect of much lower toxicity if not non-existent for cells.

[0023] These data clearly show an unexpected and surprising difference in toxicity and activity profile between TAK242 and FP7 and FP12 as TLR4 antagonists, which is probably linked to the different mechanism of action of the molecules, being FP7 and FP12 ligands of MD-2 that interfere with the process of TLR4 / MD-2 dimer formation, while TAK242 is known to bind only TLR4 in a site close to cell membrane.

[0024] Further investigations clarify the rationale and broaden the scope of the invention. FP7 and FP12 were designed to bind the MD-2 / TLR4 extracellular binding region, preventing TLR4 homodimerization and activation of the downstream pathway, with the goal of disrupting a profibrotic positive feedback loop in which fibrosis progression promotes TLR4 activation and inflammation, which in turn sustains and amplifies fibrotic processes. To support target relevance in human disease, bioinformatic analyses of published single-cell RNA-seq datasets for idiopathic pulmonary fibrosis (I PF) and Crohn’s disease (CD) show that the TLR4 / MD-2 dimer is overexpressed in fibrosis-associated fibroblast / myofibroblast populations in diseased tissues. A functional role for TLR4 in the fibrotic phenotype is further supported by experiments on lung fibroblasts isolated from WT versus Tlr47“ mice, where the knock-out cells show strongly reduced induction of fibrosis markers upon TGF-pi stimulation, consistent with TLR4 being mechanistically involved rather than a bystander marker.- 4 - SIB BW1373R

[0025] The antifibrotic effect of FP7 / FP12 was then validated using complementary experimental readouts and clinically relevant treatment paradigms. In MRC5 cells, fibrosis was induced with TGF-pi and the effect of FP7 / FP12 was assessed under pretreatment and post-treatment schedules across multiple levels, including metabolic activity and viability / cytotoxicity assays, RT-qPCR profiling of activation / extracellular matrix (ECM) inflammatory markers, intracellular protein accumulation (aSMA) and ECM deposition, as well as soluble collagen production.

[0026] At effective, non-toxic concentrations (e.g. ~5 pM), FP7 and FP12 inhibit fibroblast proliferation associated with fibrotic activation, downregulate fibrosis markers, reduce soluble collagen release, and decrease deposition of major ECM components. Washout experiments indicate that even a short exposure to FP7 / FP12 can produce a durable inhibition of intracellular fibrosis markers for an extended period after compound removal, suggesting that continuous dosing may not be required in vivo. Analyses of the molecular mechanisms further support the disruption of the profibrotic feedback circuitry, consistent with an early TGF-p / SMAD activation phase followed by later TLR4 / NF-KB involvement during fibrosis establishment, which is suppressed by FP7 / FP12 treatment.

[0027] Importantly, the effect of FP7 and FP12 treatment is not limited to lung fibrosis. In intestinal fibrosis models, FP7 / FP12 mitigate fibrotic activation in human colon fibroblasts CCD-18Co induced by TGF-pi and show inhibitory trends also under a more disease-mimicking inflammatory cytokine stimulation setting. Antifibrotic trends are further supported in primary human intestinal fibroblasts. Moreover, in a preliminary ex vivo system using surgically isolated intestinal specimens from a Crohn’s disease patient, FP7 reduces key fibrotic transcripts (e.g., COL1A1 and TGF-pi), supporting translational potential in a complex organ environment beyond monoculture systems. Overall, these data demonstrate that FP7 and FP12 are effective, low-toxicity antifibrotic agents capable of preventing and partially reversing fibrotic phenotype through specific modulation of the TLR4 / MD-2 axis, with multi-organ applicability including pulmonary and intestinal fibrosis.

[0028] Therefore, the object of the present invention is a compound of formula I:

[0029] < >

[0030] >

[0031] >

[0032]

[0033] Formula I- 5 - SIB BW1373R

[0034] wherein R1is a saturated Cn or C13 saturated alkyl group, and wherein R2is a C11 or C13 saturated alkyl group,

[0035] for use in the treatment of a fibrotic disease in a patient in need thereof,

[0036] a pharmaceutical composition comprising a compound for use according to the present invention and at least one pharmaceutically acceptable carrier or excipient, for use in the treatment of a fibrotic disease in a patient in need thereof.

[0037] DETAILED DESCRIPTION OF THE FIGURES

[0038] Figure. 1. RT-qPCR assay of TGF-pi -induced fibronectin (encoded by the FN1 gene) production in MRC5 cells. TGF-pi was administered 1h after treatment with FP7 and FP12 (5 pM). The TLR4 antagonist TAK 242 was administered, always in pre-treatment, as a positive control.

[0039] Figure 2. RT-qPCR assay of TGF-pi -induced aSMA (encoded by the ACTA2 gene) production in MRC5 cells. TGF-pi was administered 1h after treatment with FP7 and FP12 (5 pM). The TLR4 antagonist TAK 242 was administered, always in pre-treatment, as a positive control.

[0040] Figure 3. RT-qPCR assay of TGF-pi-induced collagen I (COL1A1 gene) production in MRC5 cells. TGF-pi was administered 1 h after treatment with FP7 and FP12 (5 pM). The TLR4 antagonist TAK 242 was administered, always in pretreatment, as a positive control.

[0041] Figure 4. RT-qPCR assay of TGF-pi-induced production of the inflammatory cytokine IL-6 (IL6 gene) in MRC5 cells. TGF-pi was administered 1 h after treatment with FP7 and FP12 (5 pM). The TLR4 antagonist TAK 242 was administered, always in pretreatment, as a positive control.

[0042] Figure 5. RT-qPCR assay of TGF-pi-induced fibronectin (FN1) production in MRC5 cells. TGF-pi was administered 24 h before FP7 and FP12 treatment (5 pM). The TLR4 antagonist TAK 242 was administered, always post-treatment, as a positive control.

[0043] Figure 6. RT-qPCR assay of TGF-pi-induced collagen (COL1A1) production in MRC5 cells. TGF-pi was administered 24 h before FP7 and FP12 treatment (5 pM). The TLR4 antagonist TAK 242 was administered, always post-treatment, as a positive control.- 6 - SIB BW1373R

[0044] Figure 7. RT-qPCR assay of TGF-pi-induced aSMA (ACTA2) production in MRC5 cells. TGF-pi was administered 24 h before FP7 and FP12 treatment (5 pM). The TLR4 antagonist TAK 242 was administered, also post-treatment, as a positive control.

[0045] Figure 8. RT-qPCR assay of TGF-pi-induced IL-6 (IL6) production in MRC5 cells. TGF-P1 was administered 24 h before FP7 and FP12 treatment (5 pM). The TLR4 antagonist TAK 242 was administered, also post-treatment, as a positive control.

[0046] Figure 9. TAK242 worsens aSMA expression in TGFb-treated fibroblasts Very mild effect of FP7 and FP12 though. RT-qPCR assay of TGF-pi-induced aSMA (Acta2) production in murine 3T3 fibroblast cells. TGF-pi was administered 1 h after treatment with FP7 and FP12 (5 pM). Cells were collected 48 hrs after the start of TGF pi treatment. The TLR4 antagonist TAK 242 was administered, always in pre-treatment, as a positive control.

[0047] Figure 10. Toxicity of the TAK242 which is inducing a phenotype in the normal fibroblasts (not TGFb-activated). RT-qPCR assay of TGF-pi-induced aSMA (ACTA2) and collagen I (COL1A1) production in CCD-18Co fibroblast cells. TGF-pi was administered 1h after treatment with FP7 and FP12 (5 pM). Cells were collected 48 hrs after the start of TGF pi treatment. The TLR4 antagonist TAK 242 was administered, always in pre-treatment, as a positive control.

[0048] Figure 11. TLR4 and MD2 expression in human lung-derived mesenchymal cells across Idiopathic Pulmonary Fibrosis (IPF), Chronic Obstructive Pulmonary Disease (COPD) and control patient groups, (a) Global LIMAP plot depicting distinct cell populations, including fibroblasts / myofibroblasts, smooth muscle cells (SMCs), and pericytes, identified by their transcriptional profiles, (b) LIMAP projections showing the distribution of these cell subsets across Control, IPF, and COPD samples, (c-d) Expression patterns of TLR4 and LY96 (MD2) genes mapped onto the LIMAP space across the three conditions. Color intensity corresponds to normalized gene expression levels as indicated by the color scale.

[0049] Figure 12 Activation phenotype of WT and Tlr4 knock-out mouse lung fibroblasts, (a) Schematic of the experimental design, (b-d) Relative transcript levels of Acta2, Col1a1, and Tgfbl analyzed by RT-qPCR in mouse lung fibroblasts isolated from WT and Tlr4 knock-out (Tlr4- / -) mice after treatment with 10 ng / mL TGFpi and sample collection at- 7 - SIB BW1373R

[0050] 24 (b), 48 (c), and 72 (d) hours. Each sample was normalized to its respective untreated WT control. Data represent mean ± SEM of n=4 biological replicates.

[0051] Figure 13. Activation panel of MRC5 cells showing transcriptional changes of fibrotic and inflammatory markers, (a-c) RT-qPCR analysis of MRC5 fibroblasts treated with 10 ng / mL TGFpi and collected at 24, 48, and 72 hours. mRNA was extracted, reverse-transcribed, and analyzed by RT-qPCR. Each sample was normalized to its respective untreated control at the same time point. Analyzed genes include: (a) activation markers (ACTA2, FAP, TGFB1, TGFBRI, TGFBRII), (b) extracellular matrix (ECM) production (COL1A1, COL3A1, COL5A1, FN1), (c) inflammation (IL6, IL6R). Data are presented as mean ± SEM of n=3 biological replicates. Statistical significance calculated by paired two-tailed Student’s t test (treated vs. untreated): *P < 0.05; **P < 0.01.

[0052] Figure 14. IL-6 and fibronectin release from MRC5 cells, (a-b) ELISA assays to measure IL6 (a) and fibronectin (FN) (b) secretion by MRC5 cells treated with 10 ng / mL TGFpi for the indicated time points. Data are shown as mean ± SEM of at n=3 biological replicates. Statistical significance (treated vs untreated): *P < 0.05; ***P < 0.001.

[0053] Figure 15. Dose-dependent response of MRC5 cells to FP7 and FP12 treatment. Cells were pre-treated with the indicated 2-fold concentrations of FP7 and FP12 (range from 20 pM to 0.156 pM), followed by evaluation of biological effects using three different assays after 72 hours treatment, (a) Schematic representation of the treatment schedule, (b) CCK8 assay measuring cytoplasmic dehydrogenase activity, (c) MTT assay assessing mitochondrial dehydrogenase activity. Data are presented as mean ± SEM of n=3-4 biological replicates. Statistical significance calculated by using one-way ANOVA Dunnett’s multiple comparisons test: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, comparing FPs-treated vs TGFpi or TGFpi vs untreated.

[0054] Figure 16. Cytoxicity and proliferation assessment in activated MRC5 cells pre-treated with FPs compounds, (a) Schematic representation of the pre-treatment schedule. FPs compounds were administered after 24 hours of serum starvation in low-serum DMEM. After 1 hour of pre-treatment, recombinant human TGFpi was added, and analyses were performed at the indicated time points, (b) Cytoxicity was evaluated by LDH release assay at the indicated time points, (c) Proliferation was evaluated by MTT assay at the indicated time points. Data represent means ± SEM from n=3 biological replicates. Statistical analysis: RM one-way ANOVA with Dunnett’s multiple comparisons test- 8 - SIB BW1373R

[0055] comparing each sample to the respective untreated control at each time point. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Grey asterisks indicate comparisons with vehicle; black asterisks indicate comparison to TGFpi -treated samples.

[0056] Figure 17. FPs administration prevents fibrotic activation in MRC5 fibroblasts, (a) Schematic representation of the pre-treatment schedule, (b-d) Relative transcript levels of ACTA2, FN1, COL1A1, and TGFB1 at 24 (b), 48 (c), and 72 (d) hours following FPs exposure in the pre-treatment schedule. Data are means ± SEM (n=3 biological replicates). Statistical significance (paired two-tailed Student’s t-test on induced samples): *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0057] Figure 18. FPs administration downregulate aSMA and TLR4 expression in activated MRC5 fibroblasts, (a) Representative images of aSMA and TLR4 immunolabeling at 72 hours of treatment, (b-d) Quantification of aSMA and TLR4 expression in MRC5 cells at 24, 48, and 72 hours of pre-treatment, expressed as percentage relative to vehicle. Data shown as means ± SEM (n=3). Statistical analysis by unpaired two-tailed Student’s t-test on induced samples: *P < 0.05; **P < 0.01; ***P < 0.001*.

[0058] Figure 19. FPs administration for only 24 hours is efficient in decreasing aSMA and Collagen I expression in activated MRC5 fibroblasts, (a-b) MRC5 cells were subjected to pre-treatment schedule under four different conditions: 1) regular pre-treatment schedule as indicated in Fig.6a; 2) TGFp and FPs refreshed every day; 3) TGFp washout after 24 hours of treatment; 4) FPs washout after 24 hours of treatment. In all conditions cells were analyzed at 72 hours from the start of the treatment. Cells were acquired with Operetta Imaging system. aSMA (a) and Collagen I (b) were quantified from 69 ROIs / well at 20* magnification, (c) Representative images of aSMA and Collagen I immunostaining from a single field among 69 acquired fields / well at 20* magnification. Data are presented as means ± SEM (n=3-6 biological replicates). Statistical analysis by unpaired two-tailed Student’s t-test: *P < 0.05; **P < 0.01; ***P < 0.001.

[0059] Figure 20. FPs treatment reduces ECM production and extracellular deposition in MRC5 lung fibroblasts (a-b) Total ECM deposition analyzed by Flamingo staining and quantified with representative images (single field of 69 ROIs per well at 20* magnification). Images were acquired with Operetta Imaging system. Quantification expressed as a percentage relative to the vehicle, (c) Immunostaining for the indicated ECM components. Images- 9 - SIB BW1373R

[0060] were acquired with Operetta Imaging system. Quantification expressed as a percentage relative to the vehicle. Data in b,d were normalized to nuclei counts (Hoechst stain). Means ± SEM (n=3 biological replicates). Statistical analysis by unpaired two-tailed Student’s t-test vs. TGFpi, and one-way ANOVA Dunnett’s multiple comparisons test vs. vehicle (grey). **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0061] Figure 21. Nuclear translocation of phosphorylated-p65 (p-p65) and SMAD2 / 3 transcription factors, and active TGFpi release following FPs administration in MRC5 cells, (a-b) Immunostaining for SMAD2 / 3 (a) and p-p65 (b) was performed on cells harvested at 30 minutes, 24, 48, and 72 hours, acquired with Operetta Imaging system and quantified from 69 ROIs / well at 20* magnification. Nuclear fluorescence was normalized to total cellular fluorescence and to untreated controls. Data are means ± SEM (n=3 biological replicates). Statistical analysis by unpaired two-tailed Student’s t-test: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Means ± SEM (n=3 biological replicates). Statistical analysis: paired two-tailed Student’s t-test. *P < 0.05; **P < 0.01.

[0062] Figure 22. Cytotoxicity and proliferation evaluation in MRC5 cells under post-treatment schedule with FPs compounds, (a) Schematic representation of the post-treatment schedule. TGFp-treated MRC5 were subjected to FPs compounds treatment after 24 hours of TGFpi stimulation in low-serum DMEM and collected at 24, 48 and 72 hours. (b) LDH release assay to assess cytotoxicity was conducted at the indicated time points. (c) MTT assay to assess proliferation was conducted at the indicated time points. Data represent means ± SEM from n=3 biological replicates. Statistical analysis: RM one-way ANOVA Dunnett’s multiple comparisons test comparing each sample to respective untreated controls. **P < 0.01; ***P < 0.001; ****P < 0.0001. Grey asterisks indicate comparison with vehicle, black asterisks indicate comparison with TGFp-induced samples only.

[0063] Figure 23. FPs administration reverses fibrotic marker activation in activated MRC5 fibroblasts, (a) Schematic representation of the treatment schedule, (b-d) Relative transcript levels of ACTA2, FN1, COL1A1, and TGFB1 at 24 (b), 48 (c), and 72 (d) hours following FPs exposure in post-treatment schedule, as analyzed by RT-qPCR. Data are represented as means ± SEM (n=3 biological replicates). Statistical analysis by paired two-tailed Student’s t-test on induced samples.- 10 - SIB BW1373R

[0064] Figure 24. FPs post-treatment reverses aSMA and TLR4 upregulation in activated MRC5 fibroblasts, (a) Representative images of aSMA and TLR4 immunostaining at 72 hours. Cells were acquired with Operetta Imaging system, (b-d) Quantification of aSMA and TLR4 in MRC5 subjected to post-treatment schedule and analyzed at 24, 48, and 72 hours, presented as percentage of aSMA and TLR4 intensity / area relative to the vehicle. Data are presented as means ± SEM (n=3 biological replicates). Statistical analysis by unpaired two-tailed Student’s t-test on induced samples: *P < 0.05.

[0065] Figure 25. FPs post-treatment reverses ECM production and extracellular deposition in activated MRC5 lung fibroblasts.

[0066] (a-b) Total ECM deposition assessed by Flamingo staining: (a) quantification and (b) representative images (single field of 69 fields / well, 20* magnification) . (c) Immunostaining for single ECM components at the indicated treatment conditions. Images were acquired with Operetta Imaging system. Data in b-d normalized to nuclei counts (Hoechst). Data are represented as means ± SEM (n=3 biological replicates). Statistical analysis by unpaired two-tailed Student’s t-test vs TGFpi and one-way ANOVA Dunnett’s test vs vehicle (grey). **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0067] Figure 26. TLR4 expression across mesenchymal cells isolated from fibrostenotic Crohn’s disease (CD)-derived intestinal specimens, (a) LIMAP representation of clustered mesenchymal cells from different lesions of the terminal ileum of CD patients (relative to scRNA-seq dataset published in Ke et al., JCI 2024171). (b) Heatmap showing relative TLR4 expression in each mesenchymal subset.

[0068] Figure 27. TLR4 and MD2 (LY96) expression across the TAMMA database, (a-b) Boxplots show Iog2-transformed expression levels of TLR4 (a) and LY96 (b) in colon, ileum, and rectum tissues from control, ulcerative colitis (UC), and Crohn’s disease (CD) groups, (c) Boxplots show Iog2-transformed expression levels of TLR4 and LY96 in CD-derived colon fibroblasts.

[0069] Figure 28. FPs administration limits fibrotic activation markers and reduces ECM production and extracellular deposition in CCD18-Co colon fibroblasts, (a) Schematic representation of the experimental design, (b) Relative transcript levels of ACTA2, FN1, COL1A1, and TGFB1 at 48 hours following FPs exposure in pre-treatment schedule, (c-d) Evaluation of total ECM by immunostaining for Flamingo (b) and evaluation of specific ECM components by Collagen I and III immunostaining (c) of CCDI8-C0 fibroblasts- 11 - SIB BW1373R

[0070] subjected to a modified pre-treatment schedule. After FPs administration, cells were stimulated with a cytokine mix to promote a more in vivo-like fibrotic phenotype. Quantification of all three staining was conducted on 69 acquired fields / well at 20* magnification and presented as relative quantification with respect to the vehicle group. Images were acquired with Operetta Imaging system. Data normalized to nuclei counts (Hoechst). Data are presented as mean ± SEM (n=3 biological replicates). Statistical analysis in a by paired two-tailed Student’s t-test. Statistical analysis in b by unpaired two-tailed Student’s t-test vs. TGFpi and one-way ANOVA Dunnett’s multiple comparisons test vs. vehicle (grey). **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0071] Figure 29. Efficacy of FPs compounds on human derived activated intestinal fibroblasts, (a) Schematic representation of the experimental design, (b-c) Relative transcript levels of fibrotic genes in human-derived healthy intestinal fibroblasts subjected to pretreatment schedule and harvested 48 (b) and 72 (c) hours from the start of the treatment. Each sample was normalized to its respective vehicle control at the same time point. Data represent mean ± SEM of n=3 biological replicates. Statistical significance by paired two-tailed Student’s t-test: *P < 0.05; **P < 0.01.

[0072] Figure 30. Efficacy of FP7 anti-fibrotic treatment in ex-vivo intestinal samples. COL1A1 and TGFB1 relative transcript levels as assessed by RT-qPCR on colon tissue surgical specimens from Crohn’s disease (CD) donors, including stenotic and healthy control portions, cultured ex vivo in the presence of vehicle or 5 pM FP7 for 24 hours. Each sample was normalized to the healthy vehicle control. Data are presented as mean ± SEM (n=1 biological replicate).

[0073] GLOSSARY

[0074] The terms used in this description are as generally understood by those skilled in the art, unless otherwise indicated.

[0075] At any point in the description, the term “comprising” may be replaced with the term “consisting of”.

[0076] In the present description, the term “TLR4 receptor antagonist” denotes a compound that selectively binds to the MD-2 coreceptor thus inhibiting or totally blocking the formation of the TLR4 / MD-2 dimer and the subsequent formation of the (TLR4 / MD-2)2 “dimer of dimer”, which from the cell surface initiates the TLR4 signal cascade leading to the final synthesis of pro-inflammatory cytokines (mainly TNF and various interleukin types). In the present description, the term “Fibrosis” or “fibrotic disease” refers to a pathological- 12 - SIB BW1373R

[0077] wound healing in which connective tissue replaces normal parenchymal tissue to the extent that it goes unchecked, leading to considerable tissue remodeling and the formation of permanent scar tissue.

[0078] In the present description, the compounds identified as FP7 and FP12 refer to a compound having the formula represented below:

[0079]

[0080] In the present description, the term “saturated alkyl group” refers to a hydrocarbon chain consisting only of single bonds between carbon atoms, where each carbon atom is fully saturated with hydrogen atoms. This means that the alkyl group does not contain any double or triple bonds, and all the carbon atoms are connected by single bonds (C-C), with hydrogen atoms filling any remaining valencies (C-H).

[0081] Herein the word treatment refers to the use of interventions, therapies, medications, procedures, or other strategies aimed at improving a patient's health condition. This includes curing or alleviating symptoms of a disease, as well as ameliorating (make better or less severe) the disease or condition, or avoiding worsening or progression of the disease. Medical treatment can involve addressing the root causes of illness, managing its symptoms, preventing complications, and enhancing the patient's overall well-being, with the goal of stabilizing or improving health outcomes overtime.

[0082] DETAILED DESCRIPTION

[0083] The present invention relates to a compound of formula I:

[0084]

[0085] Formula I

[0086] wherein R1is a saturated Cn or C13 saturated alkyl group, and wherein R2is a C11 or C13 saturated alkyl group, for use in the treatment of a fibrotic disease in a patient in need thereof. In fact, the authors of the present inventions have surprisingly discovered that a compound with said formula has a particularly efficient antifibrotic activity and a- 13 - SIB BW1373R

[0087] surprising low toxicity profile with respect to known TLR4 antagonists (for example TAK242).

[0088] In one embodiment, within the compound having formula I, R1is equal to R2(R1=R2) . In a preferred embodiment, both R1and R2are Cn saturated alkyl group or C13 saturated alkyl group.

[0089] According to the present description one or more of R1, R2, or R3 is substituted or unsubstituted.

[0090] In an embodiment of the invention, at least one of R1, R2, and R3 is substituted.

[0091] When or more of R1, R2, and R3 chains are substituted, according to an embodiment of the invention said chain can be free from -OH substituents on position C2. The absence of hydroxyls in position C2 advantageously allows a shorter and more efficient synthetic route, eliminating various protection and de-protection steps of the hydroxyl groups thereby, reducing the costs of synthesis and making this synthetic process scalable and industrializable for drugs production.

[0092] According to one embodiment of the invention, at least one between R1and R2is free from -OH substituents on position C2. Position C2 refers to the second carbon position in the alkyl chain counting from left to right and / or from top to bottom.

[0093] In another embodiment, at least one of said chains is free of any substituent. This means that no substituent shall be present on the entire length of the chain.

[0094] In one embodiment, the compound is an a anomer of the compound of formula I. An a anomer is a carbohydrate in which the group bonded to the anomeric carbon is trans to the CH2O group on the other side of the pyranose or furanose ring ether oxygen atom. In another embodiment, the compound is a p anomer of the compound of formula I. A p-anomer is a carbohydrate in which the group bonded to the anomeric carbon is cis to the CH2O group on the other side of the pyranose or furanose ring ether oxygen atom. According to one embodiment of the invention, thanks to its antifibrotic activity and low toxicity profile, the compound of the present invention is used in the treatment of fibrotic diseases such as intestinal fibrosis, idiopathic pulmonary fibrosis (I PF) and fibrotic conditions generated by inflammatory diseases such as Crohn’s Disease.

[0095] An object of the present invention is also a pharmaceutical composition comprising a compound as described in any of the embodiments above mentioned and at least one pharmaceutically acceptable carrier or excipient, for use in the treatment of a fibrotic disease in a patient in need thereof.

[0096] All the embodiments described above apply mutatis mutandis to the pharmaceutical composition of the invention.- 14 - SIB BW1373R

[0097] As regards pharmaceutically acceptable excipients, the skilled person will be able to choose the most suitable one or those based on the type of formulation to be created, as well as any further additives, based on his knowledge of pharmacopoeia without the need for inventive contributions.

[0098] The pharmaceutical carrier may be selected to assist release of the component(s) over an extended period of time from the composition. The carrier may include a water-soluble or water-insoluble substance.

[0099] A water-soluble substance is a substance which plays a role in controlling infiltration of water into the interstices of the drug dispersion.

[0100] One water-soluble substance, or a combination of two or more water-soluble substances may be used.

[0101] The water-soluble substance specifically may be selected from one or more of the groups consisting of synthetic polymers (eg. polyethylene glycol, polyethylene polypropylene glycol), sugars (eg. sucrose, mannitol, glucose, sodium chondroitin sulfate), polysaccharides (e.g. dextran), amino acids (eg. glycine and alanine), mineral salts (eg. sodium chloride), organic salts (eg. sodium citrate) and proteins (eg. gelatin and collagen and mixtures thereof).

[0102] In addition, when the water-soluble substance is an amphiphilic substance, which dissolves in both an organic solvent and water, it has an effect of controlling the release of, for example, a lipophilic drug by altering the solubility thereof. An amphiphilic substance includes, but not limited to, one or more selected from the group consisting of polyethylene glycol or a derivative thereof, polyoxyethylene polyoxypropylene glycol or a derivative thereof, fatty acid ester and sodium alkylsulfate of sugars, and more specifically, polyethylene glycol, polyoxy stearate 40, polyoxyethylenepolyoxypropyleneglycol, polyoxyethylene-polyoxypropylene-glycol, polyoxyethylene- polyoxypropyleneglycol, sucrose esters of fatty acids, sodium lauryl sulfate, sodium oleate, sodium chloride, sodium desoxycholic acid (or sodium deoxycholic acid (DCA)) of which mean molecular weights are more than 1500.

[0103] In addition, the water-soluble substance may include a substance selected from one or more of the groups consisting of drugs, peptides, proteins, glycoproteins, polysaccharides.

[0104] A water-insoluble carrier, when present, may include a substance which plays a role in controlling infiltration of water into the interstices of the drug dispersion. One waterinsoluble substance, or a combination of two or more water-insoluble substances may be used.- 15 - SIB BW1373R

[0105] The water-insoluble substance specifically may be selected from one or more of the groups of water insoluble polymers, resins and latexes including water-insoluble acrylates, methacrylates and other carboxy polymers, waxes, lipids including phospholipids and lipoproteins.

[0106] The skilled person knows the amount of carrier and optional further eccipients commonly used in a pharmaceutical composition.

[0107] In an embodiment, the pharmaceutical carrier may constitute from approximately 1% to 20% by weight, preferably approximately 10% to 20% by weight, based on the total weight of the pharmaceutical composition.

[0108] The composition according to the invention may comprise one of the compounds as defined and claimed herein, or a mixture thereof.

[0109] The composition of the invention may be prepared in the form of a single mixture of active principle and anti-fibrotic compound or in the form of different mixtures for a concomitant or sequential administration of the components.

[0110] In a particular embodiment, the compound of formula I described in the present invention is the sole active principle present in the pharmaceutical composition.

[0111] The composition may further comprise one or more additional therapeutically active principle.

[0112] Said pharmaceutical composition can also be formulated in the form of an association of a plurality of active principles.

[0113] The pharmaceutical composition may be formulated for oral, parenteral, nasal, aerosol, sublingual, rectal, vaginal, topical, endovenous or systemic administration. suitable conventional carriers and / or excipients for suspension, emulsion, ointment, cream, spray, granulate, powder, solution, capsule, pill, tablet, lyophilized product, lozenge, aerosol, nebulization, injection, or others can be selected by the person skilled in the art.

[0114] According to a non-limiting example, the composition can be formulated for oral, parenteral, nasal, aerosol, sublingual, rectal, vaginal, topical or systemic administration. The pharmaceutical composition of the invention may comprise as sole active principles one or more compounds of formula I according to any one of the embodiments provided in the description or in the claims, or could also comprise additional pharmaceutical active principles (APIs), preferably suitable for treating fibrotic diseases and at least one pharmaceutically acceptable carrier or excipient. A non-limiting example of anti-fibrotic active principles may comprise Pirfenidone, Nintedanib, Colchicine, Glucocorticoids (e.g., Prednisone), Vitamin D Analogues (e.g., Paricalcitol), TGF-p Inhibitors (e.g., Fresolimumab), Antisense Oligonucleotides (e.g., RG-012), Cilengitide, Bromodomain- 16 - SIB BW1373R

[0115] and Extra-Terminal (BET) Inhibitors (e.g., JQ1), Cysteamine (Procysbi), FGF-21 (Fibroblast Growth Factor 21) Agonists, N-Acetylcysteine (NAC), Autophagy Modulators (e.g., Rapamycin), Farnesoid X Receptor (FXR) Agonists (e.g., Obeticholic Acid).

[0116] Thanks to the demonstrated anti-fibrotic effect of the compound of formula I, the pharmaceutical composition of the present invention is advantageously used in treatment of a fibrotic disease. The compound of the invention can be used alone or in combination with other active principles already known for treating fibrotic diseases. According to the present invention, the fibrotic disease is selected from intestinal fibrosis, idiopathic pulmonary fibrosis (IPF) and Crohn’s Disease.

[0117] In particular, the Authors have surprisingly found that the compound of formula 1 and the pharmaceutical composition comprising such compounds are particularly effective in the treatment or adjuvating the treatment of fibrosis when the cell lines to be treated shows, before the treatment with the compound or with the pharmaceutical composition, overexpression of genes ACTA2, COL1A1 and / or FN1 with respect to one or more healthy control samples. The healthy control samples can be samples obtained from healthy patients or from suitable cell lines. ACTA2 (aSMA), COL1A1 (collagen I) and / or FN1 (fibronectin), preferably of all said genes which are main fibrotic markers, produced in high quantities by the cells leading to an intracellular and extracellular accumulation with consequent pathological progression of fibrosis.

[0118] In jurisdictions where claims to therapeutic treatments are allowed, the subject-matter of the invention is a method of treatment of a fibrotic disease such as intestinal fibrosis, idiopathic pulmonary fibrosis (IPF) and Crohn’s Disease, including the administration of the compound having formula I or of the pharmaceutical composition comprising the compound having formula I and at least one pharmaceutically acceptable carrier according to any of the embodiments of the invention.

[0119] In compliance with Art. 170bis of the Italian patent law it is herein declared that all experiments involving cells were carried out on commercially available cells and murine models, the obligations deriving from the national or Ell regulations, and in particular, from the provisions referred to in paragraph 6 of Legislative Decree No. 206 of 12 April 2001 and 8 July 2003 no. 224, have been fulfilled.- 17 - SIB BW1373R

[0120] EXAMPLES

[0121] The antifibrotic activity data on MRC5 cells (human lung embryonic fibroblasts) were obtained by monitoring the expression of fibrotic markers in the presence and absence of TLR4 antagonists by RT-qPCR. In the MRC5 cell line, the fibrotic phenotype is induced by the administration of TGF-Beta 1 at a concentration of 10ng / mL. The analysis shown in the figures takes into account the main fibrotic markers such as ACTA2 (aSMA), COL1A1 (collagen I), FN1 (fibronectin), which in fibrotic conditions are produced in high quantities by the cells leading to an intracellular (aSMA) and extracellular (Collagen I and fibronectin) accumulation with consequent pathological progression of fibrosis.

[0122] In the same cells, the variation in production of the inflammatory marker IL-6 was also evaluated.

[0123] Cells were seeded at 80,000 cells / well (in 6-well plates) and after 24h they were starved for another 24h to make them more sensitive to TGF-Beta1 stimulation. Cells were harvested and mRNA was extracted using the Triazole-chloroform-ethanol extraction protocol. mRNA was reverse transcribed into cDNA and expression analysis was performed using RT-qPCR with SYBERgreen mix.

[0124] Figures 1-4 were obtained by pre-treating with FP7 and FP12 molecules and subsequently (after 1h) with TGF-pi which induces fibrosis. Cells were then harvested 24, 48 and 72 hours after the start of TGFb treatment. Figures 5-8 were obtained by first treating with TGF-pi and then (after 24 hours) with the molecules FP7 and FP12. The cells were then collected 24, 48 and 72 hours after the beginning of the treatment with the antagonists. In all experiments the compound TAK242, a known antagonist of TLR4, was used as a positive control.

[0125] A significant inhibition of the production of all fibrotic markers is observed when the cells are pre- or post-treated with FP7 and FP12 at a concentration of 5 pM, compared to untreated cells (green line). The positive control TAK-242 (TLR4 antagonist) is also active in inhibiting fibrotic markers in the two conditions of pre- and post-treatment. The antifibrotic activity data on 3T3 cells (mouse embryonic fibroblasts) and CCD-18Co cells (human colon fibroblasts) were obtained by monitoring the expression of fibrotic markers in the presence and absence of TLR4 antagonists by RT-qPCR (Figures 9 and 10). In both cell lines, the fibrotic phenotype is induced by the administration of TGF-Beta 1 at a concentration of 10ng / mL. The analysis shown in the figures takes into account the fibrotic markers ACTA2 (aSMA) and COL1A1 (collagen I). Cells were seeded at 80,000 cells / well (in 6-well plates) and after 24h they were starved for another 24h to make them more sensitive to TGF-Beta1 stimulation. Cells were harvested and- 18 - SIB BW1373R

[0126] mRNA was extracted using the Triazole-chloroform-ethanol extraction protocol. mRNA was reverse transcribed into cDNA and expression analysis was performed using RT-qPCR with SYBERgreen mix. Figure 9 (relative to CCD-18Co cells) and Figure 10 (relative to 3T3 cells) were obtained by pre-treating with FP7 and FP12 molecules and subsequently (after 1h) with TGF-pi which induces fibrosis. Cells were then harvested 48 hours after the start of TGFb treatment.

[0127] The aim of the work was to evaluate the antifibrotic capacity of two TLR4 antagonists previously synthesized and characterized in the research group of Francesco Peri, FP7 and FP12. Both compounds were projected to interact with the MD-2 TLR4 binding pocket, thereby preventing TLR4 homodimerization and subsequent pathway activation. The rationale is to determine whether these compounds, through TLR4 modulation, can prevent or disrupt the profibrotic positive feedback loop in which fibrosis progression induces TLR4 activation and its associated inflammatory response, which in turn promotes further profibrotic processes.

[0128] To test their antifibrotic activity, two human cell lines have been selected: MRC5 for studying lung fibrosis and CCD18-Co for investigating intestinal fibrosis. In both cases, the feasibility of the objectives was assessed through bioinformatic analyses of two published scRNA-seq datasets, one for I PF and one for Crohn’s Disease (CD), to evaluate the presence and expression variation of the receptor of interest in healthy and diseased patients. Subsequently, the model has been validated through TGFpi administration to MRC5 cells, monitoring principal fibrotic markers by RT-qPCR. Then, it has been analyzed the impact of FP7 and FP12 using two different treatment schedules: pre-treatment and post-treatment (Figure 22), to assess the compounds' ability to prevent and block / reverse the fibrotic phenotype, respectively.

[0129] These studies were conducted at multiple levels, examining dehydrogenase activity (both cytoplasmic and mitochondrial) to evaluate metabolic impact, RNA expression levels, and protein levels including both intracellular accumulation and extracellular deposition. Additionally, lung-derived fibroblasts isolated from WT and TLR4' / _mice have been evaluated in a pre-treatment schedule to exclude possible off-target effects. Concurrently, intestinal fibrosis have been investigated using human colon CCDI8-C0 cells. In this case, the pre-treatment schedule at RNA expression level and ECM deposition has been evaluated. Moreover, the post-treatment effect of FP7 compound has been analyzed in an ex vivo setup utilizing human intestinal specimens surgically isolated from a Crohn’s Disease patient, assessed by RT-qPCR analysis.- 19 - SIB BW1373R

[0130] 3.3 Materials and Methods

[0131] Cells lines.

[0132] MRC-5 human embryonic lung fibroblasts were purchased from ATCC (#CCL-171), and cultured in Dulbecco Modified Eagle’s Medium high glucose (Sigma, #D5671) supplemented with 10% of fetal bovine serum (FBS) (Euroclone, #ECS0180L), 1mM Glutamine StablelOOX (Euroclone, #ECB3004D), 1mM of Penicillin / Streptomycin (P / S) (Euroclone, #ECB3001). Cells were used between passages 7 and 14. CCD-18Co human colon fibroblasts were purchased from ATCC (#CRL-1459) and were cultured in Dulbecco Modified Eagle’s Medium-F12 medium (Euroclone, #ECM0090L) supplemented with 10% FBS, 1 mM Glutamine StablelOOX, 1 mM sodium pyruvate, 2 mM sodium bicarbonate, HEPES, and 1 mM of P / S. Cells were used between passages 7 and 11.

[0133] All cell types were maintained at 37°C in a humidified 5% CO2 incubator and routinely tested for mycoplasma using a PCR assay.

[0134] Primary fibroblasts isolation.

[0135] Human colon fibroblasts were isolated from the biopsy of three healthy patients upon informed consent. Isolated cells were maintained in M160 media (Gibco, #10043083) supplemented with LSGS, 1% Glutamine StablelOOX and 1% penicillin-streptomycin-amphotericin.

[0136] Murine lung fibroblasts were isolated from all 5 lung lobes of WT and Tlr4 / _C57BL / 6 mice provided by Francesca Granucci (Bicocca University, Italy). Collected lungs were placed in cold Tissue Trasport Medium (PBS 1X + 1% P / S) and kept on ice. Lungs were then transferred onto 100-mm Petri dish and cut in smaller pieces 1 / 2 mm3). The obtained pieces were then digested in DMEM-1%P / S-2mMGIut-3%BSA containing 30mg of collagenase IV (Sigma, C51138) to which 2 mL DMEM high glucose serum free was added. After 30’ of incubation at 37°C in rotating wheel, the remaining tissue pieces were mechanically disrupted by pipetting. Fibroblast medium (DMEM high glucose supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin / Streptomycin and 2mM Glutamine) was added to neutralize the collagenase activity. After centrifugation the cells pellet were resuspended and plated in 2 T75 flasks with fibroblast media. Isolated murine fibroblasts were maintained in DMEM high glucose supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin / Streptomycin and 2mM Glutamine.

[0137] Ex vivo instestinal culture.- 20 - SIB BW1373R

[0138] Intestine specimens were collected from a surgically resected CD patient’s tissue, upon informed consent. Disease samples were collected from the stenotic portion while healthy specimens were collected in the distal area of the pathological tissue macroscopically free from any disease. The entire tissue specimen is transported in cold wash solution (HBSS 1X - / -, 20mM HEPES, P / S 1X, Gentamycin 50ug / mL). Each tissue (healthy and disease) was cut preserving tissue structure and washed four times in cold HBSS1X- / - wash solution and blot on paper towel to remove feces and mucus. The collected tissues were then cut into two identical pieces of 1-1.5 cm, each containing all the intestinal laminas. After washing, both healthy and CD specimens were cultured for 24 hours in a 48-well plate with 300 pL of DM EM supplied with 1% PSA, 10% FBS, and 1% Glut, in the presence of 5uM FP7 or vehicle treatment. Ethical authorization for the use of human-derived samples was previously obtained at Humanitas Research Hospital.

[0139] In vitro treatments with FP compounds.

[0140] The TLR4 antagonists FP7 and FP12 were synthesized from commercially available D-glucose by multistep organic synthesis, as previously reported. Nuclear magnetic resonance and mass spectrometry analysis were utilized to assess the molecule's purity. A 5 mM stock solution of FP7 and FP12 was prepared in a 1:1 mixture of ethanol and DMSO and stored at -20°C. The TLR4 inhibitor TAK242 was purchased from InvivoGen (InvitroFit™, #CLI-095).

[0141] Fibroblast cells were seeded in 6-well plates and the following day starved overnight in their respective media containing 1% FBS. Cells were then subjected to two treatment schedules. In the pre-treatment schedule, cells were first treated with FP7, FP12, and TAK242 for 1 hour and subsequently treated with 10 ng / ml human recombinant TGF-pi (Invivogen, #rcyc-hTGFp 1). Cells were collected at 24, 48 and 72 hours after TGF-pi treatment. In the post-treatment schedule, after starvation, cells were first treated with 10 ng / ml human recombinant TGF-pi and 24 hours later treated with FPs and TAK242 compounds for 24, 48, and 72 hours.

[0142] RNA extraction and quantitative real-time PCR analysis.

[0143] Cells were homogenized in TRIzol reagent (Invitrogen, #15596018) and total RNA was extracted according to the manufacturer's directions. cDNA synthesis was performed using the Applied Biosystems cDNA synthesis kit according to the manufacturer's directions (Applied Biosystems #4368814). Quantitative PCR was performed to analyze the gene expression profiles of the listed genes using the Fast SYBR Green PCR Master- 21 - SIB BW1373R

[0144] Mix (Applied Biosystem, #4385612) and utilizing the ViiA7 Sequence Detector System (Applied Biosystems). Measurements were standardized to the expression of the GAPDH housekeeping gene. The expression data is presented as fold change (2’AACt) with the control group normalized to a fold of 1. The ACt was used to measure the statistical significance of observed changes. Genes and primer sequences are reported in Table 1.

[0145] Table 1. Human and mouse primers utilized in the real-time PCR analysis.

[0146]

[0147] - 22 - SIB BW1373R

[0148]

[0149] Cell viability assay (MTT).

[0150] MRC5 cells were seeded in a 96-well plate (1,000 cells / well), treated with vehicle orTGF-P1 , and FP7, FP12 or TAK242 at 5pM in both the treatment schedules. At the collection time points, cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, #298-93-1) according to the manufacturer's instructions.

[0151] CCK8 assay.

[0152] MRC5 cells were seeded in a 96-well plate (1,000 cells / well), treated with vehicle orTGF-P1, and FP7, FP12 or TAK242 at different concentration in pre-treatment schedule. At the 72h, cells were incubated with WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] ( Cell Counting Kit-- 23 - SIB BW1373R

[0153] 8, Sigma Aldrich #96992) for 2h at 37°C and then the absorbance was read at 450nm using microplate reader.

[0154] Cytotoxicity assay.

[0155] The cell death and toxicity of both treatment schedules were tested on MRC5 by measuring the lactate dehydrogenase (LDH) leakage into the medium using the CytoTox 96 non-radioactive cytotoxicity assay (Promega, #G1780), following the manufacturer’s protocol. Measurements were performed on the supernatant of cell culture at the different experimental conditions as described for the MTT assay.

[0156] Sircol assay.

[0157] MRC5 were plated in 6-well plates and subjected to pre-treatment and post-treatment schedules with TGFpi, FPs, and TAK242 compounds. Supernatants were collected at 48 and 72-hour time points and subjected to the Sircol 2.0 soluble collagen assay (Biocolor, SIRC2), according to the manufacturer’s instructions. Briefly, the soluble collagen that accumulates in 1ml of growth medium was concentrated overnight at 4°C with Collagen Concentration Regent provided in the kit. The day after samples were centrifuged to induce collagen precipitation and aqueous suspension was removed. Thus the precipitated was resuspended in acid-acetic solution and evaluated according to the manufacturer's directions with soluble collagen concentration correction. Indeed, the amount showed is the real amount of soluble collagen present in the starting 1mL samples.

[0158] Immunofluorescence.

[0159] MRC5 cells were seeded (1*103cells / well) into PhenoPlate 96-well microplates (PerkinElmer, #6055500), where they were exposed to pre- or post-treatment schedules. At the end of the time course treatment (24, 48 and 72 hours), cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, #F8775) and permeabilized with 0.5% Triton X-100(Sigma-Aldrich #9036-19-5). solution in PBS. Then, blocking was performed using PBS containing 5% BSA and 0.3% Triton X-100. Subsequently, cells were labeled with anti-actin a-smoot muscle-Cy3 antibody, mouse mAb (Merk C6198-100UL), and TLR4 Monoclonal Antibody (76B357.1) (Invitrogen, #MA5-16216, diluted 1:500). Cells were then tagged with goat anti-mouse IgG (H+l) Alexa Fluor Plus 647 secondary antibody (Invitrogen, #A32728) to allow target visualization, cytoplasm was stained with DyLight™ 488 Phalloidin (Cell Signaling, #12935, diluted 1:100) for 30’ at 37°C. Nuclei were counter-labeled with PhenoVue Hoechst 33342 Nuclear Stain (PerkinElmer, #CP71) for- 24 - SIB BW1373R

[0160] 5’ at room temperature (RT). In the wash-out experiment cells were stained with rabbit anti-human collagen I antibody (Novus Biologicals, #NB600-408, diluted 1:500), and mouse alpha-smooth muscle actin staining (Novus Biologicals, NB300-978, diluted 1 :500) and then with secondary antibodies including Cy5-conjugated donkey anti-rabbit IgG antibody (Jackson ImmunoResearch, #711-175-152, diluted 1:500) and Cy3-conjugated donkey anti mouse (Jackson ImmunoResearch, #715-165-150, diluted 1:500) Images were acquired using the Operetta CLS High-Content Analysis System. 69 ROIs / well were acquired on a 96-well plate, at 20* magnification using a water immersion objective and analyzed by using the Harmony 4.5 / 5.2 software (PerkinElmer). The target intensity was normalized on single cell area. Each biological replicate was analyzed utilizing n=2-4 technical replicates.

[0161] Measurement of ECM deposition.

[0162] Cells (5 xio3 / well for 96-well plates) were plated into PhenoPlate 96-well microplates (PerkinElmer, #6055500). Following 72h of incubation in both pre and post treatment conditions, cells were washed in PBS and stained with PhenoVue Hoechst 33342 Nuclear Stain (PerkinElmer, CP71) in growth media for 15’ at37°C 5% CO2. Nuclei count was performed exploiting Operetta. After nuclei count cells were washed 3 times with PBS and lysed with 20pl of 0.25 M ammonium hydroxide in 25 mM Tris for 15 min at 37°C. The matrix was washed 3 times in PBS, fixed using 50p I of 100% methanol (unless otherwise stated) for 30 min at -20 °C and stained overnight using 1X Flamingo fluorescent staining (BioRad #1610490). For single ECM components the matrix after fixing was was labelled with primary antibodies, including goat anti-human collagen III alpha 1 antibody (1:500, NBP1 -26547, Novus Biologicals), rabbit anti-human collagen I antibody (1:500, NB600-408, Novus Biologicals diluted 1:500), and sheep anti-human fibronectin antibody (R&D Systems, AF1918, diluted 1:500) at 4°C O / N, and then labelled with secondary antibodies, including Cy3-conjugated donkey anti-goat IgG antibody (Jackson ImmunoResearch, #705-165-003, diluted 1:500), Alexa Fluor 647-conjugated donkey anti-sheep IgG antibody (Jackson ImmunoResearch, #713-605-147, diluted 1:500), and Briliant Violet anti-rabbit IgG antibody (BioLegend, #406410, diluted 1:150).

[0163] Plates were scanned on the Operetta CLS High-Content Analysis under the Confocal profiling at 20* water objective, NA 1.0 objective with 2 x 2 binning (2160x 2160 pixels / field). 69 ROIs / well were acquired on a 96-well plate. To define the amount of ECM deposition, using the Harmony 4.5 / 5.2 software (PerkinElmer Inc., Milan, Italy), we measured the total intensity of the target of the entire image as the object and normalized- 25 - SIB BW1373R

[0164] on nuclei count number to have an indication of how much ECM was secreted by single cells. Then the amount of ECM deposition was reported as 100% compared to the vehicle sample. Each biological replicate was analyzed utilizing n=2-4 technical replicates.

[0165] Nuclear Translocation

[0166] MRC5 cells were seeded (1*103cells / well) into PhenoPlate 96-well microplates (PerkinElmer, #6055500), where they were exposed to pre-treatment schedules. At the end of the time course treatment (30’, 24, 48 and 72 hours), cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, #F8775) and permeabilized with 0.5% Triton X-100(Sigma-Aldrich #9036-19-5). solution in PBS. Then, blocking was performed using PBS containing 5% BSA and 0.3% Triton X-100. Subsequently, cells were incubated with Phospho-NF-KB p65 (Ser536) (93H1) Rabbit mAb (Cell Signaling, #3033) overnight at 4°C. The following day, cells were incubated with PhenoVue Fluor 568 conjugated anti-rabbit secondary antibody (PerkinElmer, #2GXRB568C1) for 1h at 37°C temperature. Then cells were incubated with (Phospho-SMAD2 (Ser465 / 467) / SMAD3 (Ser423 / 425) (D27F4) Rabbit mAb (Cell Signaling, # 8685S) O / N at 4°C. The day after they were incubated for 1h RT with PhenoVue Fluor 633 conjugated anti-rabbit secondary antibody (Invitrogen, #A21070) and cytoplasm was stained with DyLight™ 488 Phalloidin (Cell Signaling, #12935, diluted 1:100) for 30’ at 37°C. Nuclei were counter-labeled with PhenoVue Hoechst 33342 Nuclear Stain (PerkinElmer, #CP71) for 5’ at room temperature (RT). Images were acquired using the Operetta CLS High-Content Analysis System. 69 ROIs / well were acquired on a 96-well plate, at 20* magnification using a water immersion objective. Imageswere analyzed by using the Harmony 4.5 / 5.2 software (PerkinElmer) normalizing the nuclear detected intensity on the entire cell intensity. Each biological replicate was analyzed utilizing n=2-4 technical replicates.

[0167] Statistical analysis.

[0168] Data comparisons between two groups has been performed utilizing unpaired two-tailed Student’s / test or one-way ANOVA Dunnett’s multiple comparisons test as indicated in each figure legend. RT-qPCR statistical analysis between two groups has been performed utilizing paired two-tailed Student’s t test or RM one-way ANOVA Dunnett’s multiple comparisons test. Data are presented as mean ± standard error of the mean (S.E.M.). Statistical analysis has been performed using GraphPad Prism software (version 8.0.2. (263)). Statistical significance is defined as P< 0.05. All experiments were- 26 - SIB BW1373R

[0169] conducted on at least n=3 biological replicates analyzed by using n=2-4 technical replicates.

[0170] 3.4 Results

[0171] 1. TLR4 is expressed in IPF-associated human fibroblasts and is functionally involved in the fibrotic phenotype

[0172] We initially evaluated the expression of TLR4 and its partner MD-2 in healthy and idiopathic pulmonary fibrosis patients by exploiting the public database I PF Cell Atlas (https: / / www.ipfcellatlas.com / ). Based on scRNA-seq data present in the Kaminski / Rosas database169, in which Uniform Manifold Approximation and Projection (UMAP) represents 312,928 cells from 32 IPF, 18 COPD, and 28 control donor lungs, we obtained, a TLR4 and MD-2 expression panel in mesenchymal cells.

[0173] The bioinformatic analysis - conducted in collaboration with KU Leuven University -revealed that both TLR4 and, more notably, MD-2 are overexpressed in IPF compared to the healthy control group of mesenchymal cells obtained from patients lungs (Figure 11 a-d). In particular, there is increased expression of TLR4 and MD-2 in IPF-associated fibroblast and myofibroblast populations (Figure 11 c-d).

[0174] To verify whether TLR4 exert a functional role in the fibrotic context, we isolated lung fibroblasts from WT and Tlr4 full body knock-out mice (Tlr4 “), in collaboration with Francesca Granucci’s laboratory at Bicocca University (Milan). Cultured WT and Tlr44 “ lung fibroblasts were stimulated with 10 ng / ml TGF-pi for 24, 48 and 72 hours and the activation of fibrosis markers was tested via RT -qPCR (Figure 12a). From data analysis, it emerged that WT fibroblasts are well activated over the course of the treatment, as shown by the upregulation of Acta2, Col1a1 and TGF / 3 1 transcripts (Figure 12b-d), with an optimal peak at 48h (Figure 12c), in which all three markers are more expressed following TGF-pi stimulus. Conversely, Tlr4 “ did not show the same activation, with relative expression remaining near to the non-treated WT and Tlr4 “ cells (Figure 12b-d).

[0175] These data support previous evidences pointing to a role for TLR4 in the fibroblast populations, thus supporting a rationale for considering TLR4 as a promising therapeutic target in the fibrosis context.

[0176] 2. Generation and validation of an in vitro pulmonary fibrosis model

[0177] Having confirmed TLR4 expression and functional role in human lung fibroblasts, for our studies we selected the MRC5 cell line, an embryonic human lung fibroblast line. To- 27 - SIB BW1373R

[0178] induce a fibrotic phenotype, we treated MRC5 fibroblasts with 10 ng / mL of human recombinant TGFpi after 24 hours of serum starvation at 1% FBS to increase TGFpi sensitivity. Cells were then harvested at different time points (24, 48, 72h) to evaluate the acquisition of a fibrotic phenotype and its maintenance over a 3-days time. The analysis was conducted by RT-qPCR, examining the following fibrotic and inflammatory markers: ACTA2, FAP, TGFB1, TGFBRI, TGFBRII (fibroblast activation), COL1A1, COL3A1, COL5A1, FN1 (extracellular matrix), IL6, IL6R (inflammation).

[0179] As shown in Figure 13, TGFpi effectively activates MRC5 cells, with upregulation of activation markers (Figure 13a) and expression of ECM genes (Figure 13b) already evident after 24h from the start of the treatment. Additionally, our results demonstrate that the activation is sustained over time, having these markers dysplayed upregulation up to 72 hours. Interestingly, the TGFp positive feedback loop which is activated in the treated cells and demonstrated by the upregulation of the TGFB1 transcript, is sustained by the upregulation of the TGFp receptor I (TGFBRI) rather than by the receptor II (TGFBRII) (Figure 13a).

[0180] Interestingly, an increase in the production of the inflammatory cytokine IL-6 is also evident, accompanied by a parallel decrease in IL-6 receptor (IL6R) expression (Figure 13c). This highlights the acquisition of an IL6-dependent inflammatory phenotype by the activated fibroblasts with a potential paracrine effect on the surrounding cells enhanced by their own downregulation of the cognate inflammatory receptor.

[0181] In addition to the RNA expression evaluation, secreted protein levels of IL-6 and FN1 were also measured by specific ELISA assays performed at 24, 48 and 72 hours from the start of the TGFp treatment (Figure 14a-b). ELISA results sustain and strengthen the already observed increase of IL-6 and FN1 transcripts, confirming the fibrotic phenotype and inflammatory pathway activation and supporting the validity of our experimental model. Once the MRC5 TGFpi stimulation system was validated, we proceeded to investigate the effects of FP7 and FP12 on the induced fibrotic phenotype of MRC5 fibroblasts.

[0182] 3. Viability, cytotoxicity and dose-response assessment of anti-TLR4 compounds In order to propose pharmacological targeting of lung fibrosis by treatment with the developed TLR4 antagonists an assessment of cell cytotoxicity and dose-response analysis has been conducted to detect any potential cytotoxic effect and determine the best in vitro treatment dose. FP7 and FP12 compounds were tested on MRC5 cells at a range of sequential two-fold concentrations, ranging from 20 pM to 0.156 pM. Cells were- 28 - SIB BW1373R

[0183] challenged with the pre-treatment schedule, consisting in FPs treatment at the above described concentrations one hour before TGFp treatment. Cells were collected 72 hours later and subjected to the CCK-8 and MTT assays to assess cell viability (Figure 15a). The two assays are routinely employed to measure cell viability in cytotoxic assays and differ on the fact that CCK-8 is based on the activity of cytoplasmic dehydrogenases while MTT on mitochondrial dehydrogenases.

[0184] Results show that TGF-pi stimulation, which was previously shown consistently promoted the fibrotic phenotype, was accompanied by increased dehydrogenase activity indicating enhanced metabolism, possibly required to sustain the increased ECM production. FP7 utilized at 5, 2.5 and 1.25pM range reduced the dehydrogenase activity compared to the TGFp-treated cells and did not alter its activity compared to the untreated control. Lower FP7 concentrations (0.625, 0.312 and 0.156 pM) had only a minor effect compared to the TGFp -treated group. Conversely, higher concentration (7.5, 10 and 20 pM) displayed a reduction of the dehydrogenase activity even compared to the untreated control, possibly indicating a cytotoxic effect (Figure 15b).

[0185] FP12 showed slightly different results, with concentration range 1.25-0.156 pM inducing no effect (level comparable to the untreated control), concentrations 7-2.5 pM decreasing the dehydrogenase activity compared to the TGFp-treated and also the untreated control. No effects were observed with the 10 and 20 pM treatment concentrations (Figure 15b).

[0186] While performing the experiments, we noticed a substantial precipitation of both FPs when utilized at high concentrations (10 and 20 pM) and concluded that the inconsistent effect of FP7 and FP12 compounds at such high doses could be due to the observed solubility limits.

[0187] In parallel, we conducted a MTT assay and observed a more pronounced dose-response effect in terms of reduction of the mitochondrial dehydrogenase activity which was proportional to the treatment concentration utilized (Figure 15c).

[0188] To exclude any possible cytotoxic effect, we performed an LDH release assay at multiple time points to assess cell integrity (Figures 16a-b). MRC5 cells challenged with the pretreatmentschedule (Figure 16a) in which 5uM concentration of FP7 and FP12 was used. Cells were collected and 24, 48 and 72 hours pre-treatment and subjected to measurement of the percentage of the LDH enzyme release, which is indicative of compromised cell membrane integrity and therefore cell death.- 29 - SIB BW1373R

[0189] Data obtained clearly indicate that there is no cell death induction in all treatment conditions (TGFp stimulation or not, co-treatment with FP7 / FP12 or only FP7 / FP12 with no TGFp stimulation) as we did not observe any LDH release increment compared to the untreated control. This result was observed at all three time points analyzed (Figure 16b). Having excluded toxic effects, we repeated the MTT assay applying the same treatment schedule and FP7 / FP12 concentration (5 pM) to evaluate MRC5 cell viability over 72 hours and assess their impact in terms of cell proliferation. Data show that while TGF-pi promotes increased proliferation of MRC5 fibroblasts over time, this phenomenon is blocked by the pre-treatment with FP7 and FP12 (Figure 16c). In this experiment we also included TAK242, a commercially available TLR4 antagonist small molecule which acts by binding at Cys747 of the intracellular domain of TLR4, disrupting the receptor's interaction with adaptor proteins TIRAP and TRAM, so preventing TLR4 downstream signaling activation170. TAK242 was also able to reduce cell proliferation in TGFp -treated fibroblasts, although to a lesser extent compared to FP7 / FP12 (Figure 16c).

[0190] Taken together, the results of all these assays indicate that our TLR4 antagonist molecules are safe and are able to inhibit fibroblast proliferation over time, which aligns with their capability to inhibit the establishment of the fibrotic phenotype.

[0191] 4. FP7 and FP12 treatment inhibits fibroblasts activation

[0192] Two critical aspects of fibrosis-associated fibroblasts were also investigated: acquisition of a myofibroblast phenotype and the production and deposition of extracellular matrix proteins. MRC5 cells subjected to the pre-treatment schedule with FP7 or FP12 utilized at 5 uM concentration and collected at multiple time points were subjected to RNA extraction and transcriptional profiling by quantitative real-time PCR (RT-qPCR) (Figure 17a). Specifically, following fibrotic markers have been analyzed: ACTA2, FN1, COL1A1, and TGFB1.

[0193] RT-qPCR results showed that pre-treatment with FP7 and FP12 can prevent increased expression of all these markers. Starting from 48h of treatment, the decreasing trend is evident, confirming the capability of our compounds to decrease the fibrotic phenotype establishment (Figure 17b-d).

[0194] More in details, ACTA2, which is encoding for the contractile protein a-SMA, is a well-known indicator of fibroblast activation. From the data it is evident that starting from 24h, FP7 and FP12 administration prevents its expression, sustaining the idea that with these compounds we can prevent fibrotic phenotype induction, and this is maintained and more- 30 - SIB BW1373R

[0195] evident over time (Figure 17b-d). Additionally, starting from 48h, the other fibrotic markers such as FN1 and COL1A1 show decreased expression levels (Figure 17c-d).

[0196] Moreover, a strong indication arose from the decrease of the TGF-pi expression itself (Figure 17c-d), which clearly indicates the interruption of the TGFp-dependent positive feedback loop and therefore fibrosis progression.

[0197] To further confirm the impact of FPs treatment on the fibrotic phenotype, protein production was also considered at different levels, both from intracellular and extracellular perspectives. Moreover, we assess newly secreted collagen (i.e. soluble collagen) as well as the deposited one to have a comprehensive characterization of the anti-fibrotic effect of FPs compounds.

[0198] First, fibroblast activation was followed by monitoring the intracellular protein expression of a-SMA along with TLR4 modulation per se, by co-immunofluorescence staining (Figure 18a-d).

[0199] MRC5 fibroblasts subjected to the pre-treatment schedule and collected at 24, 48 and 72 hrs, were co-stained with aSMA and TLR4 antibodies and their protein expression was assessed by immunofluorescence detection via the high-content Operetta CLS imaging system (Perkin Elmer). For each condition, multistack imaging was performed and for each well, 69 regions of interest (ROIs) were acquired and analyzed. To obtain the correct protein quantification, the mean of the total fluorescence for each marker was normalized to the area of each individual cell analyzed.

[0200] Results show that both a-SMA and TLR4 levels increased in presence of the pro-fibrotic TGFp stimulus, and the administration of FP7 and FP12 compounds prevented this increment (Figure 18a-d). This result was appreciable for both markers. Indeed both a-SMA and TLR4 display an increased expression when cells are stimulated with TGF-pi , while their protein level is maintained at basal in TGFp -stimulated cells in the presence of FPs compounds (Figure 18b-d).

[0201] Importantly, this results underlines the importance and involvement of TLR4 in fibrotic phenotype progression. In fact, we detected an upregulation of TLR4 protein expression in TGFp -treated MRC5 lung fibroblasts, which is in line with what observed in the scRNA-seq dataset of IPF-associated human lung fibroblasts (Figure 11c).

[0202] To understand how long the inhibition of TLR4 has an impact on fibrosis, a wash-out experiment was conducted. MRC5 fibroblasts were subjected to the pre-treatment schedule (first FPs treatment and one hour later TGFp treatment) and introduced slight- 31 - SIB BW1373R

[0203] modifications to the duration of the TGFp or the FPs treatment. We applied four conditions (Figure 19a-c):

[0204] 1) Single stimulus: cells were treated once with FPs+TGFp and incubated with both compounds for 72 hours when they were harvested for subsequent analysis;

[0205] 2) Treatment every 24 hrs: FPs and TGFp were refreshed every 24 hours in the medium of the MRC5 cells until collection at 72 hours from the start of the first treatment;

[0206] 3) TGFp wash-out after 24 hrs: cells were treated with FPs+TGFp and 24 hrs after TGFp was removed and only FPs were left in the media for additional 48 hours;

[0207] 4) FPs wash-out after 24 hrs: cells were treated with FPs+TGFp and 24 hrs after FPs were removed and only TGFp was left in the media for additional 48 hours.

[0208] At 72 hours since the start of the first treatment, cells in all conditions were harvested for immunofluorescence staining with aSMA and Collagen I antibodies. Cells were imaged and quantified with the high-content Operetta imaging system.

[0209] Our data show that FP7 / FP12 treatment for 24 hours followed by 48 hrs of wash-out (condition 4) is sufficient to maintain a decrease of the aSMA and Collagen I intracellular levels compared to the standard schedule (condition 1) (Figure 19a-c). Such inhibition was also observed when both TGFp and FPs were refreshed in the cell medium every day (condition 2) (Figure 19a-c). Contrary to what expected, it was observed that refreshing TGF-pi every 24h did not further enhance the activation status of the MRC5 fibroblasts compared to cells receiving TGFp only once. This may be due to the fact that the chosen TGFp dose of 10 ng / ml allows to reach already the maximum activation capacity, therefore refreshing the TGFp every day did not lead to an additional benefit in terms of cell activation. Moreover, it emerged that only 24h of stimulus with TGF-pi followed by 48h washout is not enough to maintain a consistent activation of fibroblasts, which returned to their baseline status (Figure 19a-c).

[0210] The results of this in vitro experiment constitute a preliminary indication for the future in vivo treatment of mouse models of fibrosis, suggesting that, daily FP7 and FP12 treatment might not be required as the anti-fibrotic effect last at least for 48 hours.

[0211] 5. FP7 and FP12 treatment blocks ECM deposition

[0212] After assessing the fibroblast activation status at the intracellular level, the extracellular secretion and deposition of extracellular proteins have been investigated. In this step, the investigation was directed toward mature and soluble ECM production. In fact by performing Sircol assay, the effect of FP compounds has been measured on the soluble- 32 - SIB BW1373R

[0213] collagen secreted in the extracellular environment by MRC5 fibroblasts. In addition, by immunostaining we measured the deposited ECM fibers.

[0214] Subsequently, the deposition of ECM has been analyzed by utilizing the Flamingo™ fluorescence protein staining, which allows the fluorescence-based imaging of total deposited extracellular proteins. Imaging and quantification of the Flamingo staining clearly show increased ECM deposition in TGFp -treated MRC5 fibroblasts compared to untreated cells. The amount of deposited extracellular proteins is decreased by almost 50% when cells were pre-treated with the TLR4 inhibitors (Figure 20a-b).

[0215] To further confirm these data, we stained, imaged and quantified three ECM proteins abundantly expressed by fibrotic fibroblasts: Collagen I, Collagen III and Fibronectin. Examining all these ECM components via Operetta imaging system, it emerged that all are increased in presence of TGF-pi stimulus, while pretreatment with FP7 and FP12 leads to their decrease to basal or even lower levels (Figure 20c).

[0216] Taken together, these data underline the efficacy of FP7 and FP12 TLR4 antagonists in inhibiting the establishment of fibrosis in an in vitro experimental model of lung fibrosis. Moreover, the data indicate the efficacy of these compounds to regulate both extracellular secretion and deposition of the most important ECM proteins responsible for the scaring of the organ parenchyma.

[0217] 6. FP7 and FP12 treatment are effective in blocking TLR4 signaling

[0218] To obtain molecular insights into the intracellular signaling pathways affected by anti-TLR4 treatment, high-content fluorescence imaging analysis of the TLR4 and TGFp signaling pathways has been performed by using the Operetta Imaging system. The phosphorylated form of NF-KB (p-p65) has been exploited as downstream effector of TLR4 signaling activation, and the nuclear translocation of SMAD2 / 3 as readout of TGFp activation. The fluorescence intensity of the two targets in the nucleus was normalized to the total intensity detected in the entire cells to obtain the ratio of the translocated proteins.

[0219] To follow the translocation, multiple time points have been evaluated. The early time point at 30 minutes from the start of TGFp treatment gives us the readout of TGFp signaling pathway activation. The cells have been analyzed at 24, 48 and 72 hours from the start of the treatment to evaluate the activation of the TLR4 pathway and any secondary effect of the TGFp pathway itself.- 33 - SIB BW1373R

[0220] Obtained results indicate that TGF-pi induces the activation of SMAD2 / 3, as expected, with increased translocation of the protein complex into the nucleus (Figure 21a). While p-p65 at 30’ shows to be nuclear localized, with no reduction in presence of FP7 and FP12. Then this is reduced as similar level of vehicle, indicating that TGF-pi is not able to trigger directly TLR4 and its downstream pathway. However, at 72h from TGF-pi administration, p-p65 was found to be localized in the nucleus, indicating that TLR4 signaling is now actually activated (Figure 21b).

[0221] The nuclear presence of SMAD2 / 3 was reduced compared to 30’ time point but still increased compared to vehicle and non-treated cells at 72h, and increased compared to 24 and 48h time points were we did not observe the maintenance of nuclear SMAD2 / 3 (Figure 21a). Therefore we could speculate that the nuclear SMAD2 / 3 observed at 72h may represent the results of the positive feedback loop fibroblasts self-sustain to maintain and amplify TGFp signaling and therefore their fibrotic activation.

[0222] The later activation of p-p65, consequent to TGF-pi stimulus (Figure 21b), underlines the involvement of TLR4 in late fibrosis establishment that is due not to the direct effect of TGF-pi but to a feedback loop driven by DAMPs released by cells during ECM overproduction and remodeling.

[0223] In this contest emerged the capability of FP7 and FP12 compounds to inhibit both TLR4 activation, as demonstrated by the reduction of p-p65 nuclear translocation which returned to basal levels at 72h, and to inhibit the SMAD2 / 3 activation pathway at 72 hours, thus proving to block the feedback loop of fibroblasts activation and fibrosis perpetuation (Figure 21a-b).

[0224] Overall, these data, together with those previously obtained via RT-qPCR and high-content imaging as well as the mechanistic data obtained by the analysis of p-p65 and SMAD2 / 3 nuclear translocation, clearly indicated that TLR4 modulation with FPs compounds can break the positive feedback loop between TLR4 and TGF-pi and can act to prevent fibrotic phenotype onset.

[0225] 7. Blocking TLR4 by FP7 and FP12 antagonist limits already initiated fibrosis Since in the pre-treatment schedule compounds were so active and observing their capability to take action over time and modulate protein production and ECM modeling, it has been wondered if they could be effective also in reversing / blocking an already induced fibrotic phenotype.- 34 - SIB BW1373R

[0226] For this purpose, the treatment schedule was changed to a post-treatment setup. Here, after starvation, MRC5 cells were activated for 24h in presence of 10 ng / mL of TGF-pi . Then media was changed and FP7, FP12, and TGF-pi were administered (Figure 22a).

[0227] First, toxicity and proliferation were evaluated over time as done in pre-treatment. Thus, cells were treated with FP7 and FP12 5uM and harvested at 24, 48,72h from TGFp administration.

[0228] Data obtained clearly indicate that there is no cell death induction in all treatment conditions (TGFp stimulation or not, co-treatment with FP7 / FP12 or only FP7 / FP12 with no TGFp stimulation) as we did not observe any LDH release increment compared to the untreated control. This result was observed at all three time points analyzed for FP7 (Figure 22b). However, a small increment of LDH released seems to be observed at 72h for vehicle and FP12+TGFP (Figure 22b), probably due to extensive starvation, while FP7 and the other treatments did not perturb the system.

[0229] In parallel from the same plate it has also been performed the MTT assay to evaluate MRC5 cell viability over 72 hours and assess their impact in terms of cell proliferation. Data show, as observed in pre-treatment, that while TGF-pi promotes increased proliferation of MRC5 fibroblasts over time, this phenomenon is blocked by the FP7 and FP12 administration, as well as the positive control TAK242 (Figure 22c),

[0230] Thus, these results indicated that there is a proliferation block that is not dependent on increased cell death, indicating that post-treatment with FP7 and FP12 is effective and not toxic.

[0231] Taken together, the results of these assays indicate that FP7 and FP12 are safe and retain the ability to inhibit fibroblast proliferation over time also in a yet established fibrotic model.

[0232] 8. FP7 and FP12 treatment impeeds fibroblasts activation

[0233] The two critical aspects of fibrosis-associated fibroblasts have been investigated: acquisition of a myofibroblast phenotype and the production and deposition of extracellular matrix proteins in MRC5 cells subjected to the post-treatment schedule (Figure 23a). Specifically, the following fibrotic markers have been analyzed: ACTA2, FN1, COL1A1, and TGFB1 (Figure 23b-d). Following these markers over time, a comprehensive view of the transcriptional changes occurring during fibroblast activation in the presence of FP7 and FP12 treatment can be deduced.- 35 - SIB BW1373R

[0234] RT-qPCR results showed that post-treatment with FP7 and FP12 can block the increased expression of all the analyzed markers, although we are aware that additional replicates need to be added to clarify the inconsistent pattern of some of the markers (Figure 23b-d).

[0235] Indeed, as shown in the graphs presented in Figure 23b, at 24h there is a faint indication of fibrosis inhibition. It is appreciable mainly for ACTA2 expression, which starts to show a reduction trend. This is more evident at 48h and 72h where FP7 and FP12 treatment causes a clear drop in ACTA2 expression, sustaining the idea that with these compounds we can block the activation phenotype induction, and this is maintained and more evident over time (Figure 23c-d).

[0236] Additionally, starting from 48h, the other fibrotic markers present a reduction trend that becomes strongly evident at 72h of post-treatment (Figure 23c-d). In particular, this is true for FN1 and COL1A1 expression levels, in which there is a strong decreasing trend in the presence of both molecules FP7 and FP12.

[0237] Then, the intracellular protein level was also evaluated. Again, a-SMA was followed as a fibroblast activation marker, and in parallel, the expression of TLR4 was evaluated by Operetta high-content imaging system (Figure 24a).

[0238] Similarly to what was observed by RT-qPCR (Figure 23), modulation of protein expression is more evident at 48 and 72h post-treatment (Figure 24b-d). As observed in the pre-treatment schedule, both a-SMA and TLR4 expression are increased by TGF-P1 presence. This expression was found to be downregulated in presence of FP7 and FP12 compounds. In particular, at48h post-treatment, the reduction trend is visible and is stronger at 72h (Figure 24c-d). This indicates that not only is there mRNA modulation, but this is already appreciable at the protein level, underlining the ability of our compounds to reduce and partially revert the fibrotic-established phenotype, more similar to the effect that could be shown by drug administration in pathological situation.

[0239] To ensure that FP7 and FP12 are able to impact also at the extracellular level, soluble production and mature ECM deposition were also analyzed (Figure 25).

[0240] Subsequently mature deposited ECM was analyzed via Flamingo staining and individual staining of principal ECM components. Imaging and quantification of the Flamingo staining clearly show increased ECM deposition in TGFp-treated MRC5 fibroblasts compared to untreated cells. The amount of deposited extracellular proteins is- 36 - SIB BW1373R

[0241] decreased when cells were treated with the TLR4 inhibitors (Figure 25a-c). In this case results indicated a more effective action of FP12 on total ECM deposition and strong effect on single components. On the other hand, FP7 seems to be less effective, with more reducing action on COL III and FN than on COL I (Figure 25c) and total ECM (Figure 25a-c). This indicates that both compounds are able to restore basal ECM protein expression and deposition with strong effect of FP12 on mature ECM and higher action of FP7 on soluble collagen (Figure 25a-c). Additionally, looking at the TAK242 compound there is no reducing effect at every level (Figure 25a-b), underlining the higher power of our FPs compounds to revert established fibrosis phenotype compared to this commercial available TLR4 inhibitor.

[0242] Moreover, the data indicate the efficacy of these compounds to regulate both extracellular secretion and deposition of the most important ECM proteins responsible for the scaring of the organ parenchyma.

[0243] All these data obtained by MRC5 cell line in both pre-treatment and post-treatment schedules, together with information derived from WT versus Tlr4 “ mice, underline the capability of our compounds not only to prevent fibrotic phenotype upset but also to block / reverse its progression via TLR4-specific modulation. This demonstrates once again the involvement of TLR4 in fibrosis progression and supports its potential as a target for therapeutic intervention in lung fibrosis. This approach therefore offer a new direction for treating I PF and related fibrotic diseases.

[0244] 8. TLR4 is involved in intestinal fibrosis

[0245] In collaboration with KU Leuven University (Leuven, Belgium), TLR4 expression was evaluated in Professor Matteoli’s dataset of mesenchymal clustersobtained by scRNA-seq of full thickness intestinal specimens from Crohn’s Disease (CD) patients, comparing control, proximal, inflamed, and stenotic human intestinal segments.

[0246] From the data mining, it emerged that in unaffected intestinal segments TLR4 expression is more prominent in ADAMDEC1+ and in GREM1+ and GREM-CD34+fibroblasts, the latter being the precursors of the fibrosis-associated FAP+ fibroblasts. In the inflamed and fibrotic portions, TLR4 is expressed by the FAP+ fibroblast population (Figure 26).

[0247] This differential expression may underline the dual action of TLR4, which acts as a sentinel in the proximal region where it is more expressed in ADAMDEC1+ and GREM1+ / - non-fibrotic fibroblasts, while its increase in the FAP+ fibroblasts potentially- 37 - SIB BW1373R

[0248] indicates active involvement in fibrosis progression, considering that these fibroblasts have been demonstrated to be the drivers of fibrosis in CD.

[0249] Additionally, we performed an additional investigation by searching for TLR4 and its binding partner MD-2 (LY96) expression along different intestine portions (ileum, colon and rectum) in healthy, Crohn's disease (CD), and ulcerative colitis (UC) patients by mining the TAMMA database.

[0250] As observed in IPF, also at the gut level, the expression of TLR4 and MD-2 is increased in pathological conditions (Figure 27). In fact, TLR4 is increased in both UC and CD patients compared to control, in all three intestinal tracts (Figure 27a). MD2 was also found to be upregulated in UC and CD in all analyzed intestinal tracts (Figure 27b). Moreover, as the TAMMA database includes one CD-derived fibroblast dataset, we look forTLR4 and MD2 expression in this dataset of colon fibroblasts, comparing CD- versus healthy-derived cells. In this context, TLR4 is upregulated in CD-derived fibroblasts compared to healthy ones. MD2 also shows upregulation although to a less extent compared to TLR4 (Figure 27c).

[0251] All these bioinformatic data indicated that both MD-2 and TLR4 are expressed on intestinal fibroblasts and this is higher in pathological conditions that are strongly related to intestinal fibrosis presence. This evidence extends the interest for targeting TLR4 as anti-fibrotic drug in multiple organ fibrosis diseases.

[0252] 9. Efficacy of anti-TLR4 treatment in an in vitro experimental model of intestinal fibrosis

[0253] At this point, we decided to extend our investigation wondering whether our FPs compounds acting on TLR4 modulation could be also act on fibrosis at the intestinal level or, on the contrary, their action is lung-specific.

[0254] For this purpose, we tested FP7 and FP12 on the human colon fibroblast cell line CCD-18Co. As done for MRC5 fibroblasts, CCD-18Co subjected to the pre-treatment schedule where cells were seeded, then starved for 24h before the treatment with FPs one hour before TGFp stimulation. Then, RT-qPCR was exploited to evaluate transcript levels of fibrotic markers 48 hours after the start of the treatment (Figure 28a).

[0255] Data obtained confirmed the acquisition of the fibrotic phenotype by CCDI8-C0 cells induced by TGFp. This increase, on the other hand, is mitigated by the pre-treatment with FP7 and FP12 molecules (Figure 28b). Indeed, a marked reduction of COL1A1 and TGFB1 is evident in activated CCDI8-C0 fibroblasts pre-treated with FP7 and FP12 (Figure 28b).- 38 - SIB BW1373R

[0256] Afterwards, we employed an inflammatory stimulation of CCDI8-C0 which better resemble the pathological inflammatory setting present in CD patients. We utilized a mix of the following cytokines: oncostatin M, TGF-pi, IL-ip, IL-1a, TNF-a, IFN-y. We thus subjected CCDI8-C0 fibroblasts to the pre-treatment schedule applying this cytokine mix 1h after FPs treatment and evaluated the capability of FP7 and FP12 compounds to inhibit ECM production at 72h post-treatment by performing high-content Operetta imaging (Figure 28c-d).

[0257] In CCD-18C0 fibroblasts the cytokine mix induces a strong increase of total ECM production correlated with higher expression and deposition of single components COL I and COL III (Figure 28c-d). Having conducted the investigation on only two biological replicates so far, an inhibitory trend was observed by both compounds with generally higher impact of FP7 (Figure 28c-d). Additional replicates are definitely required to have a full picture and conduct a proper statistical analysis.

[0258] All these results indicate the capability of FP7 and FP12 to modulate the phenotypic fibrotic onset in these intestinal fibroblasts both in experimental conditions where stimulus is promoted by TGF-pi alone or in more real-world conditions induced by the cytokine mix. This indicates that our compounds, through modulation of TLR4, are able to influence fibrosis not only in fibroblasts that are lung-related but also in intestinal fibroblasts, underlining their multi-organ efficacy. These findings underscore the role of TLR4 in fibrosis progression and support its potential as a target for therapeutic intervention in fibrosis.

[0259] 10. FP7 and FP12 are effective in reducing the experimentally-induced activation status of human-derived intestinal fibroblasts

[0260] In addition to CCD-I8C0, we also evaluated FP7 and FP12 anti-fibrotic action on colon fibroblasts isolated from healthy individuals undergoing routine endoscopic screening for colon cancer risk. In these fibroblasts fibrosis was experimentally induced with treatment with 10 ng / mL TGF-pi. FP7 and FP12 compounds were administered at 5 pM for 1h and then TGF-pi stimulus was added (i.e. pre-treatment schedule, Figure 29a). Analysis was done on samples harvested at 48h and 72h post-stimulus via RT-qPCR.

[0261] Preliminary data indicate fibrotic activation of these healthy colon fibroblasts induced by TGF-pi stimulus, for all analyzed markers (Figure 29b-c). Here, it is evident at both 48h and 72h a reduction trend of mRNA fibrosis-related expression. In particular, at 48h of pre-treatment, a strong and significant reduction arose from FP12 administration, while at 72h, the reduction of ACTA2 is already statistically relevant.- 39 - SIB BW1373R

[0262] However, from all these analyses, it emerged thatTAK-242 in this context seems to be more efficient than our compounds (Figure 29b-c), differently from what was obtained in MRC5 and CCD-I8C0 cell lines. This is probably due to higher variability in response of patients to the treatment and the different interference that our compounds have compared to TAK-242. In fact, our compounds modulate TLR4 by acting on the external portion of the receptor, interacting in the TLR4 / MD-2 binding pocket, while TAK-242 interferes with TLR4 activation via an intracellular mechanism in which it prevents intracellular phosphorylation and consequent intracellular cascade.

[0263] Although these data certainly need to be integrated with additional human samples, they preliminary indicate the efficacy of our compounds also in this cell type with a reduction trend of fibrotic marker expression, confirming the observations obtained in CCD-18C0 cell line.

[0264] 11. Targeting TLR4 by ex-vivo treatment with FP7 is a promising strategy to reduce intestinal fibrosis

[0265] In addition to testing FP compounds on experimentally-activated human-derived intestinal fibroblasts, FP7 compound on pathological conditions have also been assessed. To do this, an ex vivo system based on ex-vivo culture of surgical resections of intestinal segments from CD patients has been exploited. Specifically the ex vivo culture was conducted on a specimen isolated from the surgically removed fibrotic area as well as from an unaffected (i.e. healthy) portion. These surgical samples were cut into small pieces and incubated in fibroblast media in the presence of 5 pM FP7 or its vehicle (DMSO+EtOH in a 1:1 ratio). After 24h of in vitro treatment, tissues were collected, homogenized and RNA extracted in order to perform RT-qPCR analysis for fibrotic markers.

[0266] From the first trial that we conducted, we obtained very promising preliminary results. The analysis in fact showed that our compound FP7 is able to decrease the expression of COL1A1 and TGFB 1 (Figure 30). This results indicates that FP7 is able to exert its antifibrotic function also in a more complex system which does not only contain a single pure cell population, such as fibroblasts, but the entire organ structure of intestine with its multiple cell types. We are now working in collaboration with the IBD Center of Humanitas Research Hospital to include additional samples, upon informed consent of the patients. If these preliminary results are confirmed, we will have a strong proof of FP7 efficacy to reduce fibrosis and its potential clinical application as therapeutic strategies for fibrotic complications in CD patients.- 40 - SIB BW1373R

[0267] Moreover, these results underline their potential as a therapeutic strategy to act on fibrosis, both in the lung disease as showed by the MRC5 model but also at the intestinal fibrotic disease. This approach may therefore offer a new direction for treating multiple fibrotic diseases.

[0268] 3.5 Discussion

[0269] The extensive set of results we have generated robustly supports the central role of TLR4, and its co-receptor MD-2, in fibrotic disease progression, specifically in idiopathic pulmonary fibrosis (IPF) and intestinal fibrosis contexts. The initial bioinformatic evidence extrapolated from different databases highlighted significant overexpression of TLR4 as well as MD-2 in fibrosis-associated mesenchymal cells, especially fibroblasts and myofibroblasts. The functional involvement of TLR4 in the fibrotic process of fibroblasts emerged from an investigation conducted on Tlr4 knock-out mouse lung fibroblasts which did not reach the same activation phenotype as WT fibroblasts when stimulated with TGFp. These data, together with previous published evidences, established a strong rationale for targeting the TLR4 / MD-2 complex pharmacologically.

[0270] Experimental validation using the MRC5 lung fibroblast model confirmed successful induction of a fibrotic phenotype via TGF-pi administration, demonstrated by sustained upregulation of canonical fibrotic markers (ACTA2, COL1A1, FN1, and TGF-pi) along with activation of inflammatory pathways (IL-6 increase together with IL-6R downregulation). ELISA and immunofluorescence corroborated transcript level findings with protein expression and ECM remodeling analyses, affirming the model's validity. TLR4 antagonist compounds FP7 and FP12, developed in Prof. Peri’s laboratory, were previously shown to be effective at targeting TLR4 and inhibiting its dependent inflammatory pathway. Here it has been investigated these FPs compounds for their capacity to impact the process of fibrosis. Collected data clearly show their capacity to effectively inhibit hallmark fibrotic features in activated fibroblasts by preventing TGF-pi-driven fibroblast proliferation, down regulating fibrotic gene expression, and normalizing production and extracellular deposition of ECM proteins. Pre-treatment experiments, in which FP7 or FP12 were administered one hour prior to TGFp treatment, demonstrated substantial inhibition of a-SMA and TLR4 upregulation, collagen secretion and various ECM proteins deposition, confirming their anti-fibrotic potency. Cytotoxicity, viability and dose-response studies indicated that FPs compounds showed optimal safety and efficacy at 5 pM dosage.

[0271] Mechanistic insights revealed that TGF-pi initiates early canonical SMAD2 / 3-mediated pathway activation, but TLR4-driven NF-KB (p-p65) nuclear translocation occurs 72- 41 - SIB BW1373R

[0272] hours later, indicative of a TLR4 activation phase driven by DAMPs released in the cell medium because of ECM overproduction. Both pathways were significantly suppressed by compounds FP7 and FP12, which therefore disrupt the positive feedback loop that fibrotic fibroblast establish where their activation status is associated with self-production of TGFp, which further sustains the fibrotic phenotype, including the release of ECM proteins that can act as DAMPs and activate the TLR4 signaling pathway. This way, FPs act by blocking fibrosis amplification and progression.

[0273] The washout experiments conducted revealed the capability of FPs to maintain a durable inhibitory effect on fibrosis markers even when removed after a short 24h treatment. This result lays the ground for the future in vivo testing of these compounds suggesting that a daily administration may not be required and intermittent dosing regimens in vivo without loss of efficacy could be exploited.

[0274] Post-treatment studies demonstrated the capacity of the FPs compounds to reduce already established fibrotic phenotypes by blocking proliferation and down regulating mRNA and protein levels of fibrosis markers. This represents a reversal of fibrosis progression closer to clinically relevant treatment scenarios, where intervention occurs after disease establishment. This underlines the central role of TLR4 in fibrotic phenotype progression and supports its validity as a therapeutic target.

[0275] Of note, in comparison with the commercially available TLR4 antagonist TAK242, the FPs compounds display increased efficiency in reducing the fibrotic phenotype of lung fibroblasts and in blocking the extracellular release of Collagens and TGFp, both in pre-and in post-treatment regimen.

[0276] Extending this approach to intestinal fibrosis, studies in the human colon fibroblast CCD-18Co cell line and primary intestinal fibroblasts from healthy individuals validated the broad applicability of TLR4 modulation in fibrosis beyond the lung. Both TGF- i alone and a pro-inflammatory cytokine mix induced fibrotic phenotypes that were effectively inhibited by FPs compounds.

[0277] Preliminary ex vivo experiments on a patient-derived intestinal tissue reinforced the translational potential of these compounds, demonstrating the anti-fibrotic activity of FP7 in complex organotypic environments beyond monoculture systems. These results indicate that FP7 can reduce fibrosis not only when applied at cell-specific level but also when administered to the whole tissue. Although this preliminary result has to be integrated and confirmed by increasing the sample size, the obtain data represents a positive encouragement to the pre-clinical advancement of the FPs molecules.

[0278] Together, these data comprehensively establish TLR4 / MD-2 as key drivers and therapeutic targets in fibrosis. Compounds FP7 and FP12 exhibit promising- 42 - SIB BW1373R

[0279] pharmacological profiles by modulating both fibroblast activation and ECM dynamics in lung and intestinal fibrosis models, indicating their multi-organ capability to act on fibrotic processes. These findings support advancing these molecules into in vivo preclinical evaluation and further development as novel anti-fibrotic agents. This approach offer therefore a new direction for treating I PF, intestinal fibrosis, and potentially other fibrotic diseases.

[0280] By investigating the novel antagonist compounds FP7 and FP12, developed in Prof. Peri’s lab, it not only confirms the ability to pharmacologically inhibit TLR4 / MD-2 but also demonstrates superior potency compared with the established TLR4 antagonist TAK242 in multiple preclinical models. The inclusion of ex vivo assays using patient-derived intestinal tissue represents a key translational step showing efficacy beyond cell monocultures, highlighting multi-organ anti-fibrotic potential. Of particular novelty, this work reveals detailed temporal pathway interplay between TGF-p canonical SMAD signaling and later TLR4-driven NF-KB activation driven by ECM-derived DAMPs, uncovering a fibrotic positive feedback loop disrupted by these novel compounds. Together, these innovations provide new mechanistic and translational insights while substantiating and extending current understanding of TLR4 / MD-2 roles in fibrosis.

[0281] The present experimental work demonstrated that small-molecule TLR4 antagonists, FP7 and FP12, markedly attenuate TGF-pi -induced fibroblast activation and ECM deposition in human pulmonary and intestinal fibroblasts, mirroring pathogenic processes observed in idiopathic pulmonary fibrosis (I PF) and Crohn's disease. These compounds efficiently inhibited nuclear translocation of phosphorylated NF-KB and SMAD2 / 3, thereby disrupting the profibrotic feedback loop activated by ECM-associated damage-associated molecular patterns (DAMPs) triggering TLR4. The reduction in classical fibrosis markers ACTA2, COL1A1, and FN1, alongside diminished soluble collagen and ECM deposition, highlights robust anti-fibrotic effects without compromising cell viability. These results indicate that via TLR4 modulation, it is possible to effectively prevent and partially reverse the fibrotic established phenotype. Consistent with transcriptomic analyses, TLR4 and its co-receptor MD-2 are markedly upregulated in mesenchymal cells from fibrotic lung and intestinal tissues, reinforcing their pathogenic involvement and underlining their potential as therapeutic targets.

Claims

- 43 - SIB BW1373RCLAIMS1. A compound of formula I:Formula Iwherein R1is a Cn or C13 saturated alkyl group, and wherein R2is a C11 or C13 saturated alkyl group,for use in the treatment of a fibrotic disease in a patient in need thereof.

2. The compound for use according to claim 1, wherein R1=R2.

3. The compound for use according to any one of claims 1 or 2, wherein R1=R2= Cn saturated alkyl group, or R1=R2= C13 saturated alkyl group.

4. The compound for use according to any one of claims 1 to 3, wherein at least one between R1and R2is free of any -OH substituent in position 2.

5. The compound for use according to any one of claims 1 to 4, wherein at least one between R1and R2is free of any substituent.

6. The compound for use according to any one of claims 1 to 5, wherein said compound is an a anomer of the compound of formula I .

7. The compound for use according to any one of claims 1 to 5, wherein said compound is a p anomer of the compound of formula I .

8. The compound for use according to any one of claims 1 to 7, wherein said fibrotic disease is intestinal fibrosis, idiopathic pulmonary fibrosis (IPF) and Crohn’s Disease.

9. A pharmaceutical composition comprising a compound as defined in any of claims 1 to 8 and at least one pharmaceutically acceptable carrier or excipient, for use in the treatment of a fibrotic disease in a patient in need thereof.- 44 - SIB BW1373R10. The pharmaceutical composition for use according to claim 9, in the form of a suspension, emulsion, ointment, cream, spray, granules, powder, solution, capsule, pill, tablet, freeze-dried product, lozenge, aerosol, nebulization or injection.

11. The pharmaceutical composition for use according to claim 9 or 10, in a form suitable for oral, parenteral, nasal, aerosol, sublingual, rectal, vaginal, topic or systemic administration.

12. The pharmaceutical composition for use according to any one of claims 9 to 11, further comprising one or more APIs.

13. The pharmaceutical composition for use according to claim 12 wherein said one or more further APIs are seleceted from Pirfenidone, Nintedanib, Colchicine, Glucocorticoids, Vitamin D Analogues, TGF-p Inhibitors, Antisense Oligonucleotides, Cilengitide, Bromodomain and Extra-Terminal Inhibitors, Cysteamine , Fibroblast Growth Factor 21 Agonists, N-Acetylcysteine, Autophagy Modulators, Farnesoid X Receptor Agonists.

14. The pharmaceutical composition for use according to any one of claims 9 to 13, wherein said fibrotic disease is intestinal fibrosis, idiopathic pulmonary fibrosis (I PF) and Crohn’s Disease.

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