WO2019038156A1

WO2019038156A1 - Use of an ep4 antagonist for the treatment of arthritis

Info

Publication number: WO2019038156A1
Application number: PCT/EP2018/072132
Authority: WO
Inventors: Alexandra RAUSCH; Jens Nagel
Original assignee: Bayer Pharma AG
Current assignee: Bayer Pharma AG
Priority date: 2017-08-22
Filing date: 2018-08-15
Publication date: 2019-02-28
Anticipated expiration: 2020-02-22

Abstract

The present invention relates the use of compound 1 for the treatment of arthritis especially rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, as well as other other immune- mediated rheumatic diseases.

Description

Use of an EP4 antagonist for the treatment of arthritis

The present invention relates to the use of an EP4 antagonist for the treatment and/or prophylaxis of arthritis especially rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, as well as other immune-mediated rheumatic diseases, and/or other pathological conditions associated with increased activation of EP4 receptors by prostaglandin E2 (PGE2).

BACKGROUND OF THE INVENTION

Arthritis is not a single disorder, but is a common term for immune-mediated rheumatic diseases, which comprise a group of more than 100 diseases that affect the joints, like for instance rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA). Arthritis is characterized by progressive inflammation, severe pain, swelling and stiffness of the affected synovial joints (Koenders & van den Berg, 2016). Whereas RA mainly affects the joints in a symmetrical manner and is often associated with production of autoantibodies (e.g. rheumatoid factor), PsA and AS are considered as seronegative rheumatic diseases. In contrast to RA, the joint involvement in PsA affects the periphery in an asymmetrical manner, whereas AS is a disease of the axial skeleton mainly affecting the spine and sacroiliac joint in the pelvis leading to complete fusion and rigidity of the spine (due to ossification).

In general, the outcome of these progressive disorders are cartilage and bone/joint destruction, and thereby loss of function, in addition to a decreased life expectancy and increased morbidity amongst sufferers. Up to 1 % of the people in the western world are affected e.g. by RA and although the etiology of the arthritis is unknown, there are a number of environmental and genetic risk factors associated with disease incidence (smoking, obesity, HLA type, etc.) {Sparks and Costenbader, 2014).

A characteristic hallmark of arthritis is the activation of immune cells that target self-tissue leading to inflammation upon production of pro-inflammatory cytokines. Once the disease is triggered, immune cells (e.g. antigen-presenting cells like dendritic cells and monocytes/macrophages) migrate into the joints, where they secrete large amounts of cytokines and chemokines leading to the activation and intense recruitment of further immune cells (e.g. T cells, B cells) into the joints. Thereby, hyperplasia of the normally thin synovial membrane occurs and fluid collects in the narrow joint space causing swelling and impaired mobility (Firestein, 2003). The arising joint inflammation can differ in the severity between patients, but is chronic and progressive in nature causing joint damage and bone erosion (Pablos and Canete, 2013).

Despite the differences in pathogenesis and clinical presentation of RA, AS and PsA, the treatment of these inflammatory arthritic disorders is very overlapping. Nonsteroidal antiinflammatory drugs (NSAIDs) are used to reduce pain and inflammation in arthritis and represent an effective therapy (Koenders & van den Berg, 2016). However, due to the risk of side-effects under long term treatment this treatment option exists only for a short term treatment employing doses as low as possible. Additionally, conventional disease-modifying anti-rheumatic drugs (DMARDs) such as methotrexate (MTX) are still prescribed for the treatment of arthritis despite their side-effects. Since biologic agents such as tumor necrosis factor (TNF) inhibitors have become available they are used for patients not responding to DMARDs. However, based on the results of clinical trials, more than half of arthritis patients with moderate to severe disease (target population) treated with TNF blockers do not achieve a substantial response. Furthermore, some arthritis patients treated with cytokine inhibitors lose their responsiveness over time, develop intolerabilities or side effects and need to switch to other agents. Therefore, a high unmet medical need remains and alternative strategies as long-term treatment options with reduced side-effects are needed to fight arthritic disorders and to further improve patients' lives.

The receptor EP4 (PTGR₄) is one of the 4 human receptors that are activated by endogenously formed prostaglandin E2 (PGE2). EP4 belongs to the family of membrane- bound G-protein coupled receptors (GPCR) and is mainly provided with a Gs coupling, which after activation leads to an accumulation of the intracellular signal molecule cyclic AMP (cAMP). The expression of the receptor was detected on different immune cells, such as T cells, dendritic cells, and macrophages {Yao, Sakata, Esaki et al., 2009). For these cell types, especially TH17 (T helper 17) cells, great importance was demonstrated in connection with the pathogenesis in arthritis (Paulissen, van Hamburg, Davelaar et al., 2013; Koenders & van den Berg, 2016). Binding of PGE2 to its EP4 receptor participates and promotes TH 17 cell differentiation/maturation, expansion and function {Yao, Sakata, Esaki et al., 2009; Boniface et al., 2009; Sheibanie et al., 2004; Khayrullina et al., 2008). PGE2 thereby increases the IL-17 production from TH17 cells through the EP2/EP4 - cAMP signaling pathway {Yao, Sakata, Esaki et al., 2009; Boniface et al., 2009). Furthermore, PGE2-EP4 signaling indirectly promotes TH17 cell expansion, due to the fact that it stimulates interleukin (IL)-23 production by dendritic cells {Yao, Sakata, Esaki et al., 2009; Chizzolini et al., 2008; Boniface et al., 2009; Napolitani et al., 2009). Next to this, Sheibanie et al. showed that PGE2 exacerbates collagen-induced arthritis in mice via the IL-23/IL-17 axis {Sheibanie, Khayrullina, Safadi, Ganea et al. 2007). Additionally, PGE2 has been associated with the edema and the erosion of cartilage and bone in arthritis {McCoy, Wicks, Audoly, 2002). Furthermore, it has been shown by McCoy et al. that EP4 receptor-deficient mice show a decreased incidence and severity of arthritis related to reduced IL-6 and serum amyloid A levels. Histopathological evaluation of these EP4 receptor-deficient mice revealed reduced bone destruction, proteoglycan loss, and type II collagen breakdown in cartilage compared to wildtype mice (McCoy, Wicks, Audoly, 2002). Antagonists of the EP4 receptor might therefore provide novel agents for the treatment of arthritis as well as other immune- mediated rheumatic diseases and/or other pathological conditions caused by increased activation of EP4 receptors by prostaglandin E2 (PGE2).

Up to now, no EP4 antagonist has been licensed as a medicament. However, EP4 antagonists of different structural classes have been described, Thus in WO20050121508, WO2005102389 and WO2005105733 (Pfizer), for example, /V-benzylarylamides, N- benzylheteroarylamides and [(1 H-benzimidazol-1 -yl)phenylethyl]aryl- and [(1 H-benzimidazol- 1 -yl)phenylethyl]heteroarylsulphonylcarbamates are described for use in the case of pain, inflammation, osteoarthritis and rheumatoid arthritis. Pfizer also describes in WO2002032422, WO2002032900 and WO2003086371 structures that include generic benzimidazoles. Thiophene-/V-benzylamides in WO2008017164 and WO2009020588, indole-N-benzylamides in WO2007121578 and N-{[(6,8-dihydro-7H-pyrrolo[3,4-g]quinolin-7- yl)-aryl]methyl}sulphonylamides in WO2008104055 are addressed for nearly the same indication spectrum by Merck-Frosst. In WO2004067524 (Pharmagene Laboratories), furan derivatives for the treatment of headache and migraine are described.

EP2172447 (Astellas Pharma) claims generically in a very broad manner compounds that can consist of two heterocycles connected directly to one another, for the indications renal insufficiency and diabetic nephropathy.

In WO2014086739 (Bayer Pharma) novel benzimidazole-5-carboxylic acid derivatives and their use for treatment of endometriosis are disclosed. The disclosure of this application is incorporated herewith in its entirety.

As mentioned above there is an urgent need for medicaments which are effective in the treatment of arthritis especially rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, as well as other immune-mediated rheumatic diseases and/or other pathological conditions caused by increased activation of EP4 receptors by prostaglandin E2 (PGE2), which do not have the disadvantages of the prior art.

The underlying problem of the present invention therefore lies in the provision of medication for treatment of arthritis especially rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, as well as other immune-mediated rheumatic diseases and/or other pathological conditions associated with increased activation of EP4 receptors by prostaglandin E2 (PGE2). SUMMARY OF THE INVENTION

It was found that compound 1 (2-(9-Ethyl-6-methyl-9H-carbazol-3-yl)-1 -(2-methoxyethyl)-4- methyl-1 H-benzimidazole-5-carboxylic acid)

is a highly potent, competitive human EP4 receptor antagonist with inverse agonist activity (Fig. 1 ). Secondary in vitro assays revealed a high functional potency on EP4 receptors of mouse, rat and cynomolgus. Compound 1 was highly active in inhibiting the PGE2-mediated augmentation of IL-17 secretion during maturation of TH17 cells in vitro (Fig. 2).

Next to this, compound 1 showed robust efficacy in murine arthritis studies in vivo. Here, the compound attenuated the disease severity, which was assessed by the disease activity score (Fig. 3), as well as the joint swelling (Fig. 4). This anti-inflammatory reduction in the in- life parameters after treatment with compound 1 were confirmed by histopathological evaluation of joints (Fig. 6). Additionally, hyperalgesia was reduced by treatment with compound 1 in arthritic rats (Fig. 5). Evaluation of clinical relevant pro-inflammatory biomarkers revealed an inhibition of the erythrocyte sedimentation rate (ESR), the levels of CRP (C-reactive protein) and the cytokine IL-6 after treatment with compound 1 during experimental arthritis (Fig. 7 A, B, C). Therefore the use of compound 1 for the treatment of arthritis especially rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis, as well as other immune-mediated rheumatic diseases, and/or other pathological conditions associated with increased activation of EP4 receptors by prostaglandin E2 (PGE2) provides a solution for the underliying problem of the invention.

DESCRIPTION OF THE FIGURES

Figure 1 : Characterization of binding

This figure shows the binding characteristics of compound 1 . Experiments were performed to show that the binding is antagonistic (Part A), agonistic (part B) or inverse agonistic (Part C). The results show that compound 1 is a highly potent, competitive human EP4 receptor antagonist with inverse agonist activity. In figure 1A square (A), B (triangle), C (circle) and D (diamond) show the results of the same experiment. In figure 1 B square stands for PGE2 at 1 x ECso (HCS-QZY) =14.0 nM; triangle for PGE2 at 4 x EC₈₀ (HCS-QZZ) = 56.0 nM; circle for PGE2 at 10 x EC₈₀ (HCS-RAA) = 100.0 nM and diamond for PGE2 at 16 x EC₈₀ (HCS- RAB) = 150.0 nM. In figure 1 C square (A) and B (triangle) show the results of the same experiment.

Figure 2: Dose-dependent inhibitory effect of compound 1 on PGE2-facilitated IL-17 production

Total CD4⁺ T cells isolated from human PBMCs were stimulated with anti-CD3/anti-CD28 plus IL-23 in the presence or absence of exogenous PGE2 and compound 1 for 3 days. Compound 1 was applied to stimulated cell culture in the indicated concentrations ranging from 1 pM up to 1 μΜ. Data are shown as means ± SD. These data from one donor are representative of at least four independent donors. Statistical analysis was performed by ANOVA with Dunett^'s Test, ^*p < 0.05; ^**p < 0.01 ; ^***p < 0.005; ^****p < 0.001 compared with anti-CD3/anti-CD28 + IL-23 + PGE2-stimulated CD4⁺ T cells. (rhlL-23 stands for recombinant human IL-23).

Figure 3: Effect of compound 1 on disease severity in the rat AIA model

Disease activity score was evaluated on the day of CFA injection (day 0) and daily onwards showing first symptoms (> day 8). Treatment with compound 1 (0.2 mg/kg = open triangle, 1 mg/kg = grey triangle and 5 mg/kg = filled triangle, once daily dose (QD) p.o.), or Etanercept (1 .5 mg/kg, grey square, QD s.c.) started with the induction of arthritis. Healthy control group (ctrl. = open circle) and AIA group (AIA= filled circle) were treated with vehicle only. Data are shown as mean ±SD of n= 8 rats per group. ANOVA with Dunett^'s Test, ^*p < 0.05; ^**p < 0.01 ; ^***p < 0.005; ^****p < 0.001 compared with AIA group. The figure clearly shows that compound 1 reduced the severity of adjuvant-induced arthritis in rats significantly.

Figure 4: Inhibition of joint swelling by compound 1 in rat AIA model

Joint thickness, measured sagittal x transversal (in mm²), was evaluated by an automated calipar before the arthritis induction (day 0) and afterwards on days 8, 10, 13, 15, 17 and 20. Treatment with compound 1 (0.2 mg/kg = open triangle, 1 mg/kg = grey triangle and 5 mg/kg = filled triangle, once daily dose (QD) p.o.), or Etanercept (1.5 mg/kg, grey square, QD s.c.) started with the induction of arthritis. Healthy control group (ctrl = open circle) and AIA group (AIA = filled circle) were treated with vehicle only. Data are shown as mean ±SD of n= 8 rats per group. ANOVA with Dunett^'s Test, ^*p < 0.05; ^**p < 0.01 ; ^***p < 0.005; ^****p < 0.001 compared with AIA group (AIA). Figure 4 shows that compound 1 significantly reduced the swelling and thereby the inflammation within the joints after arthritis induction.

Figure 5: Inhibitory effect of compound 1 on hyperalgesia by increase of grip strength in rat AIA

Hyperalgesia, measured via loss of grip strength, was detected by a grip strength test meter before the arthritis induction (day 0) and afterwards on days 8, 10, 13, 15, 17 and 20. Treatment with compound 1 (0.2 mg/kg = open triangle, 1 mg/kg = grey triangle and 5 mg/kg = filled triangle, once daily dose (QD) p.o.), or Etanercept (1.5 mg/kg, grey square, QD s.c.) started with the induction of arthritis. Healthy control group (ctrl. = open circle) and AIA group (AIA= filled circle) were treated with vehicle only. Data are shown as mean ±SD of n= 8 rats per group. ANOVA with Dunett^'s Test, ^*p < 0.05; ^**p < 0.01 ; ^***p < 0.005; ^****p < 0.001 compared with AIA group (AIA). Figure 5 shows that high doses of compound 1 significantly increased the grip strength in rat AIA indicating an inhibitory effect on hyperalgesia.

Figure 6: Compound 1 improves joint pathology in rat AIA model

Histopathological evaluation of joints from rat AIA model collected at day 20. Treatment with compound 1 (2.5 mg/kg = grey triangle and 5 mg/kg = dark grey triangle, once daily dose (QD) p.o.) started with the induction of arthritis at day 0. Healthy control group (ctrl. = open circle) and AIA group (AIA = filled circle) were treated with vehicle only. Histopathology score grading arthritis severity: 0 = normal, 1 = mild, 2 = moderate, 3 = marked, 4 = severe. Data are shown as mean ±SD of n= 8 rats per group. ANOVA with Dunett^'s Test, ^*p < 0.05; ^**p < 0.01 ; ^***p < 0.005; ^****p < 0.001 compared with AIA group (AIA). Figure 6 represents the significant efficacy of compound 1 treatment with regard to the prevention of joint destruction after blinded evaluation of the joint histopathology in AIA rats.

Figure 7: Anti-inflammatory effects of compound 1 on inflammatory biomarkers in rat AIA model

Erythrocyte sedimentation rate (ESR) was quantified in plasma, whereas both C-reactive protein (CRP) and pro-inflammatory cytokine IL-6 were quantified in joint tissue extract from day 20. Treatment with compound 1 (2.5 mg/kg = grey triangle) and 5 mg/kg QD p.o. = dark grey triangle) started with the induction of arthritis. Healthy control group (ctrl.= open circle) and AIA group (AIA= filled circle) were treated with vehicle only. Data are shown as mean ± SD of n= 8 rats per group. ANOVA with Dunett^'s Test, ^*p < 0.05; ^**p < 0.01 ; ^***p < 0.005; ^****p < 0.001 compared with AIA group (AIA = filled circle). Figure 7 clearly shows a decrease in levels of pro-inflammatory mediators ESR, CRP and IL-6 in plasma and respectively joint tissue of AIA rats.

The data presented in several in vitro and in vivo models demonstrates the effectiveness of compound 1 for treatment and/or prophylaxis of arthritis especially rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, as well as other immune-mediated rheumatic diseases, and/or other pathological conditions associated with increased activation of EP4 receptors by prostaglandin E2 (PGE2).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that compound 1 (2-(9-Ethyl-6-methyl-9H- carbazol-3-yl)-1 -(2-methoxyethyl)-4-methyl-1 H-benzimidazole-5-carboxylic acid) is highly potent in the treatment of arthritis especially rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, as well as other immune-mediated rheumatic diseases, and/or other pathological conditions associated with increased activation of EP4 receptors by prostaglandin E2 (PGE2). Therefore subject matter of the present invention is directed to the use of compound 1 for treatment of arthritis especially rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, as well as other immune-mediated rheumatic diseases, and/or other pathological conditions associated with increased activation of EP4 receptors by prostaglandin E2 (PGE2).

DEFINITION

The therapeutically active dose is dependent on the body weight, administration route, individual behaviour, and the type of preparation and time or interval at which administration takes place. A typical dose range for a woman of 70 kg body weight is between 1 -500 mg/day, preferably between 5 and 30 mg/day.

A further subject of the present invention relates to medicaments containing compound 1 according to the invention and at least one or more other active substances, in particular for the treatment and/or prophylaxis of arthritis. Suitable combination with active substances that may be mentioned by way of example and preferably are: inhibitors of mitogen activated protein (MAP) kinase and inhibitors of MAP kinases (Mkk3/6, Mek1/2, Erk1/2), inhibitors of protein kinases B (ΡΚΒα/β/γ; Akt1/2/3), inhibitors of phosphoinositide-3 kinase (PI3K), inhibitors of cyclin-dependent kinase (CDK1/2), Inhibitors of the hypoxia-induced signal pathway (HIF1 alpha inhibitors, activators of the prolylhydroxylases), histone deacetylase (HDAC) inhibitors, prostaglandin F receptor (FP) (PTGFR) antagonists, neurokinin 1 receptor antagonists, paracetamol, selective COX2 inhibitors and/or non-selective COX1/COX2 inhibitors; immunomodulatory and immunosuppressive compounds such as cyclosporine, Methotrexate® (MTX), Jak/STAT inhibitors (e.g Baricitinib, Tofacitinib, GLPG0634), TNF antagonists (e.g. Humira®, etanercept, infliximab, certolizumab), IL-1 inhibitors (e.g. anakinra, canakinumab, rilonacept), IL-17 inhibitors (e.g. secukinumab, brodalumab, ixekizumab), IL-6/IL-6 receptor antagonists (e.g. tocilizumab, sarilumab, siltuximab), IL-12/IL- 23 inhibitors (e.g. ustekinumab), phosphodiesterase inhibitors (e.g. Apremilast®), leflunomid, teriflunomid, cyclophosphamide, rituximab, belimumab, tacrolimus, rapamycin, mycophenolate mofetil, interferons, corticosteroids (e.g. prednisone, prednisolone, methylprednisolone, hydrocortisone, betamethasone), cyclophosphamide, azathioprine and sulfasalazine, paracetamol, non-steroidal anti-inflammatory substances (NSAIDS) (aspirin, ibuprofen, naproxen, etodolac, celecoxib, colchicine).

Compound 1 can be employed for the treatment of arthritis especially rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis, both after oral as well as parenteral administration.

Compound 1 according to the invention can act systemically and/or locally. For this purpose, it can be administered in a suitable manner, such as e.g. orally, parenterally, pulmonarily, nasally, sublingually, lingually, buccally, rectally, dermally, transdermally, conjunctivally, otically or as an implant or stent.

For these administration routes, the compound according to the invention can be administered in suitable administration forms.

The dosage of compound 1 in these preparations should be 0.01 % - 20 %, in order to achieve an adequate pharmacological action.

The dosage of the active substances can vary, depending on administration route, age and weight of the patient, nature and severity of the disease to be treated and similar factors. The treatment can be carried out by means of individual doses or by a plurality of doses over a relatively long period. In the case of oral administration, the amount per day is approximately 0.01 to 100 mg/kg of body weight. The amount of compound 1 to be administered varies within a wide range and can cover any effective amount. A typical dose range for a woman of 70 kg body weight is between 1 - 500 mg/day, preferably between 5 and 30 mg/day. Nevertheless, it can optionally be necessary to deviate from the amounts mentioned, namely, depending on body weight, administration route, individual behaviour towards the active substance, nature of the preparation and time or interval at which administration takes place. Thus, in some cases it can be adequate to manage with less than the aforementioned minimum amount, while in other cases the upper limit mentioned must be exceeded. In the case of the administration of relatively large amounts, it can be advisable to divide these into a number of individual doses over the course of the day.

As carrier systems, surface-active excipients such as salts of the bile acids or animal or vegetable phospholipids can also be used, but also mixtures thereof and liposomes or their constituents.

The formulations and administration forms described above are likewise the subject of the present invention.

If, in addition to the compound according to the invention, further active substances are contained, these can be formulated in a common administration form or optionally also administered as a combination preparation. For oral administration, administration forms functioning according to the prior art, releasing compound 1 to be used according to the invention rapidly and/or in modified form, which contain compound 1 according to the invention in crystalline and/or amorphized and/or dissolved form, are suitable, such as, e.g., tablets (non-coated or coated tablets, for example, having enteric or slowly dissolving or insoluble coatings, which control the release of the compound to be used according to the invention), tablets or films/wafers disintegrating rapidly in the oral cavity, films/lyophilizates, capsules (for example, hard or soft gelatine capsules), coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions.

Parenteral administration can take place circumventing an absorption step (e.g. intravenously, intraarterially, intracardially, intraspinally or intralumbarly) or with inclusion of an absorption (e.g. intramuscularly, subcutaneously, intracutaneously, percutaneously or intraperitoneally). For parenteral administration, suitable administration forms are, inter alia, injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilizates or sterile powders.

For the other administration routes, e.g., pharmaceutical forms for inhalation (inter alia powder inhalers, nebulizers), nose drops, solutions or sprays, tablets to be administered lingually, sublingually or buccally, films/wafers or capsules, suppositories, ear or eye preparations, tinctures, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, crystal suspensions, aqueous and oily injection solutions, depot preparations, ointments, fatty ointments, gels, creams, transdermal therapeutic systems (e.g. patches), milk, pastes, foams, dusting powders, implants, or stents are suitable.

Oral or parenteral administration is preferred, in particular oral and intravenous administration. Topic application is also an option.

Compound 1 to be used according to the invention can be converted into the administration forms mentioned. This can take place in a manner known per se by mixing with inert, nontoxic, pharmaceutically suitable excipients. These excipients include, inter alia, carrier substances (for example, microcrystalline cellulose, lactose, mannitol), solvents (e.g. liquid polyethylene glycols), emulsifiers and dispersants or wetting agents (for example sodium dodecylsulphate, polyoxysorbitan oleate), binding agents (for example polyvinylpyrrolidone), synthetic and natural polymers (for example albumin), stabilizers (e.g. antioxidants such as, for example, ascorbic acid), colourants (e.g. inorganic pigments such as, for example, iron oxides) and taste and/or odour corrigents.

A further subject of the present invention is a medicament that contains compound 1 according to the invention, customarily together with one or more inert, non-toxic, pharmaceutically suitable excipients, and their use for the previously mentioned purposes. The percentages in the following tests and examples are, if not stated otherwise, percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios and concentrations of liquid/liquid solutions in each case relate to the volume.

The subject matter of the present invention is also directed to a method for alleviating pathological conditions associated with PGE2 induced increased EP4 receptors activation by antagonising the activation of EP4 receptors by compound 1.

EXAMPLES

EXAMPLE 1 : Synthesis of compound 1 :

The synthesis of the intermediates as well as of compound 1 is disclosed in WO2014086739 (example 141 ). The disclosure of this application is incorporated herewith in its entirety Briefly, in analogy to Example 4/Variant B of WO2014086739, methyl 2-(9-ethyl-6-methyl-9H- carbazol-3-yl)-1 -(2-methoxyethyl)-4-methyl-1 H-benzimidazole-5-carboxylate was first prepared from 1 .0 g (4.2 mmol) of methyl 3-amino-4-[(2-methoxyethyl)amino]-2- methylbenzoate and 1 .0 g (4.2 mmol) of 9-ethyl-6-methyl-9H-carbazole-3-carbaldehyde (CAS No 122060-05-3).

1 H-NMR (400 MHz, DMSO-d6), δ [ppm] = 1 .35 (t, 3H), 2.50 (s, 3H), 2.89 (s, 3H), 3.12 (s, 3H), 3.69 (t, 2H), 3.87 (s, 3H), 4.49 (q, 2H), 4.55 (t, 2H), 7.35 (dd, 1 H), 7.58 (t, 2H), 7.75 (d, 1 H), 7.83 (d, 1 H), 7.90 (dd, 1 H), 8.06 (s, 1 H), 8.59 (d, 1 H).

This was subsequently reacted analogously to Example 2 of WO2014/086739 to give 2-(9- ethyl-6-methyl-9H-carbazol-3-yl)-1 -(2-methoxyethyl)-4-methyl-1 H-benzimidazole-5-carboxylic acid. (920 mg, 50%).

1 H-NMR (300 MHz, DMSO-d6), δ [ppm] = 1 .35 (t, 3H), 2.49 (s, 3H), 2.89 (s, 3H), 3.12 (s, 3H), 3.69 (t, 2H), 4.43 - 4.59 (m, 4H), 7.34 (dd, 1 H), 7.56 (m, 2H), 7.75 (d, 1 H), 7.81 - 7.93 (m, 2H), 8.07 (s, 1 H), 8.59 (d, 1 H), 12.25 - 12.95 (br., 1 H).

EXAMPLE 2: Binding experiments to determine mode of action

Model description I:

A. Principle of detection:

Binding of PGE2 to the Gas coupled human EP4-receptor leads to the stimualtion of adenylate cyclase activity and the formation of cAMP. The amount of cAMP produced following agonist stimulation is detected with the help of a competition assay based on a fluorescently labelled cAMP tracer (cAMP-d2) and Eu-cryptate labelled anti-CAMP antibody. Maximum signal (obtained via fluorescence resonance energy transfer; FRET) is obtained in the absence of cellular cAMP. Upon cell lysis, any cAMP produced (and accumulated in the presence of IBMX) competes for binding of the tracer to the antibody. An appropriate plate reader (RubyStar, PheraStar, ViewLux) is used to induce and measure FRET emissions at 620 and 665nm following excitation at 337 nm. Any signal-decrease shows activation of the GPCR (i.e. change in well-Ratio; defined as 665nm / 620nm^*10000). Antagonists result in signal increase. B. Standard antagonist assay protocol:

Four microliters of a cell suspension containing 625 cells/μΙ (2500 cells/well; by this time also containing the cAMP-D2 tracer) were added to a testplate already containing the test compounds (50 nl; 100 % DMSO). Following a 20 minute pre-incubation at room temperature two microliters of a 3xECso PGE2 agonist solution were added per well and the plate was incubated in the presence of a I xECso concentration of agonist for 60 minutes (total volume: 6 μΙ). Finally the whole reaction was stopped via the addition of 2 μΙ of lysisbuffer also containing the Eu-cryptate labelled anti-CAMP antibody (final volume: 8 μΙ). Another 20 minutes later the cell lysate containing plate was transferred to TR-FRET compatible reader in order to quantify the results (see model description II for a more detailed description of all generic steps involved). (See Fig. 1A)

C. Standard assay protocol:

Four microliters of a cell suspension containing 625 cells/μΙ (2500 cells/well; by this time also containing the cAMP-D2 tracer) were added to a testplate already containing the test compounds (50 nl; 100 % DMSO). Following a 20 minute preincubation at room temperature two microliters of cell medium solution were added per well and the plate incubated for another 60 minutes (total volume: 6 μΙ). Finally the whole reaction was stopped via the addition of 2 μΙ of lysisbuffer also containing the Eu-cryptate labelled anti-CAMP antibody (final volume: 8 μΙ). Another 20 minutes later the cell lysate containing plate was transferred to TR-FRET compatible reader in order to quantify the results (see model description II for a more detailed description of all generic steps involved). (See Fig. 1 B).

Model description II:

Test for Inverse Agonism: STANDARD ASSAY PROTOCOL

1 . Principle of detection:

Constitutive hEP4-R activity results in the formation of cAMP even in the absence of an agonist and these basal cAMP levels are detected when using 10 000 hEP4-R positive HEK293 cells per well. Upon cell lysis, any cAMP produced by now competes with a fluorescent cAMP tracer (cAMP- d2) for binding to a Europium labelled anti-cAMP antibody. Thus, a maximum fluorescence resonance energy transfer (FRET) signal is obtained in the absence of cellular cAMP. Following excitation at 337 nm any reduction of the FRET induced emission of the cAMP-d2 tracer at 665nm is indicative of cAMP production. A second FRET induced emission at 620nm (resulting from the Eu-labelled anti cAMP antibody) is used for well internal referencing. Inverse agonists inhibit basal receptor activity therefore the signal should increase. (Measurement instrument: RubiStar; read-out: well-Ratio defined as 665nm / 620nm ^* 10000). (see Fig. 1 C). 2. Preparation of assay ready cells from frozen cells stocks:

Table 1 represents the steps of assay ready cells from frozen cell stocks.

3. Materials:

Cells: hEP4-R stably transfected into HEK cells (clone hEP4-C1 cells), Read plates: Polystyrol; 384well, SV white, Greiner # 784075, Dimethylsulphoxide, DMSO: Sigma-Aldrich # D-2650, 3-lsobutyl-1 -methylxanthine, IBMX: Sigma-Aldrich # 1-7018, cAMP HTRF-Assay Kit: Cisbio International 62AM6PEJ high range.

3.1 . Preparation of cAMP-HTRF detection reagents:

Step 1 : Reconstitution of both, cAMP-d2 & anti-CAMP cryptate with water (5 ml) according to suppliers manual.

Step 2: Further 1 :39 dilution with Conjugate & Lysisbuffer. Conjugate & Lysisbuffer prepared according to cAMP kit protocol (Cisbio).

The binding experiments revealed that compound 1 is a highly potent, competitive human EP4 receptor antagonist with inverse agonist activity.

EXAMPLE 3: in vitro IL-17 inhibition experiments in stimulated human TH17 cells

To study the inhibitory effect of the inventive compound 1 on the PGE2-mediated promotion of the production of pro-inflammatory cytokine IL-17 (interleukin-17, synonyme IL-17A), a key cytokine in the pathogenesis of arthritis (Lubberts et al., 2015; Marinoni et al., 2014; Isailovic et al., 2015), human CD4⁺ T cells were used.

For this purpose, human PBMCs (peripheral blood mononuclear cells) were isolated from anti-coagulated human blood by gradient centrifugation using Histopaque^®-1077 (Sigma, # 1077-1 ). After centrifugation of the blood at 800 g for 15 min at room temperature, plasma including thrombocytes was discarded. Then the mononuclear cell layer (PBMCs) was carefully collected and washed three times with PBS (phosphate buffered saline; w/o Ca²7Mg²⁺) (Gibco, # 14190-094). The supernatant was discarded and PBMCs were resuspended in culture medium (RPMI 1640 GlutaMax [Gibco, # 61870], supplemented with 10% FCS (fetal calf serum), 50 U/ml Penicillin and 50 g/ml Streptomycin [PAA, # P1 1 -010]). Afterwards, CD4⁺ T cells were isolated out of PBMCs by magnetic cell separation (CD4+ T cell Isolation Kit, Miltenyi Biotech, # 130-096-533) via a column (LS column, Miltenyi Biotech, # 130-042-401 ). Total CD4⁺ T cells were plated in 1 x10⁵ cells/well in a flat bottom 96-well microtiter plate (Costar, # 3599). Compound 1 was serial diluted out of a constant volume of 100% DMSO and was used in 8 different concentrations (from 1 μΜ to 1 pM) in the stimulation assay. Each of the 8 tested concentrations contained 0.1 % DMSO. Following a 30 min pre-incubation time with the compound 1 , human CD4⁺ T cells were differentiated over 3 consecutive days towards IL-17-producing TH17 cells by adding a recombinant human (rh) IL-23 (10 ng/ml; eBioscience, # 14-8239-63) and the activation stimulus with plate-bound anti-CD3 (5 μg ml; purified mouse monoclonal lgG1 ; R&D Systems, # MAB100) and anti-CD28 (1 g/ml; purified mouse monoclonal lgG1 ; R&D Systems, # MAB342) in the presence or absence of PGE2 (10 nM; Cayman, # 14010). After 3 days of culture in an incubator, supernatants were collected and analyzed for the cytokine IL-17A by using a commercially available ELISA (human IL-17a MSD; Mesoscale, # L451 RFA-1 ). The results of compound 1 with regard to the inhibition of PGE2-promoted IL-17A secretion of TH17 cells are shown in the figure 2.

IN VIVO CHARACTERIZATION EXAMPLE 4: Effect of compound 1 on adjuvant-induced arthritis (AIA) in rats

A subcutaneous injection of complete Freund^'s adjuvant (CFA) in rats leads to the onset and progression to polyarticular inflammation, thereby representing an experimental arthritis model for human arthritis indications such as psoriatic arthritis, rheumatoid arthritis, reactive arthritis as well as ankylosing (Bendele et al., 2001 ; McCann et al., Arthritis Res Ther, 2010). The anti-inflammatory effects of compound 1 in the adjuvant-induced arthritis (AIA) in rats were investigated.

Male Lewis rats (100 to 125 g body weight, Charles River Laboratories, Germany) were subcutaneously (s.c.) injected with 100 μΙ of complete Freund's adjuvant (CFA) solution (M. tuberculosis H37Ra [Difco Lab, # 231 141] diluted in incomplete Freund's adjuvant [Difco Lab, # 263910]) at the tail base on day 0. Animals were randomized with n=8 animals per treatment group. As controls a healthy and a disease group, treated with vehicle only, were included in the studies. Compound 1 was once daily (QD) applied per os (p.o.) from day 0 onwards in different dosages (0.2 mg/kg, 1 mg/kg and 5 mg/kg or 2.5 mg/kg and 5 mg/kg) using an appropriate vehicle (0.5% CMC [carboxylmethyl cellulose] in water; m/v), which allowed sufficient exposure of the animals with the compound. Assessment of arthritis severity as well as treatment effects of compound 1 were determined by the disease activity score three times per week after the onset of arthritis (approximately day 8). This score graded the extent of joint inflammation (including presence of erythema and extent of joint swelling) from 0 to 4 (0 = no; 1 = slight; 2 = moderate, 3 = marked, 4 = severe). The disease activity score was determined for both hind paws and added up. Furthermore, both hind paw volume using a plethysometer (IITC Life Science Inc., USA) and joint thickness (sagittal x transversal in mm², hind paws) were detected. Next to this, grip strength as indicator for hyperalgesia was analysed by using an automated grip strength test meter (IITC Life Science Inc., USA). At the end of the study (day 20) blood serum was collected and analysed for pro- inflammatory biomarkers (e.g. CRP, BD Biosciences, # 55825; ESR). In addition, joint biopsies were obtained and the extent of joint damage was evaluated by histopathology using a scoring system grading synovial hyperplasia, immune cells infiltration, erosion of cartilage/bone, etc.. Next to this, pro-inflammatory cytokines were analysed in the joint biopsies after generation of tissue extracts via pulverization of joints with a cryomill at minus 196°C (CryoMill; Retsch GmBH, Germany). For this purpose, 200 mg of joint tissue extract were mixed with 0.5 ml RPMI 1640 medium (Gibco, #61870) for cytokine analysis (Proinflammatory Panel 1 ; Mesoscale, # K15059D-1 ). Statistical analysis of data was performed by using one-way ANOVA (Analysis of variance) and multiple comparisons to the disease control group via the Dunnett^'s test.

Treatment with compound 1 significantly inhibited the adjuvant-induced arthritis in rats as shown e.g. by reduction in disease severity (Fig. 3) and histopathology (Fig. 6). In some experiments a clinical relevant reference compound, like the anti-TNF antagonist Etanercept (1 .5 mg/kg s.c. once daily, starting day 0), was included as competitor for efficacy in the arthritis model.

Therefore the use of compound 1 for the treatment of arthritis especially rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis, as well as other immune-mediated rheumatic diseases, and/or other pathological conditions associated with increased activation of EP4 receptors by prostaglandin E2 (PGE2) is subject matter of the present invention. Also subject matter of the present invention is the use of compound 1 for the preparation of a medicament for treatment of arthritis especially rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, as well as other immune-mediated rheumatic diseases selected from a list comprising of infectious arthritis (like Lyme disease, reactive arthritis, PAPA syndrome), juvenile rheumatoid arthritis (e.g. Still^'s disease), crystall-induced arthritis (like gout), fibromyalgia, osteoarthritis, and rheumatic diseases caused by autoimmunity (e.g. systemic lupus erythematosus, relapsing polychondritis, polymyositis, Behcet^'s disease, Sjogren syndrome, scleroderma).

Also subject matter of the present invention is a medicament comprising compound 1 in combination with one or more further active compounds, especially with immunomodulatory and immunosuppressive compounds such as cyclosporine, Methotrexat® (MTX), Jak/STAT inhibitors (e.g Baricitinib, Tofacitinib, GLPG0634), TNF antagonists (e.g. Humira®, etanercept, infliximab, certolizumab), IL-1 inhibitors (e.g. anakinra, canakinumab, rilonacept), IL-17 inhibitors (e.g. secukinumab, brodalumab, ixekizumab), IL-6/IL-6 receptor antagonists (e.g. tocilizumab, sarilumab, siltuximab), IL-12/IL-23 inhibitors (e.g. ustekinumab), phosphodiesterase inhibitors (e.g. Apremilast®), leflunomid, teriflunomid, cyclophosphamide, rituximab, belimumab, tacrolimus, rapamycin, mycophenolate mofetil, interferons, corticosteroids (e.g. prednisone, prednisolone, methylprednisolone, hydrocortisone, betamethasone), cyclophosphamide, azathioprine and sulfasalazine, paracetamol, nonsteroidal anti-inflammatory substances (NSAIDS) (aspirin, ibuprofen, naproxen, etodolac, celecoxib, colchicine) as well as inhibitors of mitogen-activated protein (MAP) kinases and inhibitors of the MAP kinases (Mkk3/6, Mek1/2, Erk1/2), inhibitors of protein kinase B (ΡΚΒα/β/γ; Akt1/2/3), inhibitors of phosphoinositide 3-kinase (PI3K), inhibitors of cyclin- dependent kinase (CDK1/2), inhibitors of the hypoxia-induced signalling pathway (HIFI alpha inhibitors, activators of prolylhydroxylases), histone deacetylase (HDAC) inhibitors, prostaglandin F receptor (FP) (PTGFR) antagonists, neurokinin 1 receptor antagonists, paracetamol, selective COX2 inhibitors and/or non-selective COX1/COX2 inhibitors.

Also subject matter of the present invention are medicaments comprising compound 1 in combination with an inert, nontoxic, pharmaceutically suitable auxiliary.

Also subject matter of the present invention is a medicament comprising compound 1 for the treatment of arthritis especially rheumatoid, ankylosing spondylitis, psoriatic arthritis, as well as other other immune-mediated rheumatic diseases selected from a list comprising of infectious arthritis (like Lyme disease, reactive arthritis, PAPA syndrome), juvenile rheumatoid arthritis (e.g. Still^'s disease), crystall-induced arthritis (like gout), fibromyalgia, osteoarthritis, and rheumatic diseases caused by autoimmunity (e.g. systemic lupus erythematosus, relapsing polychondritis, polymyositis, Behcet^'s disease, Sjogren syndrome, scleroderma).

Also subject matter of the present invention is the use of compound 1 in the form of a pharmaceutical preparation for enteral, parenteral, topical and oral administration.

Also subject matter of the present invention is a method for alleviating pathological conditions associated with PGE2 induced increased EP4 receptors activation by antagonising the activation of EP4 receptors by compound 1.

Claims

1 . Use of compound 1 for the treatment of arthritis and/or pathological conditions associated with increased activation of EP4 receptors by prostaglandin E2 (PGE2).

2. Use according to Claim 1 , whereby the arthritis is rheumatoid arthritis.

3. Use according to Claim 1 , whereby the arthritis is ankylosing spondylitis.

4. Use according to Claim 1 , whereby the arthritis is psoriatic arthritis.

5. Use of compound 1 for the preparation of a medicament for treatment of arthritis especially rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, as well as other immune-mediated rheumatic diseases selected from a list comprising of infectious arthritis, juvenile rheumatoid arthritis, crystall-induced arthritis, fibromyalgia, osteoarthritis, and rheumatic diseases caused by autoimmunity.

6. Use according to claim 5, whereby infectious arthritis is Lyme disease, reactive arthritis is PAPA syndrome, juvenile arthritis is Still^'s disease, crystall-induced arthritis is gout, and rheumatic diseases caused by autoimmunity is systemic lupus erythematosus, relapsing polychondritis, polymyositis, Behcet^'s disease, Sjogren syndrome, and scleroderma.

7. Medicament comprising compound 1 in combination with one or more further active immunomodulatory and immunosuppressive compounds, whereby the compounds are selected from a list comprising cyclosporine, Methotrexat^® (MTX), Jak/STAT inhibitors (e.g Baricitinib, Tofacitinib, GLPG0634), TNF antagonists (e.g. Humira®, etanercept, infliximab, certolizumab), IL-1 inhibitors (e.g. anakinra, canakinumab, rilonacept), IL-17 inhibitors (e.g. secukinumab, brodalumab, ixekizumab), IL-6/IL-6 receptor antagonists (e.g. tocilizumab, sarilumab, siltuximab), IL-12/IL-23 inhibitors (e.g. ustekinumab), phosphodiesterase inhibitors (e.g. Apremilast®), leflunomid, teriflunomid, cyclophosphamide, rituximab, belimumab, tacrolimus, rapamycin, mycophenolate mofetil, interferons, corticosteroids (e.g. prednisone, prednisolone, methylprednisolone, hydrocortisone, betamethasone), cyclophosphamide, azathioprine and sulfasalazine, paracetamol, non-steroidal anti-inflammatory substances (NSAIDS) (aspirin, ibuprofen, naproxen, etodolac, celecoxib, colchicine) as well as inhibitors of mitogen-activated protein (MAP) kinases and inhibitors of the MAP kinases (Mkk3/6, Mek1/2, Erk1/2), inhibitors of protein kinase B (ΡΚΒα/β/γ; Akt1/2/3), inhibitors of phosphoinositide 3-kinase (PI3K), inhibitors of cyclin- dependent kinase (CDK1/2), inhibitors of the hypoxia-induced signalling pathway (HIF1 alpha inhibitors, activators of prolylhydroxylases), histone deacetylase (HDAC) inhibitors, prostaglandin F receptor (FP) (PTGFR) antagonists, neurokinin 1 receptor antagonists, paracetamol, selective COX2 inhibitors and/or non-selective COX1/COX2 inhibitors.

8. Medicament comprising compound 1 in combination with an inert, nontoxic, pharmaceutically suitable auxiliary.

9. Medicament according to Claim 7 or 8 for the treatment of arthritis especially rheumatoid, ankylosing spondylitis, psoriatic arthritisas well as other immune- mediated rheumatic diseases selected from a list comprising of infectious arthritis, juvenile rheumatoid arthritis, crystall-induced arthritis, fibromyalgia, osteoarthritis, and rheumatic diseases caused by autoimmunity.

10. Use of compound 1 in the form of a pharmaceutical preparation for enteral, parenteral, topical and oral administration.

1 1 . Method for alleviating pathological conditions associated with PGE2 induced increased EP4 receptors activation by antagonising the activation of EP4 receptors by compound 1 .