WO2018162562A1

WO2018162562A1 - Use of an ep4 antagonist for the treatment of inflammatory pain

Info

Publication number: WO2018162562A1
Application number: PCT/EP2018/055615
Authority: WO
Inventors: Jens Nagel; Stefan BÄURLE; Maik Obendorf; Markus Koch; Gernot Langer; Anne-Marie GODINHO-COELHO
Original assignee: Bayer Pharma Aktiengesellschaft
Priority date: 2017-03-10
Filing date: 2018-03-07
Publication date: 2018-09-13
Also published as: TW201836604A

Abstract

The present invention relates the use of compound 1 for the treatment of inflammatory pain.

Description

Use of an EP4 antagonist for the treatment of inflammatory pain

The present invention relates to the use of an EP4 antagonist for the treatment and/or prophylaxis of inflammatory pain and/or other pathological conditions related to increased activation of EP4 receptors by prostaglandin E2 (PGE2).

BACKGROUND OF THE INVENTION

According to Basbaum (Basbaum et al (2009). Cell 139, 267-284) inflammatory pain is one form of nociceptive pain which is typically accompanied by an immune response and mediated by pro-inflammatory molecules which are released by tissue due to a trigger which endangers or destroys cell integrity or signals endangered cell integrity. These conditions lead to accumulation of endogenous factors released from activated nociceptors (peripheral nerve endings of the pain pathway) or non-neural cells that reside within or infiltrate into the injured/endangered area (including mast cells, basophils, platelets, macrophages, neutrophils, endothelial cells, keratinocytes, and fibroblasts). Collectively, these factors, referred to as the "inflammatory soup," represent a wide array of signaling molecules, including neurotransmitters, peptides (substance P, CGRP, bradykinin), eicosinoids and related lipids like prostaglandins which include the endogenous activators of the EP4 receptor, thromboxanes, leukotrienes, endocannabinoids, neurotrophins, cytokines, and chemokines, as well as extracellular proteases and protons. Nociceptors express one or more cell-surface receptors capable of recognizing and responding to each of these pro- inflammatory or pro-algesic agents. Such interactions enhance excitability of the nerve fiber, thereby heightening its sensitivity to temperature or touch leading finally to hyperalgesia. Ongoing or frequently occurring inflammatory events lead in many cases to chronic inflammatory pain which severely affecting the quality of life of patients. Inflammatory pain of varied origins is associated but not limited to osteoarthritis, rheumatoid arthritis, rheumatic disease, tenosynovitis, gout, ankylosing spondylitis, endometriosis, injuries, various skin diseases and bursitis.

Currently inflammatory pain can be treated with nonsteroidal anti-inflammatory drugs (NSAID). 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. Combining all available treatment modalities, recent data confirm a significant degree of unmet medical need since up to 70% of treated patients have persistent symptoms like chronic pelvic pain that are not managed (Bayer market research, 2009, 21.700 women in 8 countries). Therefore, new and long-term treatment options with reduced side-effects and high efficacy are urgently needed. 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 cAMP. The expression of the receptor was detected on peripheral nerve endings of nociceptors, on macrophages and neutrophils. For these cell types, great importance was demonstrated in connection with inflammatory pain. It is assumed that the local inflammation of the endometriotic lesions makes a significant contribution to the genesis of the pain symptoms observed (Stratton & Berkley 2010; Giudice 2010).

Up to now, no EP4 antagonist has been licensed as a medicament. However, EP4 antagonists of different structural classes have been described, Thus in WO2005/0121508, WO2005102389 and WO2005/105733 (Pfizer), for example, N-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-A/-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 WO2014/086739 (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 inflammatory pain and/or other pathological conditions related to 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 inflammatory pain and/or other pathological conditions related to 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)

Compound 1

was highly active in several models of pain. It could be demonstrated that compound 1 was identified as a highly potent, competitive human EP4 receptor antagonist with inverse agonist activity. Secondary in vitro assays revealed a high functional potency on EP4 receptors of mouse, rat and cynomolgus.

The compound showed after p.o. administration robust effects in in vivo studies in mice and rats. The compound attenuated inflammatory pain and edema formation in various settings. The compound can be employed for long term treatment (fig. 5). The suppression of PGE2 induced proliferation of uterine tissue was shown in vitro and in vivo in mice. Therefore the use of compound 1 for the treatment of inflammatory pain and/or other pathological conditions related to 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 charactistics 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.

Figure 2: Effect of Compound 1 on CFA induced pain.

Compound 1 was given in increasing concentrations ranging from 0.2 to 25 mg/kg. The figure clearly shows that compound 1 reduced inflammatory pain significantly and dose- dependently (^*p<0.05, ^**p<0.01 , ^***p<0.005, ^****p<0.001 , ANOVA followed by post hoc test (Dunnett) vs vehicle).

Figure 3: Effect of Compound 1 on CFA induced edema

Compound 1 was given in increasing concentrations ranging from 0.2 to 25 mg/kg. The figure clearly shows that compound 1 reduced edema significantly and dose-dependently (^**p<0.01 and ^****p<0.001 , ANOVA followed by post hoc test (Dunnett) vs vehicle).

Figure 4: Effect of Compound 1 on PGE2 induced pain

Compound 1 was given in following concentrations: 1 mg/kg, 5 mg/kg and 25 mg/kg. Figure 4 shows that compound 1 significantly reduced PGE2 induced pain (^*p<0.01 vs vehicle, analysed by 1 -way ANOVA followed by Dunnett^'s post hoc test).

Figure 5: Effect of Compound 1 on PGE2 induced proliferation in vitro

Figure 5 represents the effect of compound 1 on cell proliferation. Compound 1 (100 nM) was given with (crossed bar) or without (vertical hatched bar) 10 nM PGE2, PGE2 was also given alone (horizontal hatched bar). In the presence of compound 1 the total cell number did not increase when compared with the group which contained PGE2 alone. Compound 1 together with PGE2 also did not affect the total cell number.

Figure 6: Effect of Compound 1 on PGE2 induced proliferation

Figure 6 demonstrates that stabilized PGE2, applied directly in the cavity of the uterus, induced proliferation in uterine tissue in aenesthetized mice. Compound 1 was given in concentrations ranging from 0.2 to 25 mg/kg; compound 1 dose-dependently suppressed PGE2-induced cell number increase in uterine tissue (^*p<0.05 and ^**p<0.01 compared to 2 mg/kg dm-PGE2 group, 3 outliers following Grubbs test removed, analysed by 1 -way ANOVA followed by Dunnett's post hoc test). Figure 7: Effect of compound 1 in endomet iosis dyspareunia model (rat, vaginal distension

Figure 7 shows the effect of compound 1 in an endometriosis dyspareunia model. Open bars represent the vehicle group, hatched bars represent the compound group, and crossed bars show the effect of ibuprofen. Compound 1 decreased vaginal hyperalgesia in a rat endometriosis model. + stands for: cumulative number of contraction per 0.1 ml distension and ++ for: different vehicle, these were excluded from statistical analysis

*^*p<0.01 ^*p<0.05 t-test vehicle vs compound 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that compound 1 is highly potent in the treatment of inflammatory pain and/or other pathological conditions related to 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 inflammatory pain and/or other pathological conditions related to increased activation of EP4 receptors by prostaglandin E2 (PGE2). Inflammatory pain inflammatory pain is selected from a group comprising but not limited to ankylosing spondylitis, or pain associated with endometriosis, post operative pain, pain related to skin burn injuries and osteoarthritis.

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 endometriosis. Suitable combination active substances that may be mentioned by way of example and preferably are: selective oestrogen receptor modulators (SERMs), oestrogen receptor (ER) antagonists, aromatase inhibitors, 17· -HSD1 inhibitors, steroid sulphatase (STS) inhibitors, GnRH agonists and antagonists, kisspeptin receptor (KISSR) antagonists, selective androgen receptor modulators (SAR s), androgens, 5· -reductase inhibitors, selective progesterone receptor modulators (SPRMs), gestagens, antigestagens, oral contraceptives, inhibitors of mitogen activated protein (MAP) kinase and inhibitors of MAP kinases (Mkk3/6, Mek1/2, Erk1/2), inhibitors of protein kinases B (ΡΚΒ· /· /· ; 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.

Compound 1 can be employed for the treatment of inflammatory pain, both after oral as well as parenteral administration.

Inflammatory pain includes forms of acute and chronic pain, including chronic pelvic pain, endometriosis associated pain, as well as pain associated with angina, or pain of varied origins (including but not limited to pain associated with osteoarthritis, rheumatoid arthritis, rheumatic disease, tenosynovitis, gout, bursitis, post operative pain, pain related to skin burn injuries, and ankylosing spondylitis. Therefore subject matter of the present invention is also the use of compound 1 for the treatment of inflammatory pain, whereby the inflammatory pain is selected from a group comprising but not limited to ankylosing spondylitis, or pain associated with endometriosis, post operative pain, pain related to skin burn injuries and osteoarthritis

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, between 1 -500 mg/day, preferably between 5 and 30 mg/day, where the dose can be given as an individual dose to be administered once or subdivided into 2 or more daily doses.

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, intraspinaliy 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, vaginal capsules and suppositories, tampons, intrauterine pessaries, 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, intrauterine spirals, vaginal rings 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.

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/dayNevertheless, 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.

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 WO2014/086739 (example 141 ). The disclosure of this application is incorporated herewith in its entirety Briefly, in analogy to Example 4/Variant B of WO2014/086739, 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), · [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), · [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 an 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) are 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 3XEC80 PGE2 agonist solution are added per well and the plate is incubated in the presence of a 1XEC80 concentration of agonist for 60 minutes (total volume: 6 μΙ). Finally the whole reaction is 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 is 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 figure 1 A)

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) are 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 are added per well and the plate incubated for another 60 minutes (total volume: 6 μΙ). Finally the whole reaction is 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 is 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 figure 1 B).

Model description II:

Test for Inverse Agonism: STANDARD ASSAY PROTOCOL

(10 000 cells/well - Target Details: PTGER4, NM_00958; NP_00949).

1 . Principle of detection:

Constitutive hEP4-R activity results the formation of cAMP even in the absence of 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 an 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. As inverse agonists inhibit basal receptor activity they should result in a signal increase. (Measurement instrument: RubiStar; read-out: well-Ratio defined as 665nm / 620nm ^* 10000). (see figure 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: Poiystyroi; 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 (5ml_) according to suppliers manual.

Step 2: Further 1 :39 dilution with Conjugate & Lysis Buffer. 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. IN VIVO CHARACTERIZATION

EXAMPLE 3: Effect of compound 1 on CFA induced pain and edema (rat)

Model description:

Female SD rats were treated with compound 1 or respective vehicle. 1 h later all animals got an intraplantar injection of 50 · L of 100% Complete Freund's Adjuvant (CFA) under isoflurane anesthesia. CFA injection leads within hours to a robust edema accompanied by hyperalgesia and allodynia affecting the treated paw.

Endpoint 1 : Dynamic weight bearing (DWB)

DWB analyses the weight distribution of an animal on the four paws and is therefore able to give evidence for pain-related relief posture and could be used as a method to measure spontaneous pain reaction in the freely moving animal (Gruen et al. 2014). The automated DWB device (Bioseb, Boulogne, France) consists of a Plexiglas enclosure (22x22x30cm), a floor sensor composed of captors in order to detect pressure variation and a camera placed above the enclosure in order to validate the animal position during analysis (Robinson et al., 2012; Tetreault et al., 201 1 ). The rats were able to move freely in the enclosure during 5 min while the acquisition data were transmitted to a computer. The animals were not acclimated to the enclosure before the test.

Endpoint 2: Paw weight analysis (edema)

The paw was removed from the dead animal at the ankle joint and weight of the paw was noted.

Group (1 ): Vehicle: group (2): compound 1 , 0.2 mg/kg; group (3) compound 1 , 1 mg/kg: group (4): compound 1 , 5 mg/kg; group (5): compound 1 , 25 mg/kg Time scheme see Figure 8

Both, figure 2 and 3 demonstrate that compound 1 reduced inflammatory pain and edema formation significantly and dose-dependently.

EXAMPLE 4: Effect of COMPOUND 1 on PGE2 induced pain

Model description:

After the naive baseline values for paw withdrawal thresholds were obtained (day -1 ), animals were assigned to 5 groups (incl. morphine control) and received test article or vehicle administration (day 0, T=0). Approximately 60 minutes after the treatment (day 0, T=60 min), inflammatory pain was induced by a single injection of 10 μg dmPGE2 into the plantar surface of the left hind paw. Morphine served as the positive control for the study and was administered 30 minutes prior to dmPGE2 injection (day 0, T=30 min). Paw withdrawal thresholds were then measured 30 minutes after dmPGE2 injection (day 0, T=90 min). All animals were euthanized on day 0 following the behavioral assessment and plasma was collected from animals treated with test articles. Compound 1 was given at concentrations of 0.2 (dottet bar), 1 (horizontal hatched bar) and 5 mg/kg (vertical hatched bar).

Figure 4 shows that PGE2 induced pain is mediated by EP4 receptors. Compound 1 significantly reduced PGE2 induced pain dose dependendly.

EXAMPLE 5: Inhibition of PGE2 induced proliferation,

Model description:

Cell-number assay:

Cells (Cell culture: EM42 cells (Desai et al., 1994, Fert. Steril. 61 : 760) were cultivated one passage before experiment in test media (DMEM w/o phenolred, 1 % Pen/strep, 1 % L- glutamine plus indicated concentration of FCS [e.g. :1 %])

Indicated number of cells (e.g. 1000) was plated in wells of 96well plates (Perkin Elmer #6005688). Substances were added as indicated in tetraplicates, inhibitors were added first. PGE2 was given twice daily. For incubation without PGE2 the same amount of media was added twice daily. Incubation of MTPs for 4 days at 37°C and 5% CO? was followed by a CellTiter Glo measurement at day 4.

Preparation of cell number calibration curve from cells in test media: Cells were counted for calibration and plated in duplicates in rows 1 and 2 and incubated for two hours. Celltiter Glo measurement was performed as described by vendor (Promega #G7571 ). Briefly, MTPs were adjusted to room temperature for 30 minutes. 100μ! celltiter reagent was added, followed by mixing and incubation for 10 minutes. The luminescence was measured of with POLARstar (bmg). Measurements of relative light units (RLU) and calculation of cell number equivalent was performed by employing linear part of calibration curve.

Compound 1 (100 nM) was given with (crossed bar) or without (vertical hatched bar) 10 nM PGE2, PGE2 was also given alone (horizontal hatched bar). In the presence of compound 1 the total cell number did not increase when compared with the group which contained PGE2 alone. Compound 1 together with PGE2 also did not affect the total cell number.

EXAMPLE 6: Effect of compound 1 in endometriosis dyspareunia model (rat, vaginal distension

Model description:

Female SD rats underwent endometriosis surgery in the estrus phase. 4 uterine fragments were sewn onto the mesenteric artery arc (closed to small intestine) and few other fragments (>2) were sewn onto the distal colonic wall. In parallel electrodes for later EMG recordings were implanted in the abdominal wall to allow recording of the visceral motor response.

After training viscero motor response (VMR) / vaginal distension (VD) testing was performed 5 and 6 weeks post-surgery in the proestrus phase. For vaginal distension session, the uninflated balloon was lubricated and inserted into the mid-vaginal canal, located so, that it would not touch the cervix even when inflated. Inflating the balloon with different volumes of water using a computer-controlled pump distended the vaginal canal. The pressure produced by each volume of distention (corrected for compliance characteristics of the balloon) was measured through a small-volume pressure transducer. In parallel EMG recordings were collected to measure the VMR related to the distension.

Compound 1 (5 mg/kg, p.o.) was administered daily starting from week 3 to week 5 (group 2, N=19). Group 1 (N=20) received vehicle (20 % DMSO / 20 % PEG400 / 60 % water) in the same time period (5 ml/kg, p.o.), Group 3 (N=18) received I bu prof en (100 mg/kg, p.o.). Animals of group 3 were treated once 2 hours prior VMR, VD was at week 5 and week 6).

Compound 1 decreased vaginal hyperalgesia versus vehicle-treated animals. The effect maintained in the treatment free period of week 6. Therefore compound 1 is suitable for long term treatment of the aforementioned diseases. This might point to a disease modyfing component induced by the EP4 receptor antagonism exceeding pure analgesic activity. Interestingly, daily dosed compound 1 showed a more pronounced effect than acute treatment with one analgesic dose of ibuprofenoilected to measure the VMR related to the distension.

Claims

1 . Use of compound 1 for the treatment of inflammatory pain and/or pathological conditions related to increased activation of EP4 receptors by prostaglandin E2 (PGE2).

2. Use of compound 1 for the treatment of inflammatory pain.

3. Use according to Claim 2, whereby the inflammatory pain is selected from a group comprising but not limited to ankylosing spondylitis, or pain associated with endometriosis, post operative pain, pain related to skin burn injuries and osteoarthritis.

4. Use of compound 1 for the preparation of a medicament for treatment of inflammatory pain.

5. Medicament comprising compound 1 in combination with one or more further active compounds, especially with selective oestrogen receptor modulators (SERMs), oestrogen receptor (ER) antagonists, aromatase inhibitors, 17-· -HSD1 inhibitors, steroid sulphatase (STS) inhibitors, GnRH agonists and antagonists, kisspeptin receptor (KISSR) antagonists, selective androgen receptor modulators (SARMs), androgens, 5· -reductase inhibitors, selective progesterone receptor modulators (SPRMs), gestagens, antigestagens, oral contraceptives, inhibitors of mitogen- activated protein (MAP) kinases and inhibitors of the MAP kinases (Mkk3/6, Mek1/2, Erk1/2), inhibitors of protein kinase B (ΡΚΒ· /· /· ; 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. Medicament comprising compound 1 in combination with an inert, nontoxic, pharmaceutically suitable auxiliary.

6. Medicament according to Claim 4 or 5 for the treatment of inflammatory pain.

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

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