Use of biomarkers in treatment of fibrotic conditions with PDE4B inhibitors

Abstract

A PDE4-inhibitor # imgabs0 # of formula A 'is used in a method for treating progressive fibrotic interstitial lung disease (PF-ILD), preferably IPF, in a patient, comprising the steps of: a) measuring or having measured the concentration, expression level or activity of one or more biomarkers in serum or plasma of a blood sample obtained from said patient, the present invention relates to biomarkers selected from the group consisting of Krebs von denLungen protein (KL-6), pulmonary surfactant protein D (SP-D), matrix lysolytic factor (MMP7), CA-125 (also known as MUC-16), CA19-9, E-selectin, sICAM-1, interstitial lysolytic factor (MMP3), osteopontin (OPN), connective tissue growth factor (CTGF), cartilage oligomeric matrix protein (COMP), prostate protein, von Willebrand factor (vWF), and C-reactive protein (CRP), b) comparing or having been compared the concentration, expression level or activity of the one or more biomarkers listed in step a) with a reference concentration, expression level or activity of the corresponding one or more biomarkers in a serum or plasma sample of the patient, c) determining or having determined that the concentration, expression level or activity of the respective one or more biomarkers listed in step a) is modified compared to the respective reference concentration, expression or activity of the respective one or more biomarkers, d) administering to the patient a therapeutically effective amount of the PDE4-inhibitor of formula A '.

Description

1. Background Technology

[0001] 1.1 Phosphodiesterase and its role in fibrosis

[0002] Phosphodiesterase (PDE) mediates the hydrolysis of the second messenger cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate. PDE is encoded by 11 gene superfamilies, which contain multiple genes (encoding subtypes A, B, C, etc.), and the gene superfamily also produces alternative mRNA splicing variants that lead to approximately 100 PDE isoforms. PDE4 is traditionally associated with the regulation of inflammation and the modulation of immunocompetent cells, and the three currently available selective PDE4 inhibitors support the beneficial effects of PDE4 inhibitors in inflammatory and / or autoimmune diseases (Sakkas et al., 2017, Curr. Med. Chem. 24, 3054-3067; Li et al., 2018, Front. Pharmacol. 9, 1048). Oral Roflumilast, a first-of-its-kind PDE4 inhibitor It was approved by the U.S. Food and Drug Administration in 2011 for reducing the risk of COPD exacerbations in patients with severe COPD who have chronic bronchitis and a history of acute exacerbations (U.S. Food and Drug Administration, 2013, Another compound, Apremilast, is taken orally. Approved in 2014 for the treatment of psoriatic arthritis and plaque psoriasis (U.S. Food and Drug Administration, 2017, (Apremilast) and was approved for Behcet's disease in 2019. The third PDE4 inhibitor, Crisaborole Approved in 2016 for the topical treatment of mild to moderate atopic dermatitis (U.S. Food and Drug Administration, 2016, EUCRISA TM (crisaborole). None of these showed any preferential enzyme inhibition among the four PDE4 subtypes of AD.

[0003] The general anti-inflammatory potential of PDE4 inhibition, exemplified by Roflumilast, has been well documented (Hatzelmann et al., 2010, Pulm.Pharmacol.Ther.23,235-256), and the use of PDE4 inhibitors in various inflammatory and immune-mediated diseases has been extensively studied (Sakkas et al., 2017, Curr.Med.Chem.24,3054-3067; Li et al., 2018, Front.Pharmacol.9,1048). However, in the past decade, based on animal studies and in vitro experiments evaluating the functional effects of PDE4 inhibitors in fibroblasts, it has become increasingly clear that PDE4 may also play an important role in fibrosis. PDE4 inhibitors have been shown to attenuate pulmonary fibrosis under various experimental conditions, most widely in bleomycin-induced fibrosis in rodents. In a rat model, rolipram was shown to inhibit fibrosis scores, hydroxyproline content, and serum tumor necrosis factor-α (TNF-α) (Pan et al., 2009, Respirology 14, 975-982). In this initial study, the PDE4 inhibitor was administered from the beginning of the bleomycin challenge, so it was unclear whether rolipram was active primarily due to inhibition of initial inflammation or inhibition of secondary fibrosis. However, a second early study conducted in mice and rats showed that oral roflumilast was active in both preventive and therapeutic regimens in a dose-dependent manner (Cortijo et al., 2009, Br. J. Pharmacol. 156, 534-544). In lung extracts, roflumilast inhibited histologically assessed fibrosis, hydroxyproline content, and mRNA expression of TNF-α, transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), α1 collagen, endothelin-1, and mucin 5ac. In bronchoalveolar lavage fluid (BALF), levels of TNF-α, interleukin (IL)-13, TGF-β, and mucin 5ac, lipid hydroperoxide formation, and influx of inflammatory cells such as neutrophils and macrophages were inhibited. In addition to fibrosis, roflumilast also had a positive effect on right ventricular hypertrophy and vascular remodeling (pulmonary artery). The same group later demonstrated that the metabolome associated with lung fibrosis in bleomycin-treated mice was modulated by roflumilast. Roflumilast reduced levels of the amino acids (AA) glycine and proline involved in collagen formation / structure, while lung glutathione and plasma tetrahydrobiopterin increased, indicating that roflumilast altered the oxidative balance (Milara et al., 2015, PLoS One 10, e0133453).Cilomilast, another PDE4 inhibitor, was shown to inhibit advanced lung fibrosis and tend to reduce collagen content in bleomycin-treated mice, although no effects were found on TGF-β1 and collagen type (Col) 1A1 expression (Udalov et al., 2010, BMC Pulm. Med. 10, 26).

[0004] Improvement of pulmonary fibrosis by PDE4 inhibition is not limited to the bleomycin model. In a mouse model of pulmonary fibrosis targeting type II alveolar epithelial cells in transgenic mice expressing diphtheria toxin receptor under the control of the mouse surfactant protein C promoter, roflumilast reduced lung hydroxyproline content and TNF-α, fibronectin (FN) and CTGF (Sisson et al., 2018, Physiol. Rep. 6, e13753). Interestingly, roflumilast is active in both prevention and treatment regimens, and under the latter condition, roflumilast appears to be therapeutically equivalent to pirfenidone and nintedanib. In addition, in a chronic graft-versus-host disease mouse model, oral roflumilast can reduce pulmonary fibrosis (Kim et al., 2016, Exp. Hematol. 44, 332-341. e334). Roflumilast inhibits fibrosis, collagen deposition, hydroxyproline and TGF-β1 content, cell infiltration, and IL-6 and IL-1β mRNA expression. In addition, in BALF inflammatory cells (macrophages, lymphocytes, neutrophils and eosinophils), IL-6, IL-1β and monocyte chemoattractant protein-1 are inhibited by Roflumilast. In a rabbit tuberculosis model, lung injury and fibrosis are inhibited by two PDE4 inhibitors CC-3052 (Subbian et al., 2011, Am. J. Pathol. 179, 289-301) and CC-11050 (Subbian et al., 2016, EBioMedicine 4, 104-114) from Celgene. PDE4 inhibition improves antibiotic treatment and pulmonary fibrosis by actively affecting collagen deposition and mRNA expression of various matrix metalloproteinases (including matrix metalloproteinase 12).

[0005] In addition to the lung, the beneficial effects of PDE4 inhibition on fibrosis have been confirmed in several other organs including skin, liver, kidney and colon. For example, in various preclinical mouse models of SSc, rolipram and apremilast inhibited skin fibrosis and chronic graft-versus-host disease induced by bleomycin or topoisomerase I (Maier et al., 2017, Ann. Rheum. Dis. 76, 1133-1141). The group did not find a direct inhibitory effect of PDE4 inhibition on the release of profibrotic cytokines (IL-6, IL-13, TGF-β1 / β2) in fibroblasts and M2 macrophages purified from peripheral blood of SSc patients, which may be due to the lack of exogenous cAMP triggering under the experimental conditions used. In the obstructive nephropathy model induced by unilateral ureteral obstruction in mice, rolipram was shown to inhibit renal interstitial fibrosis (Ding et al., 2017, Antioxid. Redox Signal. 29, 637-652). In vitro, TGF-β upregulated PDE4A / B in primary mouse renal tubular epithelial cells, and rolipram inhibited TGF-β-induced damage, FN expression, and mitochondrial biogenesis defects. Roflumilast inhibited diethylnitrosamine-induced rat liver fibrosis, hydroxyproline deposition, and TGF-β1 expression (Essam et al., 2019, Life Sci. 222, 245-254). Similarly, in a rat bile duct ligation-induced liver fibrosis model, rolipram inhibited collagen deposition, α-smooth muscle actin (α-SMA) staining and mRNA expression, and TGF-β1 mRNA and TNF-α protein expression, accompanied by upregulation of PDE4A, B, and D (Gobejishvili 2019). In hepatic stellate cells in vitro, rolipram inhibited the mRNA expression of α-SMA and Col1A2 (Gobejishvili et al., 2013, J. Pharmacol. Exp. Ther. 347, 80-90). For colonic tissue, rolipram inhibited collagen and TGF-β1 in a rat colitis model induced by trinitrobenzene sulfonic acid (Videla et al., 2006, J. Pharmacol. Exp. Ther. 316, 940-945), and apremilast inhibited colonic fibrosis, collagen deposition, and expression of fibrosis-related genes in a mouse ulcerative colitis model induced by sodium dextran sulfate (Li et al., 2019, Br. J. Pharmacol. 176, 2209-2226). In a murine cecal abrasion model, rolipram inhibited the fibrotic response, suggesting that PDE4 inhibition has the potential to prevent postoperative intra-abdominal adhesions (Eser et al., 2012, Dis. Colon Rectum 55, 345-350). Adhesions are thought to result from abnormal healing after laparotomy.To support this hypothesis, in mouse subcutaneous or intraperitoneal polyether-polyurethane sponge implant models, rolipram showed activity by inhibiting collagen and TGF-β1 deposition in implants (Mendes et al., 2009, Microvasc. Res. 78, 265-271). Therefore, in various animal models, it has been demonstrated that selective PDE4 inhibition has beneficial effects on fibrosis, most widely in the lungs, but also in several other organs. Although the specific targets of PDE4 inhibitors in fibrotic diseases are largely unknown, it is easy to speculate that they act indirectly by inhibiting proinflammatory cells and mediators, and / or act directly by inhibiting typical effector cells (fibroblasts, myofibroblasts) that mediate fibrosis.

[0006] 1.2 Progressive fibrosing interstitial lung disease (PF-ILD)

[0007] Interstitial lung disease (ILD) includes a group of heterogeneous lung diseases affecting the interstitium, which is different from obstructive airway diseases (such as asthma or chronic obstructive pulmonary disease (COPD)). Prolonged ILD may lead to pulmonary fibrosis, but this is not always the case. The most widely studied ILD is idiopathic pulmonary fibrosis (IPF), which is characterized by progressive pulmonary fibrosis. Non-IPF ILD may include connective tissue disease-related ILD, such as ILD related to rheumatoid arthritis and other autoimmune diseases, systemic sclerosis-related ILD (SSc-ILD) and polymyositis / dermatomyositis, and ILD (Cottin et al., Eur. Respir. Rev. 28, 180100) related to chronic sarcoidosis, chronic hypersensitivity pneumonitis, idiopathic nonspecific interstitial pneumonia and exposure-related diseases (such as asbestosis and silicosis); Kolb, M., and Vasakova, M. (2019), Respir. Res. 20, 57). Up to 40% of these ILD patients may develop a progressive fibrotic phenotype.

[0008] Progressive fibrosis ILD is associated with high mortality, and the median post-diagnosis survival of IPF patients is estimated to be 2-5 years (Raghu, G., Chen, SY, Yeh, WS, Maroni, B., Li, Q., Lee, YC, and Collard, HR (2014). Idiopathic pulmonary fibrosis in US Medicare beneficiaries aged 65years and older: incidence, prevalence, and survival, 2001-11. Lancet Respir. Med. 2, 566-572). The progression of fibrosis ILD is reflected in various parameters, including decreased lung function, decreased exercise capacity, worsening quality of life, worsening cough and dyspnea, acute exacerbation, and increased morphological abnormalities (Cottin et al., Eur. Respir. Rev. 28, 180100, 2019; Kolb and Vasakova, 2019, Respir. Res. 20, 57). In patients with IPF, forced vital capacity (FVC) is a well-established predictor of mortality, and acute exacerbations are associated with very high mortality. Although corticosteroids and / or immunosuppressive drugs are sometimes used off-label to treat progressive fibrosing ILD, the only approved treatments to slow disease progression in IPF are nintedanib and pirfenidone (Richeldi et al., 2018, Eur. Respir. Rev. 27, 180074). Nintedanib has been approved in the United States since 2014 (U.S. Food and Drug Administration, 2020, (nintedanib)), and since 2015 in Europe and Japan (European Medicines Agency, 2021b, (nintedanib)) was approved in 2011, while pirfenidone was approved in Japan in 2008, in Europe in 2011 (European Medicines Agency, 2021a, Esbriet (pirfenidone)) and in the United States in 2014 (U.S. Food and Drug Administration, 2019, Lung transplantation is the only potentially curative treatment for IPF, and the medical needs for IPF and other progressive fibrosing ILDs remain high.

[0009] However, in IPF patients with mild or moderate impairment of FVC (≥50% predicted value), the currently approved drugs pirfenidone and nintedanib can only reduce the decline in FVC, which is consistent with the slowing of disease progression, but neither can prevent or even reverse or cure the symptoms of IPF (Tzouvelekis et al., Ther. Clin. Risk Management 2015, 11, 359-370).

[0010] Nonetheless, both treatment options, pirfenidone or nintedanib, showed significant beneficial effects in slowing disease progression in IPF.

[0011] The most significant side effects associated with both nintedanib and pirfenidone are gastrointestinal events, particularly diarrhea, nausea, vomiting, abdominal pain, decreased appetite, and weight loss. In the event of gastrointestinal side effects, they are usually managed by interruption of treatment, dose reduction, or symptomatic treatment of gastrointestinal side effects (see Mazzei et al., Ther. Adv. Respir. Dis. 2015, Vol. 9 [3], pp. 121-129).

[0012] Due to these "cumulative gastrointestinal side effects" of pirfenidone and nintedanib, the combination of pirfenidone and nintedanib for the treatment of IPF is not often used. Studies have shown that the combination of pirfenidone and nintedanib leads to increased gastrointestinal side effects, especially diarrhea, nausea, vomiting and upper abdominal pain (Vancheri et al., nintedanib with Add-onpirfenidone in Idiopathic Pulmonary Fibrosis: Results of the INJOURNEYTrial. Am J Respir Crit Care Med. 2018, Feb 1; 197 (3): 356-363).

[0013] Therefore, since the two active agents pirfenidone and nintedanib approved for the treatment of IPF to date cannot prevent or cure IPF when administered alone, but can only slow down IPF disease progression to a certain percentage (Tzouvelekis et al., Ther. Clin. Risk Management 2015, 11, 359-370), and since both nintedanib and pirfenidone also show significant gastrointestinal side effects, which are cumulative when the two compounds are combined, there is still a significant medical need for improved treatment methods for IPF / PF-ILD.

[0014] 1.3 Biomarkers in PF-ILD and IPF

[0015] Biomarkers are defined as indicators of normal biological processes, pathogenic processes (e.g., predictive biomarkers), or responses to exposure or interventions (including therapeutic interventions) (e.g., pharmacodynamic biomarkers, outcome-related biomarkers). The sources of biomarkers that can inform the diagnosis, outcome, and treatment response of PF-ILD include peripheral blood, airways, and lung parenchyma. Peripheral blood is easy to obtain, and except for phlebotomy, almost no training is required to obtain it. As Bowman et al., Front.Med.8:680997, doi:10.3389 / fmed.2021.680997. Many diagnostic interstitial lung disease (ILD) biomarkers, such as CA 19-9, CA-125, sICAM-1, etc., distinguish different subtypes of ILD from non-ILD controls. In addition, Stainer et al., Int.J.Mol.Sci.2021,22,6255 focus more on idiopathic pulmonary fibrosis (IPF), and show evidence that IPF has different clinical phenotypes, characterized by a course that changes over time. Therefore, diagnosis, prognosis and therapeutic diagnostic biomarkers (outcome-related or pharmacodynamic biomarkers) can be useful tools to help promote IPF diagnosis, monitor IPF disease progression and therapeutic efficacy.

[0016] 1.4 Prior Art

[0017] In addition to the approved PDE4 inhibitors roflumilast and apremilast, many patent applications based on other PDE4 inhibitors with improved properties have been published:

[0018] - Pteridines as PDE4-inhibitors in WO 2006 / 056607, WO 2006 / 058869, WO 2006 / 058868 and WO 2006 / 058867.

[0019] - Piperazinyl-dihydrothienopyrimidines as PDE4-inhibitors in WO 2006 / 111549, WO 2007 / 118793 and WO 2009 / 050242.

[0020] - Piperidino-dihydrothienopyrimidines as PDE4-inhibitors in WO 2009 / 050248 and WO 2013 / 026797.

[0021] PDE4-inhibitor of formula A

[0022]

[0023] Where S* is the sulfur atom representing the chiral center,

[0024] And in particular a PDE4-inhibitor of formula A'

[0025]

[0026] Wherein (R) at the sulfur atom represents the chiral center at the sulfur atom in the R configuration,

[0027] It has been disclosed in WO 2013 / 026797 and proposed as a new treatment option for a variety of diseases including IPF. It has been shown that the PDE4-inhibitors of formula A preferentially inhibit the PDE4B isoform.

[0028] WO2019 / 081235 discloses a combination of nintedanib and a PDE4-inhibitor of formula A (particularly formula A') for the treatment of PF-ILD, preferably IPF, which shows a synergistic over-additive effect on fibroblast proliferation in an in vitro assay using human lung fibroblasts.

[0029] EP 21218202.6 discloses novel pharmaceutical compositions of PDE4-inhibitors of formula A and in particular of formula A' in combination with novel dosage regimens.

[0030] EP 21218207.5 discloses novel combinations of PDE4 inhibitors of the formula A and in particular of the formula A'.

[0031] However, none of the above prior art discloses any fibrosis-related biomarkers, the level, concentration or expression of which can be affected by treatment with a PDE4-inhibitor of formula A' (= "biomarkers with pharmacodynamic potential"), or which are "outcome-related" (which means that these biomarkers vary depending on the treatment outcome / efficacy of a PDE4-inhibitor of formula A').

[0032] Regarding fibrosis-related biomarkers, from Zhang et al., Curr Opin Pulm Med 212; 18(5): 441-446, high blood concentrations of KL-6 (also known as MUC1) have been shown to be a predictor of reduced survival in IPF, and high plasma concentrations of MMP-7, sICAM-1, and IL-8 are predictors of poor overall survival in IPF patients.

[0033] It is described in Stainer et al., Int J Mol Sci 2021, 22, 6255 that different variants of surfactant proteins in serum, such as SP-A and SP-D have been identified as diagnostic markers in IPF: for example, serum levels of SP-D in IPF patients and other non-IPF-ILDs are higher than those in healthy controls, and further KL-6 is increased in the serum of patients with several ILDs including IPF.

[0034] However, Zhang et al., Curr Opin Pulm Med 212; 18 (5): 441-446 or Stainer et al., Int J Mol Sci 2021, 22, 6255 do not describe whether and / or to what extent certain standard of care IPF therapeutic agents (such as nintedanib or pirfenidone) affect the plasma or serum concentration of the above-mentioned biomarkers, or whether the treatment results of nintedanib or pirfenidone treatment selection affect certain biomarker levels. In particular, in the articles of Zhang et al. and Stainer et al. above, there is no mention or discussion of the possible effects of new potential IPF therapeutic agents (such as PDE4-inhibitors of formula A') on fibrosis-related biomarkers, so it is completely unknown whether certain known fibrosis-related biomarkers show "pharmacodynamic potential" for new IPF or PF-ILD drugs (such as PDE4-inhibitors of formula A'). Furthermore, the above-mentioned prior art documents do not explain whether specific fibrosis-related biomarkers show "outcome-related potential" during treatment with specific novel PF-ILD or IPF drugs (eg PDE4-inhibitors of formula A').

[0035] In the Phase 2 study NCT04419506, the PDE4-inhibitor of formula A' was tested versus placebo in IPF patients without background treatment with antifibrotic drugs ("non-AF background") and in IPF patients receiving background treatment with antifibrotic drugs selected from nintedanib or pirfenidone ("AF background"). In both IPF patient groups - in the "non-AF background" group and the "AF background" group - those treated with 18 mg of the PDE4-inhibitor of formula A' twice daily showed a significant slowing of the decline in lung function ("forced vital capacity" (FVC)) compared to patients who received placebo.

[0036] The concentrations / levels of certain fibrosis-related biomarkers and other biomarkers in the plasma or serum of patients in the Phase 2 study were further analyzed.

[0037] The results of these analyses, as described in detail in Section 3.2.3.2 of the present application, show that in the "non-AF group", the blood concentrations / levels of the biomarkers KL-6, SP-D, CA-125 (MUC-16 or mucin-16), CA19-9, matrix lytic factor (= MMP7), COMP, prostasin, E-selectin and vWF in IPF patients who have been treated with the PDE4-inhibitor of formula A' decreased over time compared to IPF patients who have received placebo (see Tables 3 and Figure 6 , 8, 10, 14, 18, 20, 22, 24, 28). In addition, these reduced blood concentrations / levels of the above biomarkers in IPF patients treated with the compound of Formula A' occurred simultaneously with a significantly slower decline in lung function compared to IPF patients who had received placebo (see Figure 1 ).

[0038] Furthermore, it could be shown that in the "non-AF group" the blood concentration / level of the fibrosis-related biomarker C-reactive protein (CRP) increased over time in IPF patients who had been treated with a PDE4-inhibitor of formula A' compared to IPF patients who had received a placebo instead (see Tables 3 and Fig.26 In addition, the increase in CRP blood concentrations / levels in IPF patients treated with the compound of formula A' occurred concurrently with a significantly slower decline in lung function compared to IPF patients who had received placebo (see Figure 1 ).

[0039] These observations may indicate that hitherto untreated IPF patients who exhibit in their serum or plasma a biomarker selected from the group consisting of KL-6, SP-D, CA-125, CA19-9, matrix dissolving factor (MMP-7), COMP, prostate protein, E-selectin and vWF at high concentrations / levels exceeding a certain reference concentration / threshold level may particularly benefit from treatment with a PDE4-inhibitor of formula A'.

[0040] Furthermore, these observations may indicate that hitherto untreated IPF patients showing low concentrations / levels of CRP in their serum or plasma - in particular when a certain reference concentration / level is not reached - may particularly benefit from treatment with a PDE4-inhibitor of formula A'.

[0041] In addition, the results of the analysis as described in Section 3.2.3.2 of the present application showed that in the "AF group" (the group that had received background antifibrotic drugs of nintedanib or pirfenidone), the blood concentrations / levels of the biomarkers KL-6, SP-D, matrix dissolution factor (= MMP7) and E-selectin in those IPF patients that had been treated with the PDE4-inhibitor of formula A' decreased over time compared to those IPF patients that had received placebo instead (see Tables 4 and Figure 7 , 9 , 15, 29).

[0042] Furthermore, it can be seen that in the "AF group", the blood concentration / level of the fibrosis-related biomarker C-reactive protein (CRP) in IPF patients who have been treated with the PDE4-inhibitor of formula A' has increased over time compared to IPF patients who have received placebo instead (see Tables 4 and Fig. 27 ).

[0043] These observations may indicate that IPF patients who are already receiving background treatment with antifibrotic drugs (using nintedanib, pirfenidone or any pharmaceutically acceptable salt thereof) and who exhibit biomarkers selected from KL-6, SP-D, matrix dissolving factor (MMP-7) and E-selectin in their serum or plasma (particularly when a certain threshold / reference concentration / level / activity is exceeded) may particularly benefit from treatment with a PDE4-inhibitor of formula A' (alone or in combination with background treatment with an antifibrotic drug selected from nintedanib or pirfenidone).

[0044] Furthermore, these observations may indicate that IPF patients who are already receiving background treatment with antifibrotic drugs (using nintedanib, pirfenidone or any pharmaceutically acceptable salt thereof) and who exhibit low concentrations / levels of CRP in their serum or plasma, in particular when a certain threshold / reference concentration / level / activity is not reached, may particularly benefit from treatment with a PDE4-inhibitor of formula A' (alone or in combination with background treatment with an antifibrotic drug selected from nintedanib or pirfenidone).

[0045] Fig.30 It was shown that, in particular, the fibrosis-related biomarkers KL-6, SP-D and MMP-7 (matrilytic factor) were reduced during treatment with the PDE4-inhibitor of formula A', which therefore shows "pharmacodynamic potential" in IPF patients for treatment with the PDE4-inhibitor of formula A' (regardless of whether the patients were additionally subjected to background treatment with antifibrotic drugs). In addition, Fig.30 C-reactive protein (CRP) was shown to increase during treatment with PDE4-inhibitors of formula A', thus showing "pharmacodynamic potential" for treatment with PDE4-inhibitors of formula A' in IPF patients (regardless of whether the patients were additionally undergoing background treatment with antifibrotic drugs).

[0046] Fig.31 It was shown that, in particular, only in the placebo group of IPF patients, the baseline levels of KL-6, SP-D, CA-125 (= mucin-16), CA19-9 and sICAM-1 were negatively correlated with the decrease in lung function from baseline, measured as the change from baseline in FVC (CfB) and the change from baseline in Dlco% predicted value (pred.) (CfB) at week 12. Therefore, Fig.31 It was shown that, in particular, KL-6, SP-D, CA-125 (= Mucin-16), CA19-9 and sICAM-1 showed a "predictive potential" for IPF progression.

[0047] Fig.32It was further shown that in IPF patients during treatment with the PDE4-inhibitor of formula A', in particular in the "non-AF group" and the "AF group", changes in the levels of the biomarkers KL-6, SP-D, E-selectin and sICAM-1 were shown to be associated with the change from baseline in forced vital capacity (FVC) at week 12 (CfB) and the change from baseline in the diffusion capacity of the lung for carbon monoxide or transfer factor (Dlco) (CfB). Therefore, Fig.32 It was shown that, in particular, the biomarkers KL-6, SP-D, E-selectin and sICAM-1 have "outcome-related potential" during treatment with the PDE4-inhibitor of formula A'.

[0048] One can conclude from these biomarker analyses of the Phase 2 trial of the PDE4-inhibitor of formula A' that in particular those biomarkers with "pharmacodynamic potential", i.e. KL-6, SP-D and MMP-7, and those biomarkers with "predictive potential", i.e. KL-6, SP-D, CA-125, CA19-9 and sICAM-1, and those biomarkers showing a potential association with "outcome" (FVC and Dlco% predicted values ​​at week 12), i.e. KL-6, SP-D, MMP-7, E-selectin and sICAM-1, are preferably suitable for identifying IPF patients who may particularly benefit from treatment with a PDE4-inhibitor of formula A', or are preferably suitable for monitoring the progress of treatment with a PDE4-inhibitor of formula A'.

[0049] Since KL-6 and SP-D are biomarkers that combine all three, i.e., have a "pharmacodynamic potential" for treatment with a PDE4-inhibitor of formula A', a potential link to an "outcome" or treatment success (FVC and Dlco% at week 12 of treatment with a PDE4-inhibitor of formula A'), and additionally a "predictive potential" for IPF progression (see Fig.33 ), therefore the biomarkers KL-6 and SP-D are particularly suitable for identifying IPF patients who may particularly benefit from treatment with a PDE4-inhibitor of formula A', or for monitoring the progress of treatment with a PDE4-inhibitor of formula A'. 2. Summary of the invention

[0050] In a first aspect, the present invention relates to a PDE4-inhibitor of formula A'

[0051]

[0052] It is used in a method of treating progressive fibrosing interstitial lung disease (PF-ILD) in a patient who exhibits one or more biomarkers selected from KL-6, SP-D, MMP7, CA-125, CA19-9, E-selectin, sICAM-1, MMP3, OPN, CTGF, COMP, prostate protein, vWF and CRP.

[0053] In a preferred embodiment, the progressive fibrosing interstitial lung disease (PF-ILD) is idiopathic pulmonary fibrosis (IPF).

[0054] In a further preferred embodiment, the PDE4-inhibitor of formula A' is for use in a method of treating progressive fibrosing interstitial lung disease (PF-ILD) in a patient expressing one or more biomarkers selected from KL-6, SP-D, MMP7, OPN, CA-125, CA19-9, sICAM-1 and E-selectin.

[0055] In a further preferred embodiment, the PDE4-inhibitor of formula A' is for use in a method of treating progressive fibrosing interstitial lung disease (PF-ILD) in a patient expressing one or more biomarkers selected from KL-6, SP-D, MMP7 and E-selectin.

[0056] In a further preferred embodiment, the PDE4-inhibitor of formula A' is for use in a method of treating progressive fibrosing interstitial lung disease (PF-ILD) in a patient expressing one or more biomarkers selected from KL-6, SP-D and MMP7.

[0057] In a particularly preferred embodiment, the PDE4-inhibitor of formula A' is used in a method of treating progressive fibrosing interstitial lung disease (PF-ILD) in a patient exhibiting one or more biomarkers selected from KL-6 and SP-D.

[0058] In another preferred embodiment, the PDE4-inhibitor of formula A' is for use in a method of treating progressive fibrosing interstitial lung disease (PF-ILD) in a patient expressing one or more biomarkers selected from COMP, vWF and prostacyclin.

[0059] In a particularly preferred embodiment, the PDE4-inhibitor of formula A' is used in a method for treating progressive fibrosing interstitial lung disease (PF-ILD) in a patient exhibiting KL-6.

[0060] In another particularly preferred embodiment, the PDE4-inhibitor of formula A' is used in a method for the treatment of progressive fibrosing interstitial lung disease (PF-ILD) in a patient exhibiting SP-D.

[0061] In a second aspect, the present invention relates to a method for treating a patient with progressive fibrosing interstitial lung disease (PF-ILD), preferably idiopathic pulmonary fibrosis (IPF), comprising administering to the patient a pharmaceutically effective amount of a PDE4-inhibitor of formula A'

[0062]

[0063] wherein the patient exhibits one or more biomarkers selected from the group consisting of KL-6, SP-D, MMP7, CA-125, CA19-9, OPN, CTGF, E-selectin, sICAM-1, MMP3, COMP, prostate protein, vWF and CRP.

[0064] In a preferred embodiment, the PDE4-inhibitor of formula A' is administered to the patient at a dose of 18 mg twice a day.

[0065] In a further preferred embodiment, the patient exhibits one or more biomarkers selected from the group consisting of KL-6, SP-D, CA-125, CA19-9, MMP7, OPN, sICAM-1 and E-selectin.

[0066] In a further preferred embodiment, the patient exhibits one or more biomarkers selected from the group consisting of KL-6, SP-D, MMP7 and E-selectin.

[0067] In a further preferred embodiment, the patient exhibits one or more biomarkers selected from KL-6, SP-D and MMP7.

[0068] In a further preferred embodiment, the patient exhibits one or more biomarkers selected from KL-6 and SP-D.

[0069] In a particularly preferred embodiment, the patient expresses KL-6.

[0070] In another particularly preferred embodiment, the patient exhibits SP-D.

[0071] In another preferred embodiment, the patient expresses one or more biomarkers selected from COMP, vWF and prostate proteins.

[0072] In a third aspect, the present invention relates to a PDE4-inhibitor of formula A'

[0073]

[0074] It is used in a method of treating progressive fibrosing interstitial lung disease (PF-ILD) in a patient determined to express one or more biomarkers selected from KL-6, SP-D, MMP7, CA-125, CA19-9, OPN, CTGF, E-selectin, sICAM-1, MMP3, COMP, prostate protein, vWF and CRP.

[0075] In a preferred embodiment, the progressive fibrosing interstitial lung disease (PF-ILD) is idiopathic pulmonary fibrosis (IPF).

[0076] In a preferred embodiment, the PDE4-inhibitor of formula A' is administered to the patient at a dose of 18 mg twice a day.

[0077] In a further preferred embodiment, the patient exhibits one or more biomarkers selected from the group consisting of KL-6, SP-D, CA-125, CA19-9, MMP7, OPN, sICAM-1 and E-selectin.

[0078] In a further preferred embodiment, the patient exhibits one or more biomarkers selected from the group consisting of KL-6, SP-D, MMP7 and E-selectin.

[0079] In a further preferred embodiment, the patient exhibits one or more biomarkers selected from KL-6, SP-D and MMP7.

[0080] In a particularly preferred embodiment, the patient exhibits one or more biomarkers selected from KL-6 and SP-D.

[0081] In a particularly preferred embodiment, the patient expresses the biomarker KL-6.

[0082] In another particularly preferred embodiment, said patient exhibits the biomarker SP-D.

[0083] In another preferred embodiment, the patient expresses one or more biomarkers selected from COMP, vWF and prostate proteins.

[0084] In a fourth aspect, the present invention relates to a method for treating progressive fibrosing interstitial lung disease (PF-ILD) in a patient, comprising

[0085] - determining that the patient exhibits one or more biomarkers selected from the group consisting of KL-6, SP-D, MMP7, CA-125, CA19-9, OPN, CTGF, E-selectin, sICAM-1, MMP3, COMP, prostate protein, vWF and CRP, and

[0086] - administering to said patient a pharmaceutically effective amount of a PDE4-inhibitor of formula A'

[0087]

[0088] In a preferred embodiment, the progressive fibrosing interstitial lung disease (PF-ILD) is idiopathic pulmonary fibrosis (IPF).

[0089] In a preferred embodiment, the PDE4-inhibitor of formula A' is administered to the patient at a dose of 18 mg twice a day.

[0090] In a further preferred embodiment, the patient exhibits one or more biomarkers selected from the group consisting of KL-6, SP-D, CA-125, CA19-9, MMP7, OPN, sICAM-1 and E-selectin.

[0091] In a further preferred embodiment, the patient exhibits one or more biomarkers selected from the group consisting of KL-6, SP-D, MMP7 and E-selectin.

[0092] In a further preferred embodiment, the patient exhibits one or more biomarkers selected from KL-6, SP-D and MMP7.

[0093] In a particularly preferred embodiment, the patient exhibits one or more biomarkers selected from KL-6 and SP-D.

[0094] In a particularly preferred embodiment, the patient expresses the biomarker KL-6.

[0095] In another particularly preferred embodiment, said patient exhibits the biomarker SP-D.

[0096] In another preferred embodiment, the patient expresses one or more biomarkers selected from COMP, vWF and prostate proteins.

[0097] In a fifth aspect, the present invention relates to a PDE4-inhibitor of formula A'

[0098]

[0099] It is used in a method for treating progressive fibrosing interstitial lung disease (PF-ILD) in a patient, the method comprising the following steps:

[0100] a) measuring or having measured the concentration, expression level or activity of one or more biomarkers in the serum or plasma of a blood sample obtained from the patient, the biomarkers being selected from the group consisting of Krebs von den Lungen protein (KL-6), surfactant protein D (SP-D), matrix lytic factor (MMP7), CA-125 (also known as MUC-16), CA19-9, E-selectin, matrix lytic factor (MMP3), osteopontin (OPN), connective tissue growth factor (CTGF), cartilage oligomeric matrix protein (COMP), prostate protein, von Willebrand factor (vWF), soluble intercellular adhesion molecule 1 (sICAM1) and C-reactive protein (CRP),

[0101] b) comparing or having compared the concentration, expression level or activity of one or more biomarkers listed in step a) in the patient's serum or plasma sample with a reference concentration, expression level or activity of the corresponding one or more biomarkers,

[0102] c) determining or having determined that the concentration, expression level or activity of the corresponding one or more biomarkers listed in step a) is modified compared to the corresponding reference concentration, expression or activity of the corresponding one or more biomarkers,

[0103] d) administering to said patient a therapeutically effective amount of a PDE4-inhibitor of formula A'.

[0104] Preferably, the one or more biomarkers of step a) are selected from KL-6, SP-D, MMP7, MMP3, OPN, CTGF, COMP, prostatic protein, vWF, CA-125, CA19-9, sICAM-1 and E-selectin, and then step c) comprises determining or has determined that the concentration, expression level or activity of the corresponding one or more biomarkers selected from KL-6, SP-D, MMP7, MMP3, OPN, CTGF, COMP, prostatic protein, vWF, CA-125, CA19-9, sICAM-1 and E-selectin is increased compared to the corresponding reference concentration, expression or activity of the corresponding one or more biomarkers.

[0105] More preferably, the one or more biomarkers of step a) are selected from KL-6, SP-D, MMP7, CA-125, CA19-9, OPN, sICAM-1 and E-selectin, and then step c) comprises determining or has determined that the concentration, expression level or activity of the corresponding one or more biomarkers selected from KL-6, SP-D, MMP7, CA-125, CA19-9, OPN, sICAM-1 and E-selectin is increased compared to the corresponding reference concentration, expression or activity of the corresponding one or more biomarkers.

[0106] More preferably, the one or more biomarkers of step a) are selected from KL-6, SP-D, MMP7 and E-selectin, and then step c) comprises determining or has determined that the concentration, expression level or activity of the corresponding one or more biomarkers selected from KL-6, SP-D, MMP7 and E-selectin is increased compared to the corresponding reference concentration, expression or activity of the corresponding one or more biomarkers.

[0107] More preferably, the one or more biomarkers of step a) are selected from KL-6, SP-D and MMP7, and then step c) comprises determining or has determined that the concentration, expression level or activity of the corresponding one or more biomarkers selected from KL-6, SP-D and MMP7 is increased compared to the corresponding reference concentration, expression or activity of the corresponding one or more biomarkers.

[0108] Most preferably, the one or more biomarkers of step a) are selected from KL-6 and SP-D, and then step c) comprises determining or has determined that the concentration, expression level or activity of the corresponding one or more biomarkers selected from KL-6 and SP-D is increased compared to the corresponding reference concentration, expression or activity of the corresponding one or more biomarkers.

[0109] Particularly preferably, the one or more biomarkers of step a) is KL-6.

[0110] Particularly preferably, the one or more biomarkers of step a) is MMP-7.

[0111] Particularly preferably, the one or more biomarkers of step a) is SP-D.

[0112] In particular, the one or more biomarkers of step a) are KL-6, and then the reference activity of KL-6 in the patient's serum in step c) is > 1000 U / ml.

[0113] In another preferred embodiment, the one or more biomarkers of step a) are C-reactive protein (CRP), and step c) then comprises determining or has determined that the concentration of CRP is reduced compared to a corresponding reference concentration of CRP.

[0114] The progressive fibrosing interstitial lung disease (PF-ILD) to be treated is preferably idiopathic pulmonary fibrosis (IPF).

[0115] In a preferred embodiment, before steps a), b), c) and d), the patient has been treated with an anti-fibrotic compound selected from nintedanib, pirfenidone or any pharmaceutically acceptable salt thereof, and during treatment with the compound of formula A' in step d), treatment with the anti-fibrotic compound continues as background treatment.

[0116] In another preferred embodiment, before steps a), b), c) and d), the patient has been treated with an anti-fibrotic compound selected from nintedanib, pirfenidone or any pharmaceutically acceptable salt thereof, and during the treatment with the compound of formula A' in step d), the anti-fibrotic compound is not continued as background treatment.

[0117] In a further preferred embodiment, 18 mg of the PDE4-inhibitor of formula A' is administered to the patient twice a day in step d).

[0118] In another preferred embodiment, the concentration, expression level or activity of the corresponding one or more biomarkers in step c) is greater than the concentration, expression level or activity of the corresponding one or more biomarkers at the reference concentration, and is considered to be a predictor of PF-ILD progression (or preferably IPF progression).

[0119] In a sixth aspect, the present invention relates to the use of one or more biomarkers selected from KL-6, SP-D, MMP7, MMP3, OPN, CTGF, COMP, prostate protein, vWF, CA-125, CA19-9, CRP and E-selectin in a method for treating PF-ILD (preferably IPF) by administering a pharmaceutically effective amount of a PDE4-inhibitor of formula A'.

[0120] In a preferred embodiment, the one or more biomarkers described above are selected from KL-6, SP-D, MMP7, CA-125, CA19-9, OPN, sICAM-1 and E-selectin.

[0121] In a more preferred embodiment, the one or more biomarkers as described above are selected from KL-6, SP-D, MMP7 and E-selectin.

[0122] In a more preferred embodiment, the one or more biomarkers as described above are selected from KL-6, SP-D and MMP7.

[0123] In a more preferred embodiment, the one or more biomarkers as described above are selected from KL-6 and SP-D.

[0124] In a particularly preferred embodiment, the one or more biomarkers is KL-6.

[0125] In another particularly preferred embodiment, the one or more biomarkers is SP-D.

[0126] In a preferred embodiment, the one or more biomarkers as described above are selected from COMP, prostacyclin and vWF.

[0127] In another preferred embodiment, the pharmaceutically effective amount of the PDE4-inhibitor of formula A' is 18 mg twice a day.

[0128] In a seventh aspect, the present invention relates to a method for monitoring the treatment success of a PF-ILD patient, preferably an IPF patient, who has been treated with a PDE4-inhibitor of formula A'.

[0129]

[0130] It includes the following steps

[0131] a) measuring or having measured the concentration, expression level or activity of one or more biomarkers in the serum or plasma of a blood sample obtained from the patient, the biomarkers being selected from the group consisting of Krebs von den Lungen protein (KL-6), surfactant protein D (SP-D), matrix lytic factor (MMP7), CA-125 (also known as MUC-16), CA19-9, E-selectin, soluble intercellular adhesion molecule 1 (sICAM1), matrix lytic factor (MMP3), osteopontin (OPN), connective tissue growth factor (CTGF), cartilage oligomeric matrix protein (COMP), prostaglandin, von Willebrand factor (vWF) and C-reactive protein (CRP),

[0132] b) comparing or having compared the measured concentration, expression level or activity of one or more biomarkers listed in step a) in the patient's serum or plasma sample with a reference concentration, expression level or activity of the corresponding one or more biomarkers

[0133] or

[0134] comparing or having compared the concentration, expression level or activity of one or more biomarkers listed in step a) in the patient's serum or plasma sample with the concentration, expression level or activity of one or more biomarkers listed in step a) at the start of treatment with the PDE4-inhibitor of formula A',

[0135] Steps a) and b) may be performed only once or may be repeated several times.

[0136] In a preferred embodiment, the one or more biomarkers mentioned in step a) are selected from KL-6, SP-D, MMP-7, CA-125, CA19-9, OPN, sICAM-1 and E-selectin.

[0137] In a preferred embodiment, the one or more biomarkers mentioned in step a) are selected from KL-6, SP-D, MMP-7 and E-selectin.

[0138] In a further preferred embodiment, the one or more biomarkers mentioned in step a) are selected from KL-6, SP-D and MMP-7.

[0139] In a particularly preferred embodiment, the one or more biomarkers mentioned in step a) are selected from KL-6 and SP-D.

[0140] In a particularly preferred embodiment, the one or more biomarkers mentioned in step a) is KL-6.

[0141] In another particularly preferred embodiment, the one or more biomarkers mentioned in step a) is SP-D.

[0142] In a further preferred embodiment, the method of monitoring treatment success is used in PF-ILD patients, preferably IPF patients, who have been treated with 18 mg of the PDE4-inhibitor of formula A' twice daily.

[0143] 3. Phase II trial (NCT04419506)

[0144] 3.1 Detailed description of the Phase 2 clinical trial

[0145] Patients aged ≥40 years were diagnosed with idiopathic pulmonary fibrosis according to the 2018 American Thoracic Society (ATS) / European Respiratory Society (ERS) / Japanese Respiratory Society / Latin American Thoracic Association guidelines (Raghu et al., Am J Respir Crit Care Med, 2018; 198: e44-e68). Patients with common interstitial pneumonia (UIP) or possible UIP patterns consistent with idiopathic pulmonary fibrosis on high-resolution computed tomography were eligible as confirmed by central review (Raghu et al., AM j Respir Crit Care Med 2019; 200: 1089-1092). The patient's mandatory FVC was ≥45% predicted, and the diffusing capacity of the lung for carbon monoxide (DLco) corrected for hemoglobin 25- was <80% predicted. If the patient had received a stable dose for at least 8 weeks before screening, the patient was allowed to continue to receive antifibrotic drugs (nintedanib or pirfenidone). Patients with airway obstruction, recent respiratory infection, or a history of suicidal behavior within the past 2 years were excluded.

[0146] This study was conducted in accordance with the principles of the Declaration of Helsinki and the International Council for Harmonization tripartite harmonized guidelines for good clinical practice and was approved by local authorities. The clinical protocol was approved by an independent ethics committee or institutional review board at each participating center. All patients provided written informed consent before entry into the study.

[0147] 3.1.1 Research design

[0148] The study was a double-blind, placebo-controlled, parallel-design, Phase 2 study conducted at 90 sites in 22 countries. Patients were randomized in a 2:1 ratio to receive 18 mg of a PDE4 inhibitor of formula A' twice daily, or matching placebo, for 12 weeks. After completing the 12-week treatment period, patients entered a 1-week follow-up period. Patients who discontinued study drug prematurely were required to attend all planned follow-up visits to minimize missing data. Randomization was stratified according to background antifibrotic drug use at baseline (no or yes), with a goal of at least 60 patients per treatment group and a maximum of 150 patients overall. Patients, investigators, central reviewers, and personnel involved in study conduct and analysis were unaware of treatment assignment. Randomization was performed using an interactive voice response system.

[0149] The primary endpoint was the change in FVC (mL) relative to baseline at 12 weeks. Spirometry results assessed using a spirometer (ERTSpiroSphere) provided by the sponsor were centrally reviewed to meet ATS / ERS criteria (Miller et al., Eur Respir J, 2005; 26: 319-338). The secondary endpoint was the percentage of patients with adverse events during treatment. Changes in the predicted values ​​of the carbon monoxide lung diffusion capacity or transport factor (DLco%) corrected for hemoglobin relative to baseline were assessed as another lung function efficacy endpoint using the research institution's own equipment and implemented according to ATS / ERS guidelines (Macintyre et al., Eur Respir J, 2005; 26: 720-735).

[0150] 3.1.2 Statistical analysis

[0151] In patients who did not use and used background antifibrotic drugs at baseline, the primary endpoint (change in FVC at week 12 relative to baseline) was evaluated separately, and all data collected during treatment were included. The main analysis was based on a Bayesian borrowing approach, with the goal of using meta-analytic predictive priors to include historical data from the placebo group in the group that did not receive background antifibrotic drug treatment or received background antifibrotic drug treatment. These priors were robustly processed for prior-data conflicts (Schmidli et al., Biometrics 2014 70: 1023-1032). For the group using the PDE4 inhibitor of formula A' (hereinafter referred to as the compound of formula A'), an information-ambiguous prior was used. The primary endpoint analysis was performed in two steps. In the first step, a mixed model of repeated measures (MMRM) was used to analyze the data of the current trial using a method based on restricted maximum likelihood. The analysis included fixed categorical effects of treatment at each follow-up, and fixed continuous effects of baseline FVC at each follow-up. Follow-up was treated as repeated measures, and the intra-patient measurements were modeled using an unstructured covariance structure. Based on the model, the corrected mean change (and associated standard error) of FVC relative to baseline at 12 weeks in the formula A' compound and placebo groups in the patient groups that did not use and used background anti-fibrotic drugs was calculated. In the second step, the corrected mean of the placebo group was combined with meta-analysis prediction prior data based on clinical trials of the idiopathic pulmonary fibrosis clinical development project of Nintedanib. The posterior distribution of the treatment difference between the formula A' compound and placebo relative to the primary endpoint was used to evaluate the treatment effect in each group. The median of the posterior distribution of the treatment difference (and 95% confidence interval) was calculated as the primary analysis, and the posterior probability of the treatment difference being higher than different boundaries was reported.

[0152] All treated patients were included in the safety analysis, stratified by background antifibrotic medication use (no or yes), which was descriptive in nature. Safety was assessed by clinical and laboratory evaluations and by recording treatment-emergent adverse events, coded using the Medical Dictionary for Regulatory Activities, version 22.0.

[0153] Missing data for the primary analysis (continuous endpoints) were not imputed. MMRM analyses allowed for missing data, assuming they were missing at random. Sensitivity analyses were performed to investigate the potential impact of missing data and premature discontinuation of treatment by treatment strategy, and a pooled analysis combining all patients regardless of background antifibrotic therapy was performed.

[0154] Because this was an exploratory trial, confirmatory testing and multiplicity adjustment were not planned. The sample size was selected on the basis of the posterior probability estimate of the change from baseline in FVC at week 12, assuming a standard deviation of 200 mL and a treatment difference of 70 mL and 20 mL in patients who were not receiving or were receiving background antifibrotic therapy, respectively.

[0155] Descriptive statistics were planned for the change from baseline in DLco at week 12. Post hoc, analyses were performed according to the same MMRM defined for the primary endpoint.

[0156] 3.2 Phase 2 Clinical Trial Results

[0157] A total of 147 patients with idiopathic pulmonary fibrosis were randomized and treated with either the compound of formula A' or placebo. Demographic characteristics were comparable between the two groups, although patients receiving background antifibrotic therapy tended to have a longer time since diagnosis and a lower FVC % predicted at baseline.

[0158] A total of 15 patients discontinued the study. These patients were all in the group that received the compound of formula A' (5 patients were not using background antifibrotic drugs and 10 patients were using background antifibrotic drugs). The main reason for discontinuation was adverse events (3 patients were not using background antifibrotic drugs and 10 patients were using background antifibrotic drugs). In patients who were not using background antifibrotic drugs, the average treatment duration of the compound of formula A' and the placebo group was 81.4±12.3 days and 85.6±3.8 days. For patients using background antifibrotic drugs, this was 74.6±23.0 days and 84.7±1.5 days, respectively.

[0159] 3.2.1 Efficacy

[0160] The primary efficacy endpoint - change from baseline in FVC at Week 12 based on a Bayesian borrowing approach using historical data - revealed that treatment with the compound of Formula A' stabilized lung function compared to placebo, with a decrease in FVC ( Figure 4 ).

[0161] In patients not taking background antifibrotic drugs, the median change in FVC was +5.7 mL in the Compound A group and -81.7 mL in the placebo group (median difference: 88.4 mL, with a 99.8% probability that Compound A is superior to placebo based on Bayesian borrowing analysis, see Figure 4 In patients taking background antifibrotic medications, these changes were +2.7 mL and -59.2 mL, respectively (median difference: 62.4 mL, 98.6% probability that the compound of Formula A' is superior to placebo based on Bayesian borrowing analysis) ( Figure 4 ).

[0162] The beneficial effect of the compound of formula A' on FVC was also shown in the pre-specified MMRM analysis based only on the observed values ​​( Figure 4 , Figure 1 , 2 and 3). The treatment effect estimated from the MMRM analysis was similar between the groups of patients not using and using background antifibrotic drugs, with an overall treatment effect of 88.4 mL (95% confidence interval [CI] 40.7 to 136.0) ( Figure 5 In the MMRM analysis, for patients not taking background antifibrotic drugs, the mean change in FVC from baseline to Week 12 was +6.1 mL for the Compound of Formula A group and -95.6 mL for the placebo group, corresponding to a difference of 101.7 mL (95% CI 25.0 to 178.4) (see Figure 4 and Figure 1 ), and for patients on background antifibrotic medications, +2.7 mL and -77.7 mL, corresponding to a difference of 80.4 mL (95% CI 20.9 to 140.0) (see Figure 4 and Figure 2 ).

[0163] 3.2.2 Security

[0164] A summary of all treatment-emergent adverse events is shown in Table 1. Table 2 shows the most frequently reported treatment-emergent adverse events reported in > 3% of patients overall in the Formula A' compound-treated group.

[0165] For those patients not using and using background antifibrotic drugs, the proportion of patients with any adverse events was higher in the Compound of Formula A' treated group compared to the placebo group. Adverse events leading to discontinuation were reported only in the Compound of Formula A' treated group.

[0166] The most common adverse events by organ class were gastrointestinal disorders, reported by 27.1% and 16.0% of patients not taking background antifibrotic drugs and 36.7% and 32.0% of patients taking background antifibrotic drugs in the A' compound-treated and placebo groups, respectively (see Table 2). The most common adverse event by preferred term was diarrhea, which was also the most common adverse event leading to discontinuation. In each group, a higher proportion of patients with diarrhea were treated with the A' compound compared with the placebo group, regardless of the background use of antifibrotic drugs. Most cases of diarrhea were mild.

[0167] · In the A' compound-treated group and the placebo group, 4.2% and 4.0% of patients not using background antifibrotic drugs and 4.1% and 4.0% of patients using background antifibrotic drugs reported severe adverse events, respectively (see Table 1). Serious adverse events were reported by 6.3% and 20% of patients not using antifibrotic drugs and 6.1% and 0% of patients using background antifibrotic drugs, respectively (see Table 1). Fatal adverse events occurred in 2 patients in the A' compound-treated group: COVID pneumonia (no background antifibrotic drugs were used) and one case of suspected vasculitis and suspected exacerbation of IPF in a patient using background antifibrotic drugs, where the vasculitis was not confirmed by the independent data monitoring committee (using background antifibrotic drugs).

[0168] Table 1: Treatment-emergent adverse events

[0169]

[0170] *Adverse events of special interest are vasculitis and liver injury. There were no reported cases of: immediate threat to life, persistent or significant disability / incapacity, or congenital anomalies / birth defects. All results are presented as n (%).

[0171] Table 2: Most frequently reported adverse events during treatment *

[0172]

[0173]

[0174] *Reported in ≥3% of patients overall in the Formula A' compound group; Over 12 weeks, in patients treated with the compound of formula A' and placebo, mean weight losses observed in the group not using antifibrotic drugs were -0.76 and -0.31, respectively, and in the group using antifibrotic drugs were -1.41 kg and -1.07, respectively.

[0175] 3.2.3 Biomarker Analysis during Phase 2 Clinical Trials

[0176] During the clinical Phase 2 trial, blood samples were collected from patients at baseline, 2, 4, 8 and 12 weeks (post-baseline) of the "Formula A' compound treatment group" and the "placebo group", and plasma and / or serum were prepared according to methods known in the art.

[0177] Fibrosis-related biomarkers in plasma or serum were assessed using ELISA or Luminex technology. The changes in these fibrosis-related biomarkers at weeks 2, 4, 8, and 12 after baseline were analyzed using a mixed model of repeated measurements (MMRM). The fold changes of the tested biomarkers relative to baseline over time were then compared between patients in the "Formula A' compound treatment group" and patients in the "placebo group."

[0178] Biomarker values ​​were considered valid only within +2 days after the last medication intake for CRP and within +7 days after the last medication intake for all other proteins.

[0179] Some of the IPF-related protein biomarkers tested appear to be primarily associated with epithelial / endothelial barrier integrity, such as:

[0180] -Krebs von den Lungen protein (KL-6): A glycoprotein expressed on the extracellular surface of type II alveolar epithelial cells (AEC) and bronchiolar epithelial cells in the lung, it has been extensively studied in ILD due to its overexpression in affected lungs and regenerating type II AEC. KL-6 is increased in the serum of several ILDs, including IPF. Zhang et al., Front. Immunol. (2021), 12: 745233 discuss elevated levels / threshold levels of KL-6 in serum that can predict ILD / IPF progression / mortality, such as serum KL-6 levels >1273U / ml are the most reliable predictors of end-stage lung disease development, or serum KL-6 levels >933U / ml show lower survival than patients without such high KL-6 levels.

[0181] - Pulmonary surfactant protein D (SP-D) is expressed in alveolar type II and bronchiolar epithelial cells and secreted into the alveoli and conducting airways. However, SP-D is also measured in serum and is increased in patients with acute respiratory distress syndrome, pulmonary fibrosis, and alveolar proteinosis (Pan et al., Am J Physiol Lung Cell Mol Physiol 2002; 282(4): L824-32).

[0182] - CA-125 (also known as MUC-16) is a high molecular weight glycoprotein expressed on the surface of various epithelial cells in the human body (Haridas et al., The FASEB J, Vol. 28, pp. 4184-99). It is still used as an effective marker for early epithelial ovarian cancer detection.

[0183] -Carbohydrate antigen 19-9 (CA19-9) is a type of glycoprotein located in the epithelium of the pancreas and bile ducts (Wang et al., Oncotarget, 2017, 8:2164-2170).

[0184] Some of the IPF-related protein biomarkers tested appear to be primarily related to extracellular matrix (ECM) turnover:

[0185] -Matrilysin - also known as matrix metalloproteinase 7 (MMP7), is known to degrade several ECM components during normal physiological processes, such as embryonic development, reproduction, tissue remodeling, and disease processes such as arthritis and metastasis. In baseline BUILD-3 samples (BUILD = Bosentan use in Interstitial Lung Disease), only MMP-7 showed significantly elevated protein levels in IPF patients compared to samples from healthy controls, and further studies showed that MMP-7 levels also increased over time. (Bauer et al., ERJ Open Res 2017; 3:00074-2016). Compared with healthy controls (1.25 ng / mL), geometric mean serum MMP-7 concentrations and p-values ​​were: baseline IPF: 2.25 ng / mL (p<0.0001), IPF (4 months): 1.97 ng / mL (p<0.01) and IPF at the end of the study: 2.64 ng / mL (p<0.0001) (Bauer et al., ERJ Open Res 2017;3:00074-2016).

[0186] - Stromelysin - also known as matrix metalloproteinase 3 (MMP3), known to degrade several ECM components

[0187] - Cartilage oligomeric matrix protein (COMP) - also known as thrombospondin-5, is an extracellular matrix (ECM) protein primarily found in cartilage. In humans, it is encoded by the COMP gene (Udomsinprasert et al., SciRep (2021) 11: 16695).

[0188] - Prostaprotein - also known as channel-activated protease 1), is an extracellular serine protease with trypsin-like activity that cleaves synthetic substrates in vitro, preferably at the carboxyl-terminal side of arginine residues (Aggarwal et al., Biomark. 2013, 2013: 179864).

[0189] - Von Willebrand factor (vWF) is a useful biomarker for liver fibrosis and prediction of hepatocellular carcinoma development in patients with hepatitis B and C (Takaya et al., United European Gastroenterol. 2018; 6(9): 1401-1409). However, it is not known whether vWF is a biomarker for PF-ILD or IPF.

[0190] Some of the IPF-related protein biomarkers tested appear to play a role primarily in inflammation:

[0191] - C-reactive protein (CRP) has been reported to be a biomarker for predicting the severity of pulmonary exacerbations in patients with cystic fibrosis (Giron-Moreno et al., BMC Pulm Med. 2014, 14: 150).

[0192] - Soluble intercellular adhesion molecule 1 (sICAM1) has been reported to be increased in patients with idiopathic pulmonary fibrosis (Okuda et al., Springerplus 2015; 4:657).

[0193] 3.2.3.1 method

[0194] Description of ELISA(MLM)

[0195] The CLEIA test kit ( The biomarker KL-6 (Krebs von den Lungen) was measured using the CLEIA test kit from Fujirebio (www.fujirebio.com) and the Lumipulse G1200 system, the biomarker sICAM-1 was measured using the ECLIA assay (V-PLEX Plus Vascular Injury Panel 2 Human Kit) from Meso Scale Discovery (MSD) on a Meso QuickPlex SQ120 instrument, and the biomarker sICAM-2 was measured using the ELISA kit from R&D Systems (www.r&dm;s.com). ELISA assay (human MMP-7 / PARC The biomarker MMP-7 was measured using a TECAN absorbance reader (ELISA kit).

[0196] Description of Luminex Technology MyriadRBM

[0197] Biomarkers CA-125, CA19-9, COMP, CRP, E-selectin, sICAM-1, MMP-3, MMP-7, prostatectin, TIMP-1, and vWF were measured using validated microsphere-based immunomultiplex assays using Luminex technology at MyriadRBM (Rule-Based Medicine, Q2 Solutions, Inc., Austin, TX, USA, www.rbm.q2labsolutions.com).

[0198] 3.2.3.2 result

[0199] Biomarker data collected from the different treatment arms of the Phase II trial have been analyzed in three different ways:

[0200] 1. The biomarker data has been analyzed for the "pharmacodynamic potential" of each corresponding biomarker, meaning that the change in the biomarker level over time relative to baseline during treatment with the compound of Formula A' has been analyzed (CfB). Therefore, biomarkers that demonstrate "pharmacodynamic potential" during treatment with the compound of Formula A' can be used to determine and quantify the molecular and physiological effects of the compound of Formula A' during treatment.

[0201] 2. The biomarker data have been analyzed for the “predictive potential” of each respective biomarker, meaning that the biomarker levels at baseline in the placebo group have been analyzed in relation to the clinical outcomes after 12 weeks (= absolute change in FVC [mL] from baseline at week 12 and absolute change in Dlco % predicted at week 12 from baseline), which are relevant to the disease and can be used to assess disease progression (e.g. rapid progressors vs. slow progressors).

[0202] 3. The biomarker data have been analyzed for the "outcome-related potential" of each respective biomarker, meaning that the change in the biomarker level from baseline (CfB) in the Formula A' compound treatment group has been analyzed with the clinical outcome after 12 weeks (= absolute FVC [mL] change from baseline at week 12 and absolute change from baseline in predicted Dlco% at week 12). Thus, potential early prediction of both clinical endpoints and treatment effects can be achieved.

[0203] Table 3 summarizes the adjusted mean (95% confidence interval) fold changes of various protein biomarkers (based on mixed model repeated measures model) relative to baseline in the blood of patients without background antifibrotic drugs (non-AF background) after 4 and 12 weeks of treatment with the compound of formula A'.

[0204] Compared to patients from the "placebo group", lower fold changes over time relative to baseline (which means "reduction over time") can be measured for the following protein biomarkers in the "non-AF background patients" from the "Formula A' compound treated group": KL-6, SP-D, CA-125 (MUC-16), matrix lytic factor (= MMP7), COMP, prostate protein, E-selectin and vWF (see Tables 3 and Figure 6 , 8 , 10, 14, 18, 20, 22, 24, 28). After 4 and 12 weeks of treatment with the compound of formula A', a higher fold change over time relative to baseline (which means "increase over time") could be measured for the protein biomarker CRP in the "non-AF background patients" from the "compound of formula A' treated group" compared to the patients from the "placebo group" (see Tables 3 and Fig.26 ).

[0205] Table 4 summarizes the adjusted mean values ​​(95% confidence intervals) of fold changes relative to baseline for various protein biomarkers in the blood (based on mixed model repeated measures model) in patients on background antifibrotic drug therapy (AF background).

[0206] Compared to patients from the "placebo group", the "AF background patients" from the "Formula A' compound treated group" had a lower fold change over time relative to baseline (which means "reduction over time") as measured for the following protein biomarkers: KL-6, SP-D, matrix lytic factor (= MMP7), prostate protein and E-selectin (see Tables 4 and Figure 7 , 9 , 15, 21, 29). A higher fold change over time relative to baseline (which means "increase over time") in "AF background patients" from the "Formula A' compound treated group" compared to patients from the "placebo group" can be measured for the white matter biomarker CRP (see Tables 4 and Fig. 27 ).

[0207] Table 3: Fold changes of various biomarkers in the blood from baseline to week 4 and to week 12 in patients not receiving background antifibrotic drugs (non-AF background)

[0208]

[0209]

[0210] *Transformed back to original scale. Adjusted mean fold change ratios are shown for treatment comparisons.

[0211] CI = Confidence Interval

[0212] [1] Based on the MMRM model, with fixed categorical effects of treatment at each visit and fixed continuous effects of baseline protein biomarker values ​​at each visit and at each age, Table 4: Fold changes from baseline to weeks 4 and 12 in various biomarkers in the blood of patients on background antifibrotic drugs (AF background)

[0213]

[0214]

[0215] *Transformed back to original scale. Adjusted mean fold change ratios are shown for treatment comparisons.

[0216] CI = Confidence Interval

[0217] [1] Based on the MMRM model, with fixed categorical effects of treatment at each visit and fixed continuous effects of baseline protein biomarker values ​​at each visit and at each age.

[0218] 3.2.4 Based on the FVC decline from the INBUILD study, different biomarkers are used to predict disease progression. Analysis of the "potential"

[0219] As an example, the predictive potential of various biomarkers for disease progression was analyzed in the placebo group of the INBUILD study, a phase III trial testing nintedanib in patients with PF-ILD (Wells et al., Lancet RespiMed 2020, Nintedanib in patients with progressive fibrosing interstitial lung disease—Subgroup analysis by diagnosis of interstitial lung disease in the INBUILD trial: a randomized, double-blind, placebo-controlled, parallel-group trial, NCT02999178). Several biomarkers in peripheral blood, including KL-6, MMP7, sICAM-1, CA19-9, showed predictive properties for disease progression, which used the annual rate of FVC decline within 52 weeks as a measure of disease progression to predict disease progression.

[0220] As an example, the cut-off values ​​of some predictive biomarkers are shown in Table 5, and the annual rate of FVC decline within 52 weeks is given for each subgroup (mean and 95% confidence interval), showing the significant differences in the rate of FVC decline (as a measure of disease progression) of these subgroups. The cut-off values ​​will be seen in the context of the INBUILD study and its patient cohorts and are shown as an example.

[0221] Table 5: “Predictive potential” of the biomarkers KL-6, CA-19-9, MMP7, and sICAM1 in patients with PF-ILD in the INBUILD trial:

[0222]

[0223] As a further example, the predictive potential of KL-6, CA19-9, sICAM-1 and MMP7 (among others) for disease progression has been shown in various cohorts of IPF and PF-ILD patients, and in some cases cut-off values ​​have been determined, which must be viewed in the context of the respective cohorts. The listed references provide an overview:

[0224] Adegunsoye et al., Chest 2020, Circulating plasma biomarkers of survival in AF-treated patients with IPF

[0225] Alqalyoobi et al., AJRCCM 2020, Circulating plasma biomarkers of progressive Interstitial Lung Diseases

[0226] Bowman et al., FrontinMed 2021, Biomarkers in Progressive FibrosingInterstitial Lung Disease: Optimizing diagnosis, prognosis and treatment response

[0227] Choi et al., RespiRes 2022, Blood KL-6levels predict treatment response to antifibrotic therapy in patients with IPF

[0228] Clynick et al., ERJ 2021, Biomarker signatures for progressive IPF

[0229] Daccord and Maher,F1000Research 2016,Recent advances in understanding IPF

[0230] Guiot et al., Lung 2017, Blood biomarkers in IPF

[0231] Maher et al.,LancetRespiMed 2017,An epithelial biomarker signature for IPF(PROFILE)

[0232] Jee et al., PharmacolTherap 2019, Review: serum biomarkers in IPF and SSc-ILD–frontiers and horizons

[0233] Jenkins et al., LancetRespiMed 2015, Longitudinal change in collagendegradation biomarkers in IPF (PROFILE)

[0234] Neighbors et al., LancetRespiRes 2018, Prognostic and predictive biomarkers for patients with IPF (CAPACITY and ASCEND)

[0235] Richards et al., AJRCCM 2012, Peripheral blood proteins predict mortality in IPF

[0236] Rosas et al., PLoS Med 2008, MMP1 and MMP7 as potential peripheral blood biomarkers in IPF.

[0237] 3.2.5 Summary of biomarker analysis

[0238] 3.2.5.1 "Pharmacodynamic potential"

[0239] In order to analyze the "pharmacodynamic potential" of each tested biomarker in treatment with Formula A' compound, the change over time of the corresponding biomarker relative to baseline (CfB) during treatment with Formula A' compound has been determined in the "active agent group" ("treatment group") of the Phase II trial of Formula A' compound (48 patients in the "active group" of the "AF group" and 43 patients in the "active group" of the "non-AF group").

[0240] MMRM analysis of the changes in biomarkers over time during treatment with the compound of formula A' has been performed. The results of the MMRM analysis of the changes in different biomarkers over time during treatment with the compound of formula A' are shown in Fig.30 middle. Fig.30 Descriptive box plots of changes in biomarkers over time relative to baseline (CfB) during "Formula A' compound treatment" are shown. Decreases in the corresponding biomarkers over time during treatment with Formula A' compounds are depicted in purple / blue shading, and increases in the corresponding biomarkers over time during treatment with Formula A' compounds are depicted in orange / red shading. No changes in biomarkers over time are depicted in white.

[0241] The pharmacodynamic potential of the different biomarkers tested was largely clear and consistent within the “non-AF group”.

[0242] from Fig.30 It can be clearly seen that treatment with the compound of formula A' significantly reduces the levels of the biomarkers SP-D (=PSP-D), MMP7 (=matrilolytic factor, as shown in the RBM- and MLM-assays) and KL-6, regardless of whether background therapy is additionally administered. Therefore, the biomarkers SP-D, MMP7 (=matrilolytic factor) and KL-6 have a clear "pharmacodynamic potential" during treatment with the compound of formula A'. Therefore, SP-D, MMP-7 and KL-6 can be used to determine and quantify the molecular and physiological effects of the compound of formula A' during treatment.

[0243] also, Fig.30 It was shown that treatment with the compound of formula A' appears to be associated with an increase in C-reactive protein (CRP) levels.

[0244] 3.2.5.2 "Predictive potential"

[0245] To analyze the "predictive potential" of each tested biomarker, the changes in clinical endpoints (= forced vital capacity (FVC) and DLCO % predicted) at week 12 relative to baseline biomarker levels were analyzed and compared between the "AF group" (25 patients) and the placebo group of the "non-AF group" (25 patients) of the Phase II trial.

[0246] Fig.31 Contains scatter plots generated by CfB for FVC values ​​relative to baseline biomarker values ​​and CfB for DLCO% predicted values ​​relative to baseline biomarker values.

[0247] Rank-based correlation analysis was performed. Fig.31showed that the predictive signature was mostly clear in the non-AF group and that the biomarkers SP-D, KL-6, CA-125, and sICAM-1 (MLM) all showed "predictive potential" in IPF and could therefore be used to assess IPF progression (rapid vs. slow progressors).

[0248] Table 5 includes biomarker analysis data for patients from the INBUILD trial, additionally showing as examples that the biomarkers KL-6, CA 19-9, MMP7 and sICAM1 all show "predictive potential" for disease progression and can therefore be used to assess the progression of PF-ILD.

[0249] 3.2.5.3 "Result-related potential"

[0250] In order to find out which biomarkers are "outcome-related", the correlation between the changes in the biomarkers and the changes in the clinical endpoints (= CfB FVC and CfB DLCO) was analyzed, focusing on the two active groups (which means the "treatment group with the compound of formula A'" in the AF group and the non-AF group). "Outcome-related biomarkers" have the potential to be used for early prediction of both clinical endpoints and the therapeutic effect of the active agent (here: the compound of formula A').

[0251] Fig.32 A scatter plot of the CfB biomarker values ​​of all Formula A' compound treatment groups (AF group, non-AF group and combined group) relative to the CfB FVC at week 12 and the CfB DLCO at week 12 is shown. Fig.32 As shown, changes in the biomarkers KL-6, SP-D, E-selectin and sICAM-1 showed potential associations with CfB of FVC after 12 weeks of treatment with the compound of Formula A'.

[0252] 3.2.5.4 Summary of biomarker analysis

[0253] It is described in Section 1.1.2.1 that the compound of Formula A' appears to reduce the levels of biomarkers KL-6, SP-D, and MMP7. In addition, an approximately 2-fold increase in CRP levels was observed in the group treated with the compound of Formula A', regardless of background therapy. Therefore, this suggests the existence of a "pharmacodynamic potential" for the biomarkers KL-6, SP-D, and MMP7 (matrilolytic factor) during treatment with the compound of Formula A'. Therefore, KL-6, SP-D, and MMP7 (matrilolytic factor) can be used to determine and quantify the molecular and physiological effects of the compound of Formula A' during treatment (see Fig.33 ).

[0254] In Section 1.1.2.2, it was described that the biomarkers KL-6, SP-D, CA-125, and sICAM-1 appear to show "predictive potential" for IPF and can therefore be used to assess the progression of IPF (rapid vs slow progressors) (see Fig.33 ).

[0255] In Section 1.1.2.4 it is described that the biomarkers KL-6, sICAM-1, SP-D and E-selectin appear to have "outcome-associated potential", meaning that during treatment of IPF patients with the compound of Formula A', high baseline levels of any of the biomarkers KL-6, sICAM-1, SP-D and E-selectin show potential to be associated with a decline in lung function from baseline at Week 12, measured as CfB in FVC after Week 12 or as CfB in Dlco % predicted at Week 12 (see Fig.33 ).

[0256] Fig.33 summarizes the findings that so far only the biomarkers KL-6 and SP-D appear to combine all three properties: they are

[0257] "Outcome-related biomarkers"

[0258] ·Has pharmacodynamic potential

[0259] and has predictive potential.

[0260] in addition, Fig.33 The biomarker sICAM-1 was shown to have no pharmacodynamic potential, but incorporates predictive potential for IPF and is an "outcome-associated biomarker" for treatment with the compound of formula A'.

[0261] 4. In vivo study on the efficacy of the PDE4-inhibitor of formula A' in the rat bleomycin pulmonary fibrosis model

[0262] 4.1 Summarize

[0263] Lung remodeling was induced in rats by a single intratracheal administration of 1 mg / kg bleomycin. Oral administration of 2.5 mg / kg of the phosphodiesterase inhibitor (PDE4) of formula A', administered twice daily (0 and 12 hours), showed a 64% improvement in tissue volume measured by micro-computed tomography (μCT).

[0264] This was accompanied by improvements in lung function parameters of airway resistance, compliance, and airspace volume (measured at 30 cm H2O pressure), and a decrease in total osteopontin (OPN) protein levels in lung tissue.

[0265] 4.2 method

[0266] Adult, naive male Wistar rats (WI(Han); 280-300 g) were purchased from Janvier (JanvierLabs, Le Genest-Saint-Isle, France). All animal experiments were performed in accordance with German national guidelines and laws and regulations and were approved by the Ethics Committee. Approved in Tübingen (Germany) (license number: 12-012).

[0267] Bleomycin was dissolved in phosphate buffered saline (PBS) to a final concentration of 1.5 mg / mL. On day 0, animals were briefly anesthetized with isoflurane (3-4%) in oxygen. 200 μL / kg of saline or bleomycin (final dose 1 mg / kg) was administered intratracheally using a 1 ml syringe with a 22-G (22-gauge) flexible cannula.

[0268] The PDE4-inhibitor of formula A' was dissolved in 0.5% hydroxyethylcellulose containing 0.01% Tween 20 and administered by oral gavage twice daily at 06:00 and 18:00 from day 10 to day 20 at a dose of 2.5 mL / kg.

[0269] On day 18, animals were anesthetized with 3-4% isoflurane and placed on a μCT (Quantum FX) scanner mounting plate. After measurement, animals were returned to their cages for recovery. During imaging, a synchronization procedure was used to avoid motion blur (respiration, heartbeat), thereby improving image quality. Reconstruction of three-dimensional images from the raw data can then be performed.

[0270] Animals were anesthetized with pentobarbital (Narcoren) (60 mg / mL / kg body weight, ip) and ketamine (0.5-1 mg / animal, im). The trachea was exposed, a small incision was made, and the endotracheal tube was fixed in the trachea with a ligature. Spontaneous breathing was suppressed with pancuronium (0.8 mg / kg; 1 mL / kg body weight, iv), and the animals were mechanically ventilated with a tidal volume of 10 mL / kg, 90 breaths / min, and a positive end-expiratory pressure of 3 cm H2O. Airway resistance, compliance, elasticity, and pressure-volume loops were determined by the FlexiVent system supported by software version 7.2. For pulmonary function measurements, the template "FlexiVent FX-Rat Default-rel.B" with the script "Rat6basic_1loop v7.0 Lamb" was used. Deep inspiration to total vital capacity (deep inflation) was performed, and the inflation pressure was limited to 30 cm H2O. Snapshot-90 v7.0, QuickPrime-3 v7.0, PVS-V v7.0 and PVs-P v7.0 measurements were subsequently performed.After lung function measurements, animals were euthanized by an overdose of pentobarbital (0.16 g / 5 ml, 1 ml / animal iv).

[0271] The right mainstem bronchus was occluded with a ligature, and the right lung was removed.

[0272] Right lung tissue was homogenized into PBS + 1% BSA + 0.714% protease inhibitors (100 mg tissue / ml) using OmniPrep. The homogenate was centrifuged at 4500 rpm for 10 minutes at 4°C and 3 x 200 μL supernatant plus pellet was stored at -80°C.

[0273] OPN ELISA (R&D Systems, #MOST00) was performed according to the instruction manual.

[0274] All data are expressed as mean ± SEM of n animals. Statistical differences among groups were analyzed by a non-clinical statistical panel.

[0275] 4.3 result

[0276] Airway mechanics can be assessed by pressure-volume loops, which are a measure of airway volume as pressure is gradually increased and then decreased. After bleomycin challenge, pressure-volume loops show a marked volume depression ( Fig.34 A). When lung volume is measured at a pressure of 30 cmH2O ( Fig.34 B), a significant decrease in volume, which was partially restored by treatment with the PDE4-inhibitor of formula A' (31%, P<0.05).

[0277] Computed tomography (CT) uses computer processing of many X-ray images taken from different angles to produce cross-sectional (tomographic) images (virtual "slices") of specific areas of the object being scanned, allowing the user to see inside the object without cutting. This can be used to measure the volume of remodeled lung tissue and is usually expressed as a ratio of total lung volume (to correct for differences in lung size between animals). Bleomycin treatment resulted in an increased ratio ( Fig.34 C), which was partially reversed after treatment with the PDE4-inhibitor of formula A' (64%, P<0.05).

[0278] Osteopontin is known to regulate the recruitment and activation of inflammatory cells such as macrophages and neutrophils, which play an important role in the development of pulmonary fibrosis. In addition, osteopontin has been shown to promote tissue remodeling by affecting the migration, adhesion and proliferation of various cell types including fibroblasts and epithelial cells (Pardo A, Gibson K, Cisneros J, Richards TJ, Yang Y, Becerril C, Yousem S, Herrera I, Ruiz V, Selman M, Kaminski N. Up-regulation and profibrotic role of osteopontin in human idiopathic pulmonary fibrosis. PLoS Med. 2005 Sep; 2 (9): e251).

[0279] Measuring osteopontin in the bleomycin rat model may provide valuable insights into the progression of the disease and the effectiveness of potential therapeutic interventions.

[0280] After bleomycin challenge, osteopontin levels in lung tissue increased from about 200 pg / mL to about 550 pg / mL. Treatment with the PDE4-inhibitor of formula A' reduced OPN levels in lung tissue by about 49%.

[0281] 5. Effects of PDE4-inhibitors of formula A' on human small airway epithelial cells stimulated by IPF-related mixture (IPF-rc) In vitro study of the inhibitory effect of β-actin on the release of different biomarkers of SAEC

[0282] 5.1 Summarize

[0283] The PDE4-inhibitor of formula A' inhibits the release of the biomarkers MMP7 (matrix metalloproteinase 7), PAI1 (plasminogen activator inhibitor 1), OPN (osteopontin) and CTGF (connective tissue growth factor) in IPF-rc stimulated SAECs in a concentration-dependent manner.

[0284] 5.2 method

[0285] Primary human-derived small airway epithelial cells at passage 4 were seeded onto 24-well plates coated with type I rat tail collagen (30 μg / mL in PBS) in PneumaCult-Ex Plus complete medium.

[0286] After incubation for 24 h at 37° C. and 5% CO 2 , cells were pre-stimulated with different concentrations of the PDE4-inhibitor of formula A′ in PneumaCult-Ex Plus starvation medium for 30 min.

[0287] Afterwards, IPF-related cytokine mixture (Schruf E, Schroeder V, Le HQ, T, Raedel D, Stewart EL, Fundel-Clemens K, Bluhmki T, Weigle S, Schuler M, Thomas MJ, Heilker R, Webster MJ, Dass M, Frick M, Stierstorfer B, Quast K, Garnett JP. Recapitulating idiopathic pulmonary fibrosis related alveolar epithelial dysfunction in a human iPSC-derived air-liquid interface model. FASEBJ. 2020 Jun; 34(6): 7825-7846) to induce fibrotic changes. 72 hours after stimulation, the supernatant was collected and analyzed using R&D DuoSet ELISA according to the manufacturer's protocol.

[0288] 5.3 Results

[0289] To determine the inhibitory potency of the PDE4-inhibitors of formula A' on the release of biomarkers from IPF-rc stimulated primary human small airway epithelial cells, supernatants of cell cultures from 5 different donors were analyzed.

[0290] Fig.35 Concentration-dependent inhibition of IPF-rc-induced MMP7 (A), sICAM-1 (B), OPN (C) and CTGF (D) release is shown. The PDE4-inhibitor of formula A′ showed IC values ​​of 4.4 μM, 537 nM, 1.1 μM and 370 nM, respectively. 50-values ​​of inhibition of MMP7, sICAM, OPN and CTGF. The highest concentration of the PDE4-inhibitor of formula A' (10 μM) resulted in a maximum inhibition of MMP7, sICAM, OPN and CTGF of 63%, 39%, 67% and 75%, respectively ( Fig.35 AD).

[0291] The results of the biomarker analysis of the Phase 2 trial are further summarized in the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0292] Figure 1 : "Non-AF background" group (no background anti-fibrotic drugs used), "Formula A' compound treatment group" and "Placebo group"

[0293] Adjusted mean (SE) change from baseline in FVC [ml] over 12 weeks (MMRM).

[0294] After 12 weeks of treatment, the adjusted mean (SE) of the FVC change from baseline in the "Formula A' compound treatment group"

[0295] was +6.1 ml, while the "placebo group" was -95.6 ml, resulting in a difference of 101.7 ml.

[0296] Figure 2 The adjusted mean (SE) of the change in FVC [ml] from baseline during 12 weeks (MMRM) for the "AF background" group (using background antifibrotic drugs), the "Formula A' compound-treated group" and the "placebo group".

[0297] After 12 weeks of treatment, the adjusted mean (SE) of the FVC change from baseline in the "Formula A' compound treatment group"

[0298] was +2.7 ml, while the "placebo group" was -77.7 ml, resulting in a difference of 80.4 ml.

[0299] Figure 3 The adjusted mean (SE) (MMRM) of the change in FVC [ml] from baseline over 12 weeks for the "combined AF background" group (group using background antifibrotic drugs and group not using background antifibrotic drugs) of the "Formula A' compound treated group" and the "placebo group".

[0300] After 12 weeks of treatment, the adjusted mean (SE) of the FVC change from baseline in the "Formula A' compound treatment group"

[0301] was +4.6 ml, while the "placebo group" was -83.8 ml, resulting in a difference of 88.4 ml.

[0302] Figure 4: Mean change (95% confidence interval) in FVC (ml) from baseline at week 12 in the group not using background antifibrotic drugs ("non-AF background group") and the group using background antifibrotic drugs ("AF background group"), using MMRM analysis and Bayesian borrowing.

[0303] Figure 5 The adjusted mean (SE) of the change in FVC [ml] from baseline at Week 12 in the “combined AF background” group (group using background antifibrotic drugs and group not using background antifibrotic drugs) of the “Formula A′ compound treated group” and the “placebo group”.

[0304] Figure 6 Adjusted mean (95% confidence interval) of fold change (log10) of KL-6 [U / ml] relative to baseline for "Formula A' compound treated group" and "placebo group", both with "non-AF background" (no background treatment with antifibrotic drugs).

[0305] In patients with "non-AF background", the adjusted mean fold change of KL-6 relative to baseline was decreased in the "Formula A' compound treated group" compared to the "placebo group".

[0306] Figure 7 Adjusted mean (95% confidence interval) of fold change (log10) of KL-6 [U / ml] relative to baseline for "Formula A' compound treated group" and "placebo group", both with "AF background" (anti-fibrotic drug background treatment)

[0307] In patients with "AF background", the adjusted mean fold change of KL-6 relative to baseline was decreased in the "Formula A' compound treated group" compared to the "placebo group".

[0308] Figure 8 Adjusted mean (95% confidence interval) of fold change (log10) of lung surfactant protein SP-D [μg / L] relative to baseline in the "Formula A' compound-treated group" and the "placebo group", both with "non-AF background" (no background treatment with antifibrotic drugs).

[0309] In patients with non-AF background, SP-

[0310] D Corrected mean decrease in fold change relative to baseline.

[0311] Fig. 9Adjusted mean (95% confidence interval) of fold change (log10) of lung surfactant protein SP-D [μg / L] relative to baseline in the "Formula A' compound-treated group" and the "placebo group", both with "AF background" (anti-fibrotic drug background treatment)

[0312] In patients with "AF background", the adjusted mean fold change of SP-D from baseline was decreased in the "Formula A' compound treated group" compared to the "placebo group".

[0313] Fig.10 Adjusted mean (95% confidence interval) of fold change (log10) of CA-125 (also known as MUC-16) relative to baseline [U / ml] for the "Formula A' compound treated group" and the "placebo group", both with "non-AF background" (no background treatment with antifibrotic drugs).

[0314] In patients with "non-AF background", the adjusted mean fold change of CA-125 from baseline was decreased in the "Formula A' compound treated group" compared to the "placebo group".

[0315] Fig.11 Adjusted mean (95% confidence interval) of fold change (log10) of CA-125 (also known as MUC-16) relative to baseline [U / ml] for the "Formula A' compound treated group" and the "placebo group", both with "AF background" (anti-fibrotic drug background treatment)

[0316] Fig.12 Adjusted mean (95% confidence interval) of fold change (log10) of CA19.9 [U / ml] relative to baseline for "Formula A' compound treated group" and "placebo group", both with "non-AF background" (no background treatment with antifibrotic drugs).

[0317] Fig.13 Adjusted mean (95% confidence interval) of fold change (log10) of CA19.9 [U / ml] relative to baseline for "Formula A' compound treated group" and "placebo group", both with "AF background" (anti-fibrotic drug background treatment)

[0318] Fig.14 Adjusted mean (95% confidence interval) of fold change (log10) of matrix lytic factor (also known as MMP-7) [μg / L] relative to baseline in the "Formula A' compound-treated group" and the "placebo group", both with "non-AF background" (no background treatment with antifibrotic drugs).

[0319] In patients with "non-AF background", the adjusted mean fold change of MMP-7 from baseline was decreased in the "Formula A' compound treated group" compared to the "placebo group".

[0320] Fig.15 Adjusted mean (95% confidence interval) of fold change (log10) of matrix lytic factor (also known as MMP-7) [μg / L] relative to baseline in the "Formula A' compound treated group" and the "placebo group", both with "AF background" (anti-fibrotic drug background treatment)

[0321] In patients with "AF background", the adjusted mean fold change of MMP-7 relative to baseline was decreased in the "Formula A' compound treated group" compared to the "placebo group".

[0322] Fig.16 Adjusted mean (95% confidence interval) of fold change (log10) relative to baseline in interstitial lytic factor (also known as MMP-3) [μg / L] in the "Formula A' compound-treated group" and the "placebo group", both with "non-AF background" (no background treatment with antifibrotic drugs).

[0323] Fig.17 Adjusted mean (95% confidence interval) of fold change (log10) of stroma lytic factor (also known as MMP-3) [μg / L] relative to baseline in the "Formula A' compound treated group" and the "placebo group", both with "AF background" (anti-fibrotic drug background treatment)

[0324] Fig.18 Adjusted mean (95% confidence interval) of fold change (log10) of cartilage oligomeric matrix protein (COMP) [μg / L] relative to baseline in the "Formula A' compound-treated group" and the "placebo group", both with "non-AF background" (no background treatment with antifibrotic drugs).

[0325] In patients with "non-AF background", the adjusted mean fold change of COMP from baseline was decreased in the "Formula A' compound treated group" compared to the "placebo group".

[0326] Fig.19 Adjusted mean (95% confidence interval) of fold change (log10) of cartilage oligomeric matrix protein (COMP) [μg / L] relative to baseline in the "Formula A' compound treated group" and the "placebo group", both with "AF background" (anti-fibrotic drug background treatment)

[0327] Fig. 20Adjusted mean (95% confidence interval) of fold change (log10) of prostate protein [μg / L] relative to baseline for "Formula A' compound treated group" and "placebo group", both with "non-AF background" (no background treatment with antifibrotic drugs).

[0328] In patients with a "non-AF background", the adjusted mean fold change from baseline in prostate proteins was reduced in the "Formula A' compound treated group" compared to the "placebo group".

[0329] Fig.21 Adjusted mean (95% confidence interval) of fold change (log10) of prostate protein [μg / L] relative to baseline for "Formula A' compound treated group" and "placebo group", both with "AF background" (anti-fibrotic drug background treatment)

[0330] Fig. 22 Adjusted mean (95% confidence interval) of fold change (log10) of von Willebrand factor (vWF) [mg / L] relative to baseline in the "Formula A' compound treated group" and the "placebo group", both with "non-AF background" (no background treatment with antifibrotic drugs).

[0331] In patients with "non-AF background", the adjusted mean fold change of vWF from baseline was decreased in the "Formula A' compound treated group" compared to the "placebo group".

[0332] Fig.23 Adjusted mean (95% confidence interval) of fold change (log10) of von Willebrand factor (vWF) [mg / L] relative to baseline in the "Formula A' compound treated group" and the "placebo group", both with "AF background" (antifibrotic drug background treatment)

[0333] Fig.24 Soluble intercellular adhesion molecule 1 (sICAM-1) [μg / L] in the "Formula A' compound treatment group" and the "placebo group"

[0334] Adjusted means (95% confidence interval) of fold change (log10) relative to baseline, both with "non-AF background" (no background treatment with antifibrotic drugs).

[0335] In patients with "non-AF background", the adjusted mean fold changes of sICAM-1 relative to baseline were essentially the same in the "Formula A' compound treated group" and the "placebo group". This illustrates the fact that sICAM-1 has at least no pharmacodynamic potential during the treatment of IPF with Formula A' compound.

[0336] Fig.25Soluble intercellular adhesion molecule 1 (sICAM1) [μg / L] in the "Formula A' compound treatment group" and the "placebo group"

[0337] Adjusted mean (95% confidence interval) of fold change (log10) relative to baseline, both with "AF background" (background treatment with antifibrotic drugs)

[0338] Fig.26 Adjusted mean (95% confidence interval) of fold change (log10) of C-reactive protein (CRP) [mg / L] relative to baseline in the "Formula A' compound treated group" and the "placebo group", both with "non-AF background" (no background treatment with antifibrotic drugs).

[0339] In patients with "non-AF background", the adjusted mean fold change of CRP from baseline was increased in the "Formula A' compound treated group" compared to the "placebo group".

[0340] Fig. 27 Adjusted mean (95% confidence interval) of fold change (log10) of C-reactive protein (CRP) [mg / L] relative to baseline in the "Formula A' compound treated group" and the "placebo group", both with "AF background" (anti-fibrotic drug background treatment)

[0341] In patients with "AF background", the adjusted mean fold change of CRP from baseline was increased in the "Formula A' compound treated group" compared to the "placebo group".

[0342] Fig.28 Adjusted mean (95% confidence interval) of fold change (log10) of E-selectin [μg / L] relative to baseline for "Formula A' compound treated group" and "placebo group", both with "non-AF background" (no background treatment with antifibrotic drugs).

[0343] In patients with "AF background", the adjusted mean fold change of E-selectin relative to baseline was decreased in the "Formula A' compound treated group" compared to the "placebo group".

[0344] Fig.29 Adjusted mean (95% confidence interval) of fold change (log10) of E-selectin [μg / L] relative to baseline in the "Formula A' compound treated group" and the "placebo group", both with "AF background" (anti-fibrotic drug background treatment)

[0345] In patients with "AF background", the adjusted mean fold change of E-selectin relative to baseline was decreased in the "Formula A' compound treated group" compared to the "placebo group".

[0346] Fig.30 : Summary of biomarker analysis regarding the "pharmacodynamic potential" of individual biomarkers during treatment with the compound of formula A': In particular, KL-6, SP-D (shown as PSP-D) and MMP7 (shown as matrix lytic factor) decreased during treatment with the PDE4-inhibitor of formula A', thus showing pharmacodynamic potential, while C-reactive protein (CRP) increased during treatment with the PDE4-inhibitor of formula A'. During treatment with the PDE4-inhibitor of formula A', sICAM-1, E-selectin, COMP and CA19-9 also appeared to decrease, but to a lesser extent than KL-6, SP-D and MMP7, and primarily in the non-AF group.

[0347] Fig.31 : Overview of biomarker analysis regarding the “predictive potential” of individual biomarkers in IPF patients.

[0348] Baseline levels of KL-6, SP-D, CA-125 (= mucin-16), sICAM-1 and, to a lesser extent, CA19-9 appeared to be inversely correlated with the decline in lung function from baseline, measured as the change from baseline in FVC (CfB) and the change from baseline in Dlco% predicted (CfB) at week 12, in IPF patients in the placebo group only.

[0349] Thus, KL-6, SP-D, CA-125, and sICAM-1 (and to a lesser extent CA19-9) appear to have predictive potential in patients with IPF.

[0350] Fig.32 : Analysis of which tested biomarkers are "outcome-related" during treatment with the compound of formula A'.

[0351] During treatment of IPF patients with the PDE4-inhibitors of Formula A', changes in the levels of the biomarkers KL-6, SP-D, E-selectin and sICAM-1 were shown to correlate with the change from baseline (CfB) in forced vital capacity (FVC) and the change from baseline (CfB) in the diffusing capacity of the lung for carbon monoxide or transfer factor (Dlco) at Week 12 in both the "non-AF" and "AF" groups of the trial. Thus, KL-6, SP-D, E-selectin and sICAM-1 appear to have "outcome-related potential" during treatment with the PDE4-inhibitors of Formula A'.

[0352] Fig.33 : Summary of the "pharmacodynamic potential", "predictive potential" and "outcome relationship" of the biomarkers tested during treatment with the compound of formula A'.

[0353] Fig.34 Treatment with a PDE4-inhibitor of formula A' improves lung function in a therapeutic rat model of bleomycin-induced pulmonary fibrosis (see A, B and C) and leads to a decrease in the expression of osteopontin (OPN) protein in lung tissue (see D)

[0354] Fig.35 : Treatment with the PDE4-inhibitor of formula A' inhibits the expression of IPF-related proteins, such as MMP-7 ( Fig.35 A), sICAM-1( Fig.35 B) OPN( Fig.35 C) and CTGF ( Fig.35 D).

Claims

1. PDE4 inhibitors of formula A' It is used in a method for treating progressive fibrosing interstitial lung disease (PF-ILD) in a patient, the method comprising the following steps: a) measuring or having measured the concentration, expression level or activity of one or more biomarkers in the serum or plasma of a blood sample obtained from the patient, the biomarkers being selected from the group consisting of Krebs von den Lungen protein (KL-6), surfactant protein D (SP-D), matrix lytic factor (MMP7), CA-125 (also known as MUC-16), E-selectin, matrix lytic factor (MMP3), CA-19-9, osteopontin (OPN), connective tissue growth factor (CTGF), cartilage oligomeric matrix protein (COMP), prostaglandin, von Willebrand factor (vWF), soluble intercellular adhesion molecule 1 (sICAM1) and C-reactive protein (CRP), b) comparing or having compared the concentration, expression level or activity of the one or more biomarkers listed in step a) in the serum or plasma sample of the patient with a reference concentration, expression level or activity of the corresponding one or more biomarkers, c) determining or having determined that the concentration, expression level or activity of the corresponding one or more biomarkers listed in step a) is modified compared to the corresponding reference concentration, expression or activity of the corresponding one or more biomarkers, d) administering to said patient a therapeutically effective amount of said PDE4-inhibitor of formula A'.

2. A PDE4-inhibitor of formula A' according to claim 1, wherein the one or more biomarkers of step a) are selected from KL-6, SP-D, MMP7, CA-125, E-selectin, sICAM-1, MMP3, CA19-9, OPN, CTGF, COMP, prostatic protein and vWF, and wherein step c) comprises determining or has determined that the concentration, expression level or activity of the corresponding one or more biomarkers selected from KL-6, SP-D, MMP7, CA-125, E-selectin, sICAM-1, MMP3, CA19-9, OPN, CTGF, COMP, prostatic protein and vWF is increased compared to the corresponding reference concentration, expression or activity of the corresponding one or more biomarkers.

3. The PDE4-inhibitor of formula A' according to claim 1, wherein the one or more biomarkers of step a) is C-reactive protein (CRP), and wherein step c) comprises determining or has determined that the concentration of CRP is reduced compared to a corresponding reference concentration of CRP.

4. The PDE4 inhibitor of formula A' according to claims 1 to 3, wherein the progressive fibrosing interstitial lung disease (PF-ILD) is idiopathic pulmonary fibrosis (IPF).

5. The PDE4-inhibitor of formula A' according to claims 1 to 4, wherein the patient has been treated with an antifibrotic compound selected from nintedanib, pirfenidone or any pharmaceutically acceptable salt thereof before steps a), b), c) and d).

6. The PDE4 inhibitor of formula A' according to claim 5, wherein during the treatment with the compound of formula A' in step d), the treatment with an anti-fibrotic compound selected from nintedanib, pirfenidone or any pharmaceutically acceptable salt thereof before steps a), b), c) and d) is continued as background treatment.

7. The PDE4 inhibitor of formula A' according to claim 5, wherein during the treatment with the compound of formula A' in step d), the treatment with an anti-fibrotic compound selected from nintedanib, pirfenidone or any pharmaceutically acceptable salt thereof before steps a), b), c) and d) is not continued as background treatment.

8. The PDE4 inhibitor of the formula A' according to claim 1, wherein in step d) 18 mg of the PDE4 inhibitor of the formula A' are administered to the patient twice a day.

9. A PDE4-inhibitor of formula A' according to at least one of claims 1 to 8, wherein the concentration, expression level or activity of the corresponding one or more biomarkers in step c) is a concentration, expression level or activity of the corresponding one or more biomarkers that is greater than the reference concentration and is considered to be predictive of PF-ILD progression.

10. The PDE4-inhibitor of formula A' according to claims 1 to 8, wherein the concentration, expression level or activity of the corresponding one or more biomarkers in step c) is a concentration, expression level or activity of the corresponding one or more biomarkers that is greater than the reference concentration and is considered to be predictive of IPF progression.

11. A PDE4-inhibitor of formula A' according to at least one of claims 1 to 2 or 4 to 10, wherein the one or more biomarkers of step a) are selected from the group consisting of Krebs von den Lungen protein (KL-6), surfactant protein D (SP-D), matrix lytic factor (MMP7), CA-125 (also known as MUC-16), CA19-9, OPN, soluble intercellular adhesion molecule 1 (sICAM-1) and E-selectin, and wherein step c) comprises determining or has determined that the concentration, expression level or activity of these respective one or more biomarkers selected from the group consisting of KL-6, SP-D, MMP7, CA19-9, OPN, sICAM-1 and E-selectin is increased compared to the respective reference concentration, expression or activity of the respective one or more biomarkers.

12. A PDE4-inhibitor of formula A' according to at least one of claims 1 to 2 or 4 to 11, wherein the one or more biomarkers of step a) are selected from the group consisting of Krebs von den Lungen protein (KL-6), surfactant protein D (SP-D), matrix dissolving factor (MMP7) and E-selectin, and wherein step c) comprises determining or has determined that the concentration, expression level or activity of these respective one or more biomarkers selected from the group consisting of KL-6, SP-D, MMP7 and E-selectin is increased compared to the respective reference concentration, expression or activity of the respective one or more biomarkers.

13. A PDE4-inhibitor of formula A' according to at least one of claims 1 to 2 or 4 to 12, wherein the one or more biomarkers of step a) are selected from Krebs von den Lungen protein (KL-6), surfactant protein D (SP-D) and matrix lytic factor (MMP7), and wherein step c) comprises determining or has determined that the concentration, expression level or activity of these respective one or more biomarkers selected from KL-6, SP-D and MMP7 is increased compared to the respective reference concentration, expression or activity of the respective one or more biomarkers.

14. A PDE4-inhibitor of formula A' according to at least one of claims 1 to 2 or 4 to 13, wherein the one or more biomarkers of step a) are selected from Krebs von den Lungen protein (KL-6) and pulmonary surfactant protein D (SP-D), and wherein step c) comprises determining or has determined that the concentration, expression level or activity of these respective one or more biomarkers selected from KL-6 and SP-D is increased compared to the respective reference concentration, expression or activity of the respective one or more biomarkers.

15. A PDE4-inhibitor of formula A' according to at least one of claims 1 to 2 or 4 to 14, wherein the one or more biomarkers of step a) is Krebs von den Lungen protein (KL-6), and wherein step c) comprises determining or has determined that the concentration, expression level or activity of KL-6 is increased compared to a corresponding reference concentration, expression or activity of KL-6.

16. A PDE4-inhibitor of formula A' according to at least one of claims 1 to 2 or 4 to 14, wherein the one or more biomarkers of step a) is SP-D, and wherein step c) comprises determining or has determined that the concentration, expression level or activity of SP-D is increased compared to a corresponding reference concentration, expression or activity of SP-D.

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