Patent ID: 12233068

5. EXPERIMENTAL DATA

6.1 Pathogenesis of Fibrotic Processes that are Common to ILDs, PF-ILDs and IPF

Pathogenesis of fibrotic processes that are common to ILDs, PF-ILDs and IPF are presently not completely understood.

The main characteristics of IPF are changes in epithelial and mesenchymal cells as well as the interaction between these cells whereas it is currently believed that inflammatory processes play only a minor role [Lehtonen et al, Respiratory Research (2016) 17: 14]. One widely accepted hypothesis to explain the mechanisms in IPF pathogenesis postulates that an injury of the alveolar epithelium results in an excessive wound healing response with overshooting release of growth and transcription factors and cytokines subsequent activation and transformation of fibroblasts to the secreting myofibroblast phenotype resulting in excessive production of extracellular matrix (ECM) proteins, [King T E, Jr, Pardo A, Selman M., Lancet. 2011; 378:1949-1961]. The fibroblast focus, a typical histological feature of IPF, is a specific aggregate of cells, especially of fibroblasts and of myofibroblasts, covered by injured and hyperplastic epithelium, and ECM produced by myofibroblasts [Kuhn C, McDonald J A., Am J Pathol. 1991; 138:1257-1265]. Studies have revealed that IPF patients with a high number of fibroblast foci have a shortened survival [Kaarteenaho R., Respir Res. 2013; 14(1): 43]. In addition, the extent of expression of alpha smooth muscle actin (α-SMA), as a marker of myofibroblasts, in the lungs of IPF-patients, has been shown to be negatively associated with patient survival [Waisberg D R, Parra E R, Barbas-Filho J V, Fernezlian S, Capelozzi V L]. Increased fibroblast telomerase expression precedes myofibroblast alpha-smooth muscle actin expression in idiopathic pulmonary fibrosis [Clinics (Sao Paulo) 2012; 67:1039-1046].

Current paradigms of pathogenesis of fibrotic processes suggest that following exposure to endogenous or exogenous stimuli, the lung epithelium initiates an injury response resulting in the production of soluble factors such as Transforming Growth Factor beta-1 (TGF-(β1), platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), and cytokines including interleukin-4 (IL-4) and interleukin-13 (IL-13). These substances promote recruitment of inflammatory cells and mesenchymal activation which causes expansion of tissue resident post-embryonic fibroblasts which are thought to give rise to activated myofibroblasts. These cells are central to the process of wound healing but, if unmodulated, deposit excessive ECM and destroy normal lung architecture. During normal wound healing, myofibroblasts are transiently activated and direct production of granulation tissue by producing ECM and exerting traction forces. Once healing is achieved, granulation tissue is resorbed and myofibroblasts undergo programmed cell death to restore normal tissue architecture and function [Klingberg et al, J Pathol. 2013; 229: 298-309]. Disruptions at any stage in this process could cause tissue pathology. When the healing response is insufficient, as is seen in acute respiratory distress syndrome, a pathology dominated by acute injury and diffuse alveolar damage ensues. However, when the healing phase dominates, the tissue milieu shifts towards fibrosis and remodeling and a pathology dominated by the dysregulated accumulation of scar tissue is seen. Fibroblasts and activated myofibroblasts are believed to be central to this process [Moore et al Curr Pathobiol Rep. 2013 September; 1 (3): 199-208].

In a further level, fibroblasts and myofibroblasts in IPF demonstrate a pathologic phenotype characterized by uncontrolled proliferation and survival. These cells accumulate in lung interstitium where they deposit excessive amounts of collagen-I rich ECM and ultimately organize into the fibroblastic foci described above. As these regions expand and become juxtaposed to the alveolar space, they appear to first rupture and then ultimately destroy the alveolar basement membrane [White et al, J Pathol. 2003; 201: 343-354].

This expansion is largely attributed to the resistance to programmed cell death that has been described for primary fibroblasts obtained from IPF lung tissue [Maher et al, Am J Respir Crit Care Med. 2010; 182: 73-82 and Nho et al, PLoS one 2013; 8]. Several possible mechanisms are proposed for this observation including abnormalities in apoptotic pathways, aberrant Wnt signaling [Chang et al, J Biol Chem. 2010; 285; 8196-8206], and defective autophagy [Patel et al, PLoS One 2012; 7].

However, a number of well characterized cytokines, including TGF-0, have been either found in injured lungs or had been produced by inflammatory cells removed from the lung. Further, in an animal model of pulmonary fibrosis, TGF-β production was increased prior to collagen synthesis and was mainly produced by alveolar macrophages. In advanced idiopathic pulmonary fibrosis extensive TGF-β deposition can be detected by immunohistochemical staining, primarily in epithelial cells in areas of lung regeneration and remodelling. This suggests that the pathogenesis of the progressive fibrosis characteristic of lung diseases such as ILDs, PF-ILDs and IPF may be an aberrant repair process (see Khali et al Ciba Found Symp. 1991; 157: 194-207 and Cutroneo et al, J. Cell. Physiol. 211: 585-589, 2007.

From this background information on fibrosis it is clear that the pathology of fibrotic processes underlying ILDs, PF-ILDs and in particular IPF can be divided into “three different levels of pathogenesis of fibrotic processes”, whereby the chronological order especially of the second and the third level is not yet fully understood and could also partially take place in parallel.

In a first level of fibrotic processes, following exposure to endogenous or exogenous stimuli, the lung epithelium usually initiates an injury response resulting in the production of soluble factors such as Transforming Growth Factor beta-1 (TGF-(31), cytokines and of pro-fibrotic mediators/fibrotic markers such as for instance procollagen, fibronectin and MCP-1.

Then, in a second level of pathogenesis of fibrotic processes, these profibrotic mediators/fibrotic markers promote mesenchymal activation which causes expansion of tissue resident post-embryonic fibroblasts which are thought to give rise to myofibroblasts, an activated form of fibroblasts. These myofibroblasts are central to the process of wound healing, but if unmodulated, they produce excessive amounts of extracellular matrix material and collagen/scar tissue. This “myofibroblast phenotype” is further characterized by a strong α-smooth muscle actin (α-SMA) expression. The transformation/activation of fibroblasts into myofibroblast, which strongly express α-SMA protein, forms the second level of pathogenesis of fibrotic processes common to ILDs, PF-ILDs and IPF.

Consequently quantification of the α-smooth muscle actin (α-SMA) protein expression is a suitable measurement for the extent of transformation/activation of fibroblasts into myofibroblasts which corresponds to the second level of pathogenesis of fibrotic processes common to ILDs, PF-ILDs and IPF.

The third level of pathogenesis of fibrotic processes common to ILDs, PF-ILDs and IPF is characterized by uncontrolled proliferation/cell division and survival of fibroblasts and myofibroblasts, probably by their resistance to programmed cell death. Proliferating fibroblasts and myofibroblasts accumulate in lung interstitium where they deposit excessive amounts of collagen-I rich ECM and ultimately organize into the fibroblastic foci.

Quantification of cell division (for instance by quantification of incorporation of BrdU into the DNA of proliferating fibroblasts) is a suitable measurement for the extent of proliferation of fibroblasts which corresponds to the third level of pathogenesis of fibrotic processes common to ILDs, PF-ILDs and IPF.

6.2 Principle of Experimental Assays A) and B)

Lung fibroblasts of IPF-patients (IPF-LF cells) grown in 96-well plates were incubated for min with different concentrations of the PDE4 inhibitors “Compound of formula III”, “Apremilast” or “Roflumilast-N-Oxide” or with a combination of each of the aforementioned PDE4-inhibitors with Nintedanib.

After compound incubation cells were stimulated with the assay-relevant stimulus and incubated for the assay—relevant time in the presence of the test compounds.

α-SMA protein was determined by a Western-replacement assay (MSD) using monoclonal anti smooth muscle actin antibodies.

BrdU incorporated in the DNA of proliferating cells was determined by ELISA. BrdU is an analog of the DNA precursor thymidine. In proliferating cells, the DNA has to be replicated before the division can take place. If BrdU is added to the cell culture, proliferating cells will incorporate it into their DNA just like they would incorporate thymidine. The amount of BrdU in the DNA of cells can be detected with specific anti-BrdU fluorescent antibodies followed by flow cytometry or by cellular ELISA with monoclonal antibodies against BrdU.

6.3 Experiment A): α-SMA (Smooth Muscle Actin) Protein Assay (Western Replacement Assay)

Cell Seeding and Starvation

IPF-lung fibroblasts (passage 5 to 8) were seeded in 96-well cell culture plates at 4500 cells/well with 100 μL/well FBM+supplements. 24 h after seeding the cells were washed once with FBM medium without supplements and starved for 24 h.

Experiment A1)

In experiment A1) the PDE4B-inhibitor of formula III was used as a “test compound”in rising concentrations either alone (see full circles and black solid curve inFIG.1) orin rising concentrations together with a fixed concentration of 100 nMol/L of Nintedanib (see empty circles and grey solid line inFIG.1).
Experiment A2)

In experiment A2) Apremilast was used as a “test compound”in rising concentrations either alone (see full circles and black solid curve inFIG.2) orin rising concentrations together with a fixed concentration of 100 nMol/L of Nintedanib (see empty circles and grey solid line inFIG.2).
Experiment A3)

In experiment A3) Roflumilast-N-oxide was used as a “test compound”in rising concentrations either alone (see full circles and black solid curve inFIG.3) orin rising concentrations together with a fixed concentration of 100 nMol/L of Nintedanib (see empty circles and grey solid line inFIG.3).
Test Compound Dilutions

All “test compounds” (the PDE4B-inhibitor of formula III, Apremilast or Roflumilast) were prepared 1000× in 0.1 mmol/L HCl or DMSO and a 1:3.16 dilution series was performed (in 0.1 mmol/L HCl or DMSO). To obtain 2× concentrated compound-medium a 1:500 dilution (2 μl of the 1000× dilution was added to 998 μl FBM plus 2 nmol/L PGE2) was prepared.

Pre-Incubation with Test Compounds

48 h after seeding, the medium was aspirated and FBM (100 μl per well) was added. After 1 h incubation at 37° C., 90 μl medium containing 2× concentrated compounds (at different concentrations) plus 2× concentrated PGE2 (2 nmol/L) was added for 30 min Final concentration for PGE2 was 1 nmol/L.

Stimulation

30 min after test compound pre-incubation (190 μL), 10 μl of 20× concentrated TGF-β was added and the cells stimulated for 48 h at a temperature of 37° C.

For this purpose the TGF-β stock solution (20 μg/mL reconstituted in 4 mmol/L sterile HCL) was diluted 1:200 in starvation medium to reach a concentration of 100 ng/mL. 10 μL of this TGF-β medium or starvation medium was added to indicated wells. The test compound concentration was maintained during the stimulation. The final TGF-β concentration was 4 ng/mL.

Protein Lysates

48 h after stimulation supernatants were removed and stored at −80° C. for further experiments. Cells were washed once with ice cold PBS and 50 μl RIPA buffer containing 1× protease inhibitor was added per well. Lysates were incubated for 5 minutes on ice before stored at −80° C.

α-SMA Western Replacement Assay

After thawing, 25 μl of each lysate was transferred to the membrane of the multi-array 96 well plate (MSD) and incubated for 2 h at room temperature with gentle shaking. After the incubation time, plates were washed 3 times with 200 μl 1× Tris-wash buffer (MSD) and 150 μl of 3% blocking buffer was added for 1 h. After blocking, plates were washed 3 times with 200 μl 1× Tris-wash buffer and 25 μl of the antibody solution (per plate 0.75 ml 3% blocking buffer, 2.25 ml 1× Tris-wash buffer, 1.2 μl anti-α-SMA antibody (1:2500), 15 μl goat anti-mouse sulfo-tag antibody (1:200) was added for 1 h. After AB-incubation plates were washed 3 times with 200 μl 1× Tris-wash buffer and 150 μl of 1×MSD read buffer was added per well. Plates were measured with Sector Imager (MSD).

6.4 Experiment B: Cell Proliferation Assay

Cell Seeding and Starvation

IPF-lung fibroblasts (passage 5 to 8) were seeded in 96-well cell culture plates at 2500 cells/well with 100 μL/well FBM+supplements. 24 h after seeding the cells were washed once with FBM medium without supplements and then kept in this medium for 24 h starvation.

Experiment B1)

In experiment B1) the PDE4B-inhibitor of formula III was used as a “test compound”in rising concentrations either alone (see full circles and black solid curve inFIG.4) orin rising concentrations together with a fixed concentration of 100 nMol/L of Nintedanib (see empty circles and grey solid line inFIG.4).

The dashed line with the empty triangles represents the “calculated additive curve” of a combination treatment of 100 nMol/L Nintedanib with the corresponding concentration of the PDE4B-inhibitor of formula III.

Experiment B2)

In experiment B2) Apremilast was used as a “test compound”in rising concentrations either alone (see full circles and black solid curve inFIG.5) orin rising concentrations together with a fixed concentration of 100 nMol/L of Nintedanib (see empty circles and grey solid line inFIG.5).

The dashed line with the empty triangles represents the “calculated additive curve” of a combination treatment of 100 nMol/L Nintedanib with the corresponding concentration of Apremilast.

Experiment B3)

In experiment B3) Roflumilast-N-oxide was used as a “test compound”in rising concentrations either alone (see full circles and black solid curve inFIG.6) orin rising concentrations together with a fixed concentration of 100 nMol/L of Nintedanib (see empty circles and grey solid line inFIG.6).

The dashed line with the empty triangles represents the “calculated additive curve” of a combination treatment of 100 nMol/L Nintedanib with the corresponding concentration of Roflumilast-N-oxide.

Test Compound Dilutions

All test compounds were prepared 1000× in 0.1 mmol/L HCl or DMSO and a 1:3.16 dilution series was performed (in 0.1 mmol/L HCl or DMSO). To obtain 1× concentrated compound medium 1 μl of the 1000×DMSO dilution was added to 999 μl FBM.

Pre-Incubation with Test Compounds

48 h after seeding, medium was removed by suction and 90 μl compound- or starvation medium was added for 30 min

Stimulation

30 min after test compound pre-incubation (90 μL), 10 μl of 10× concentrated FGF plus IL-1β was added and the cells were stimulated for 92 h at a temperature of 37° C.

For this purpose the FGF and IL-1β stock solutions (250 μg/mL and 10 μg/mL respectively) were diluted in starvation medium to reach a concentration of 200 ng/mL and 300 pg/mL for FGF and IL-1β respectively. 10 μL of this stimulus medium or starvation medium was added to the indicated wells. The test compound concentration was maintained during the stimulation.

The final FGF concentration was 20 ng/mL. The final IL-1β concentration was 30 pg/mL.

BrdU Assay

Proliferation was determined by a colorimetric immunoassay for the quantification of cell proliferation, based on the measurement of BrdU incorporation during DNA synthesis. The assay was carried out according to the manufacturer's instructions.

72 h after stimulation a 1:100 dilution of BrdU in starvation medium (resulting concentration 100 μmol/L) was performed and 10 μl added per well (end-concentration per well 10 μmol/l). About 18 h later the BrdU medium was removed by suction. Cells were fixed and denatured for 30 min at room temperature with FixDenat reagent. The reagent was removed by tapping and the anti-BrdU-POD working solution was added (incubation time: 90 min). The plate was washed three times with 200 μL washing buffer before incubation with substrate solution for about 10 min. The reaction was stopped by adding 1 mol/L H2SO4to the substrate solution and plates were read at 450 nm in a photometer (EnVision 2104 Multilabel reader, PerkinElmer).

6.5 Data Analysis

x-fold of unstimulated control was calculated from optical density readings (OD) for BrdU assay or from MSD units (α-SMA assay).

The % inhibition-value was calculated from the x-fold of unstimulated control.

In each of the experiments for the different donors all inhibition values were determined in duplicates or triplicates.

Means of blanks were subtracted from all values.

The IC50-values of stimulated cells were determined as follows:
% inhibition-value=100−(Y/K1)*100K1=mean of ODs of stimulated, non-compound-treated control wells minus mean of ODs of non-stimulated, non-compound-treated control wellsY=OD of stimulated, compound-treated well

Non-linear regression of log (inhibitor concentration) versus % inhibition-value was calculated using three parameter fitting with variable slope of the Graph Pad Prism Software package.

To calculate the additive effect of the compound of formula III, Apremilast or Roflumilast-N-Oxide combined with Nintedanib the following formula was used
Effect of PDE4 inhibitor (EB)+effect of Nintedanib(EN)=EB+N=EB+EN−(EB*EN)=dashed curve (Poch & Holzmann, 1980).
Test Compounds

The test compounds Compound of formula III, Apremilast, Roflumilast-N-Oxide, and Nintedanib were dissolved in DMSO and stored at −20° C. A serial dilution of 7 concentrations was prepared before each experiment.

6.6 Material and Methods

MaterialOrderTest ArticleProvidernumberIPF-LF cell line (passage 5 to 8)AsterandDI16769DI16783DI19873BI209755BI210978BI212020rhTGF-βR&DSystems240-B-010rhFGF basicR&DSystems234_FSErhIL-1βR&DSystems201-LB-005rhPGE2Tocris2296Monoclonal anti smooth muscleSigmaA2547actin antibodyGoat anti-mouse sulfo-tag antibodyMSDR32AC-1Multi-Array 96-well Plate HighMSDL15XB-3Bind platesMSD Blocker AMSDR93BA-4MSD Tris Wash Buffer (10x)MSDR61TX-1MSD Read Buffer T (4x)MSDR92TC-2RIPA bufferSigmaR0278-500MLHalt Protease-Inhibitor cocktailThermoScientific78437(100x)PBSGibco10010023BrdU-AssayRoche11647229001Cell culture flask, 75 cm2, tissue-BD Falcon ™353110culture treatedCell culture flask, 175 cm2, tissue-BD Falcon ™353112culture treated96-well plate (cell culture)Nunc microwell 96F167008DMSOMerck1.02952.1000Cells-to-CT 1 Step TaqMan kitAmbionA25602
Cell Propagation Media:

FBM (fibroblast basal medium, Lonza, Cat. No: CC-3131) supplemented with insulin, FGF-2, 0.5% FBS, GA-1000 (all in FGM-2 SingleQuots, Lonza, Cat. No. CC-4126)

Reagents for Subculturing IPF-LF Cells:

Hepes buffered saline solution (Lonza, Cat. No. CC-5022)Trypsin/EDTA (0.25 mg/mL) (Lonza, CC-5012)TNS (trypsin neutralizing solution, Lonza, CC-5002)
Starvation Medium:

FBM without supplements

Stimulation Medium α-SMA Assay:

FBM plus 4 ng/mL rhTGF-β and 1 nmol/L PGE2

Stimulation medium BrdU assay:

FBM plus 20 ng/mL rhbFGF plus 30 pg/mL rhIL-1β

6.7 Interpretation of Experiments

Experiment A): Inhibition of TGF-β-Stimulated α-SMA Protein Expression of Human Lung Fibroblasts from Patients with IPF

The more a specific active agent tends to inhibit the TGF-β-stimulated α-SMA protein expression of human lung fibroblasts of IPF patients, the more this active agent will have a therapeutic effect in the second level of pathogenesis of fibrotic processes which is the transition of fibroblasts into myofibroblasts.

Consequently in this Experiment A) mimicking the second level of fibrotic processes the effect ofa) Nintedanib alone, the compound of formula III alone, Apremilast alone and Roflumilast-N-oxide alone andb) of the compound of formula III with Nintedanib, of Apremilast with Nintedanib and of Roflumilast-N-oxide with Nintedanibon the TGF-β-stimulated α-SMA protein expression of human lung fibroblasts of IPF patients was experimentally determined.

Whereas Nintedanib—administered alone—in the concentration 100 nMol/L showed in this experiment no inhibitory effect on TGF-β-stimulated α-SMA protein expression of human lung fibroblasts (supporting the fact that Nintedanib in this concentration alone shows no therapeutic effect on the second level of pathogenesis of fibrotic processes (see □ inFIGS.1,2and3: Inhibition was ≤0), all tested PDE4-inhibitors (the compound of formula III, Apremilast and Roflumilast-N-oxide)—when administered alone and also when administered together with Nintedanib in the fixed concentration of 100 nMol/L—showed—at least in certain concentrations—a concentration-dependent inhibition on TGF-β-stimulated α-SMA protein expression of human lung fibroblasts which supports a certain therapeutic effect of all these PDE4-inhibitors in the second level of pathogenesis of fibrotic processes (the activation to myofibroblasts).

From these results it can be concluded that PDE4-inhibitors—at least in certain concentration ranges—have the potential to show a concentration-depending therapeutic effect on the “fibroblast to myofibroblast transition/activation”, an event that represents the second level of pathogenesis of fibrotic processes which are common to ILDs, particularly to PF-ILDs, whereas Nintedanib in the concentration 100 nMol/L alone does not show a therapeutic effect on this very same “second level of pathogenesis” according to this experiment.

Consequently PDE4-inhibitors show in relation to Nintedanib a so-called “complementary effect” or “supplementary effect” on the “fibroblast to myofibroblast transition/activation” (=second level of pathogenesis of fibrotic processes). Therefore an administration of Nintedanib together with a PDE4B-inhibitor of formula III will show a superior effect on therapeutic efficacy compared to the IPF-treatment with for instance Nintedanib alone.

If you compare the measured inhibition on the TGF-β-stimulated α-SMA protein expression of human lung fibroblasts for the compound of formula III (FIG.1), for Apremilast (FIG.2) and for Roflumilast-N-oxide (FIG.3), it is obvious that only for the compound of formula III (FIG.1) the complete concentration/inhibition curve is located at inhibitions of “above zero”, whereas for instance for Apremilast and in particular for Roflumilast-N-oxide” low PDE4-inhibitor concentrations (either alone or in combination with Nintedanib)” lead to “negative inhibitions of TGF-β-stimulated α-SMA protein expression” (supporting the absence of a therapeutic effect on the second level of fibrotic processes for Apremilast and in particular for Roflumilast-N-oxide at these lower concentrations (whereas the compound of formula III seems to show a positive inhibition of TGF-β-stimulated α-SMA protein expression in all tested concentrations).

Experiment B): Inhibition of Fibroblast Proliferation

The more a specific active agent tends to inhibit proliferation of cultured human lung fibroblasts of IPF patients, the more this active agent will have a therapeutic effect in the third level of pathogenesis of fibrotic processes which is fibroblast proliferation.

Consequently the effect ofa) Nintedanib alone, the compound of formula III alone, Apremilast alone and Roflumilast-N-oxide alone andb) of the compound of formula III with Nintedanib, of Apremilast with Nintedanib and of Roflumilast-N-oxide with Nintedanibon the proliferation of human lung fibroblasts of IPF patients was experimentally determined in Experiment B).

In this experiment B) mimicking the third level of pathogenesis of fibrotic processes (the “fibroblast proliferation”), Nintedanib administered alone in the concentration 100 nMol/L already showed a clear inhibitory effect on human lung fibroblasts proliferation (see inhibition data points symbolized by □ inFIGS.4,5and6).

However, the results of Experiments B1) inFIG.4, B2) inFIG.5and B3) inFIG.6show that not only Nintedanib alone has an inhibitory effect on fibroblast proliferation, but that also PDE4-inhibitors such as the compound of formula III (see filled circles and black solid curve in B1,FIG.4)), Apremilast (see filled circles and black solid curve in B2,FIG.5)) and Roflumilast-N-oxide (see filled circles and black solid curve in B3 inFIG.6)) show in general a concentration-dependent inhibitory effect on fibroblast proliferation and therefore seem to have a therapeutic effect on fibroblast proliferation (third level of pathogenesis of fibrotic processes).

Since obviously both Nintedanib in the fixed concentration of 100 nMol/L and the tested PDE4-inhibitors concentration-dependently show an inhibitory effect on fibroblast proliferation, a simple “additive effect” for the inhibition of fibroblast proliferation by the combination of 100 nMol/L Nintedanib and the corresponding PDE4-inhibitor in its respective concentration should be expected.

InFIGS.4,5and6the dashed curves with the empty triangles represent these “calculated additive combination curves” which were calculated from the simple “addition” of the measured inhibition-value for 100 nMol/L Nintedanib plus the measured inhibition—value for the corresponding PDE4-inhibitor alone in variable concentrations.

However, the grey solid curves with the empty circles inFIGS.4,5and6represent the “experimentally measured inhibition-curves for the combinations comprising 100 nMol/L Nintedanib and the corresponding PDE4-inhibitor in variable concentrations”.

Surprisingly, inFIG.4which shows the results of Experiment B1) the “experimentally measured inhibition curve of fibroblast proliferation” for the combination of Nintedanib with the compound of formula III (solid grey line, empty circles) is “significantly shifted to the left” (that means towards lower concentrations of the compound of formula III) compared to the corresponding “calculated additive inhibition curve” for the combination of Nintedanib with the compound of formula III (dashed curve with empty triangles).

This significant “left-shift” is a clear indicator for an “overadditive synergistic effect” of the combination of 100 nMol/L Nintedanib with the compound of formula III. This experimentally observed “overadditive synergistic effect” for the combination of Nintedanib and the compound of formula III was completely surprising, in particular because this synergistic overadditive effect does not seem to be a “class effect”.

FIG.5shows the results of the corresponding Experiment B2), wherein the compound of formula III was exchanged by Apremilast.FIG.5shows that the “experimentally measured inhibition curve” for the combination of Nintedanib with Apremilast (solid grey line, empty circles) is not shifted to the left, but instead is even slightly shifted to the right (that means to higher Apremilast concentrations) compared to the corresponding “calculated additive inhibition curve” for the combination of Nintedanib with Apremilast (dashed curve with empty triangles). Such a “right-shift” would theoretically even be an indicator for a “less than additive inhibition of fibroblast proliferation” (an “anti-synergistic effect”) by the combination of Nintedanib and Apremilast. However, this rather slight right-shift of the “measured Nintedanib/Apremilast combination curve” compared to the “calculated Nintedanib/Apremilast combination curve” is more or less within the error bars and therefore not statistically relevant. Consequently, for the combination of Nintedanib with Apremilast more or less a normal “additive effect” as expected could be experimentally observed.

FIG.6shows the results of the corresponding Experiment B3), wherein the compound of formula III was exchanged by Roflumilast-N-oxide.FIG.6shows that the “experimentally measured inhibition curve” for the combination of Nintedanib with Roflumilast-N-oxide (solid grey line, empty circles) is also shifted to the right instead to the left compared to the corresponding “calculated additive inhibition curve” for the combination of Nintedanib with Roflumilast-N-oxide (dashed curve with empty triangles). Such a “right-shift” is an indicator for a “less than additive inhibition of fibroblast proliferation” (anti-synergistic effect) for the combination of Nintedanib and Roflumilast-N-oxide. This “right-shift” of the “measured Nintedanib/Roflumilast combination curve” compared to the “calculated Nintedanib/Roflumilast combination curve” is only for very high Roflumilast-N-oxide concentration beyond the error bar ranges. Consequently for the combination of Nintedanib with Roflumilast-N-oxide also a more or less “additive effect” as expected could be experimentally determined.

This “overadditive synergistic effect” on the inhibition of fibroblast proliferation which was exclusively observed for the combination of Nintedanib with the compound of formula III is also reflected in the large differences of the IC50-values calculated for the concentration/inhibition curvesa) measured for human lung fibroblast of IPF patients treated with the compound of formula III alone inFIG.4(solid black curve, IC50-value of 255 nMol/L) andb) measured for human lung fibroblast of IPF patients treated with the combination comprising the compound of formula III and Nintedanib inFIG.4(solid grey curve, IC50-value of 23 nMol/L).

Here the IC50-value for the inhibition curve measured for the compound of formula III administered alone is compared to the IC50-value for the inhibition curve measured for the combination of the compound of formula III with Nintedanib 11-fold larger (255 nMol/L/23 nMol/L=11).

In contrast to that, the corresponding differences in the IC50-values for the inhibition curves measured for the other PDE4-inhibitors Apremilast and Roflumilast-N-oxide administered alone compared to the inhibition curve measured for the corresponding PDE4-inhibitor/Nintedanib combinations were much smaller (1.13-fold larger for Apremilast, 0.82-fold smaller for Roflumilast-N-oxide).

This experimentally determined “overadditive synergistic effect” on the inhibition of fibroblast proliferation which was exclusively observed for the combination of the compound of formula III with Nintedanib obviously does not seem to be a “class effect”, since none of the other tested PDE4-inhibitors Apremilast or Roflumilast showed in combination with Nintedanib a corresponding similar “overadditive synergistic effect”, but instead only the expected “additive inhibitory effect” (Nintedanib/Roflumilast-N-oxide showed at large Roflumilast-N-oxide-concentrations even a “less than additive inhibitory effect”).

Consequently the combination of Nintedanib with the PDE4B-inhibitor of formula III shows due to the experimentally observed overadditive synergistic inhibitory effect on fibroblast proliferation surprisingly a clearly improved therapeutic efficacy for the treatment of PF-ILD-patients not only compared to treatment with the individual single agents, but also compared to the alternative combinations Nintedanib/Roflumilast-N-oxide and Nintedanib/Apremilast.

Consequently Experiments A) and B) have experimentally shown that the combination comprising the PDE4B-inhibitor of formula III and Nintedanib shows1.) on the “second level of pathogenesis of fibrotic processes common to PF-ILDs” (activation of fibroblasts to myofibroblasts) a clear therapeutic effect over the complete range of tested concentrations for the PDE4B-inhibitor of formula III (whereby Nintedanib alone showed no therapeutic effect on the second level) and2.) on the “third level of pathogenesis of fibrotic processes common to PF-ILDs” (fibroblast proliferation) surprisingly even an “overadditive synergistic therapeutic effect” (which the Roflumilast-N-oxide/Nintedanib- and Apremilast/Nintedanib-combinations surprisingly did not show).

Another additional advantage the combination of the PDE4B-inhibitor of formula III with Nintedanib obviously shows compared to other PDE4-inhibitor/Nintedanib combinations (such as for instance Roflumilast-N-oxide/Nintedanib) is its relatively good tolerability (in particularly with respect to gastrointestinal side effects).

It is known that Nintedanib and also Pirfenidone—the presently two only approved therapeutic agents for the treatment of IPF—show both significant gastrointestinal side effects such as diarrhea, nausea, vomiting, weight loss etc. which is the main reason why Nintedanib and Pirfenidone are usually not combined due to their additive and therefore more frequent gastrointestinal side effects.

In contrast to Nintedanib and Pirfenidone, the PDE4B-inhibitor of formula III has been shown to be relatively free of the PDE4-inhibitor-typical gastrointestinal side effects such as diarrhea in a corresponding rat experiment (see WO 2013/026797 Chapter 5.3: Experiments of “gastric emptying” and “intestinal transit” andFIG.2a(gastric emptying) and2b(intestinal transit)). In these experiments it could be shown that a rising amount of Example compound No. 2 (which is identical to the PDE4B-inhibitor of formula III in the present application) had basically no effect on the gastric emptying and on the intestinal transit of a test meal in the rat compared to non-treated rats.

However, in similar “gastric emptying” and “intestinal transit” experiments the alternative PDE4-inhibitor Roflumilast has shown a clear trend to show gastrointestinal side effects.

Additionally, it is also well known from clinical trials that Roflumilast (which is only authorized for the treatment of COPD) shows significant gastrointestinal side effects in human COPD-patients such as diarrhea, nausea, weight loss.

In http://www.rxlist.com/daliresp-drug.htm it is disclosed that Roflumilast given to COPD-patients in a dose of 500 μg daily leadin 9.5% of all patients to diarrhea (compared to only 2.7% to the patients receiving placebo)in 4.7% of all patients to nausea (compared to only 1.4% to the patients receiving placebo)in 7.5% of all patients to decreased weight (compared to only 2.1% to the patients receiving placebo) andin 4.4% of all patients to headache (compared to only 2.1% to the patients receiving placebo).

Due to the observations mentioned above the combination of the PDE4B-inhibitor of formula III with Nintedanib has a better tolerability with respect to gastrointestinal side effects compared to for example a combination of Roflumilast with Nintedanib. Additionally the combination of the PDE4B-inhibitor of formula III with Nintedanib has a better therapeutic efficacy with respect to treating ILDs, PF-ILDs and in particular IPF (seeFIG.1-6) combined with an acceptable tolerance with respect to gastrointestinal side effects (WO 2013/026797 Chapter 5.3).