Furoindazole derivative pharmaceutical dosage

Abstract

The present invention covers N-{[(2R)-1,4-dioxan-2-yl]methyl}-2-{[(2S)-1,4-dioxan-2-yl]methyl}-8-(trifluoromethyl)-4,5-dihydro-2H-furo[2,3-g]indazole-7-carboxamide (Compound 1) for use in a GPR84 depending disease in which the daily dose is between 5 and 30 mg, the unbound trough concentration of Compound 1 in plasma is higher than the IC80 of 17.9 μg / L and with a positive GPR84 target engagement defined as reduction of mean CD11b expression by at least 10 % compared to predosing level.

Description

[0001]Furoindazole Derivative Pharmaceutical Dosage The present invention covers N-{[(2R)-1,4-dioxan-2-yl]methyl}-2-{[(2S)-1,4-dioxan-2- yl]methyl}-8-(trifluoromethyl)-4,5-dihydro-2H-furo[2,3-g]indazole-7-carboxamide (Compound 1) for use in a GPR84 depending disease in which the daily dose is between 5 and 30 mg, the unbound trough concentration of Compound 1 in plasma is higher than the IC80 of 17.9 μg / L and with a positive GPR84 target engagement defined as reduction of mean CD11b expression by at least 10 % compared to pre- dosing level. Furthermore, the present invention covers Compound 1 for use in a GPR84 depending disease in which the daily dose is between 5 and 30 mg, the total cerebrospinal fluid (CSF) trough concentration of Compound 1 is higher than IC60 of 8 μg / L. Aim of the present invention is also a pharmaceutical oral formulation of Compound 1 in which the daily dose is between 5 and 30 mg, and the unbound trough concentration of Compound 1 in plasma is higher than the IC80 of 17.9 μg / L and the total CSF trough concentration of Compound 1 is higher than IC60 of 8 μg / L for the treatment or prophylaxis of diseases, in particular of inflammatory-driven pain diseases. BACKGROUND N-{[(2R)-1,4-dioxan-2-yl]methyl}-2-{[(2S)-1,4-dioxan-2-yl]methyl}-8-(trifluoromethyl)-4,5- dihydro-2H-furo[2,3-g]indazole-7-carboxamide (Compound 1) is an antagonist of the G- protein coupled receptor 84 (also known as GPR84). The relevance of GPR84 for human disease has been described and studied in several publications. Medium-chain free fatty acids (MCFFAs) are fatty acids with tails of 6 to 12 carbons and can activate GPR84 (Wang J et al., J. Biol. Chem.2006 Nov 10, 281(45): 34457-64). There are two sources of FAs for animal metabolism, exogenously-derived (dietary) FAs and endogenously-synthesized FAs. The biosynthesis of the latter is catalysed by FASN. MCFFAs stimulate release of IL6 from fibroblasts (Smith and Tasi, Nat. Prod. Rep.2007 Oct, 24(5): 1041-72) and myristic acid increases IL6 and IL8 levels in human coronary arterial smooth muscle (HCASM) and endothelial (HCEC) cells (Soto-Vaca A. et al., J. Agric. Food Chem.2013 Oct 23, 61(42): 10074-9). GPR84 belongs to the group of Free Fatty Acid (FFA) receptors (Wang J. et al., J. Biol. Chem.2006 Nov 10, 281(45): 34457-64). The group of FFA receptors consists of 4 GPCRs (FFA1-FFA2) and the new members GPR42 and GPR84. FFA receptors are involved in biological processes such as metabolic and immune function receptors (Wang J. et al., J. Biol. Chem.2006 Nov 10, 281(45): 34457-64). In contrast to all other FFA receptors which have a broader expression pattern, GPR84 has been described to be expressed primarily in various leukocyte populations and adipocytes (Wang J. et al., J. Biol. Chem.2006 Nov 10, 281(45): 34457-64; Lattin J.E. et al., Immunome Res.2008 Apr 29, 4: 5; Nagasaki H. et al., FEBS Lett.2012 Feb 17, 586(4): 368-72). Activation of GPR84 promotes a comprehensive fibrotic and inflammatory cellular response, exerted by enhanced migration of macrophages and neutrophils, promoted pro-inflammatory M1 macrophage polarization and response and secretion of key inflammatory cytokines such as IL1beta and TNFalpha (Gagnon L. et al., Am. J. Pathol. 2018 May, 188(5): 1132-1148; Muredda L. et al., Arch. Physiol. Biochem.2018 May, 124(2): 97-108; Huang Q. et al., Dev. Comp. Immunol.2014, 45(2): 252-258). Based on the involvement of GPR84 in fibrotic and inflammatory cellular response several diseases have been suggested to be GPR84 dependent. GPR84 as microglia-associated protein is expressed in neuroinflammatory conditions and is described as a potential target for the treatment of multiple sclerosis (Bouchard C. et al., Glia 2007 Jun, 55(8): 790-800) and for endometriosis associated and inflammatory pain (Sacher F. et al.2018, Conference Abstract SRI 2018). Furthermore, inhibition of activity and / or the knockout of GPR84 are also effective in the treatment of neuropathic pain in several preclinical models (Roman et al.2010, 7th Forum of European Neuroscience (FENS)). The relevance of GPR84 for inflammatory kidney diseases has been shown in experiments using Gpr84-knockout mice or GPR84 antagonist in models of kidney fibrosis and models for inflammatory liver diseases like non-alcoholic, alcoholic- and toxic fatty liver diseases (Puengel et al.2018, 2018 International Liver Congress (ILC) of the European Association for the Study of the Liver (EASL); Thibodeau J.F. et al. 2018, 51st Annual Meeting and Exposition of the American Society of Nephrology (ASN): Kidney Week 2018). As described previously for macrophages and monocytes, inflammatory changes in adipose tissue enhance expression of GPR84 in adipocytes and modulation of GPR84 regulates adipocyte immune response capabilities (Muredda et al., Archives of Physiology and Biochemistry 2017 Aug, 124(2): 1-12) indicating the relevance of GPR84 in metabolic and metabolic-endocrine disorders like metabolic syndrome, insulin resistance, diabetes mellitus type I and type II, and polycystic ovary syndrome (PCOS) through normalization of adipose tissue inflammation. Regulation of neutrophil activity and general inflammation by GPR84 was also described to be relevant for lung diseases like asthma, idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease (Nguyen et al.2018; Annual Congress Scientific Sessions of the American Heart Association (AHA 2018); Saniere L. et al. 2019; 2019 International Conference of the American Thoracic Society (ATS)). The patent application WO2021122415 discloses Compound 1, corresponding to Example 320, intended for the treatment or prophylaxis of diseases. However, the state of the art does not describe the dosage of Compound 1 to achieve the required and favorable plasma and CSF level to obtain the desired therapeutic effect. Furthermore, no hint is given in the state of the art in relation to a daily dose able to achieve a positive GPR84 target engagement. It has now been found, and this constitutes the basis of the present invention, that Compound 1 can be administered as a daily dose and said dose is between 5 and 30 mg. Furthermore, with said daily dose a positive GPR84 target engagement defined as reduction of mean CD11b expression by at least 10 % compared to pre-dosing level has been successfully achieved. It has been also found that with an oral dose between 5 and 30 mg, the unbound trough concentration of Compound 1 in plasma is higher than an IC80 of 17.9 μg / L and the total CSF trough concentration of Compound 1 is higher than an IC60 of 8 μg / L. DESCRIPTION In accordance with a first aspect, the present invention covers N-{[(2R)-1,4-dioxan-2- yl]methyl}-2-{[(2S)-1,4-dioxan-2-yl]methyl}-8-(trifluoromethyl)-4,5-dihydro-2H-furo[2,3- g]indazole-7-carboxamide (Compound 1) for use in a GPR84 depending in which the daily dose is between 5 and 30 mg, and the unbound plasma. In accordance with a second aspect, the invention covers N-{[(2R)-1,4-dioxan-2- yl]methyl}-2-{[(2S)-1,4-dioxan-2-yl]methyl}-8-(trifluoromethyl)-4,5-dihydro-2H-furo[2,3- g]indazole-7-carboxamide (Compound 1) for use in a GPR84 depending disease in which the daily dose is between 5 and 30 mg, the total cerebrospinal fluid (CSF) trough concentration of Compound 1 is higher than IC60 of 8 μg / L. A third aspect of the invention covers the use in a GPR84 depending disease of a daily dose of Compound 1 able to achieve a positive target engagement defined as reduction of mean CD11b expression by at least 10 % compared to pre-dosing level. A further aspect of the invention covers a pharmaceutical form for oral administration comprising N-{[(2R)-1,4-dioxan-2-yl]methyl}-2-{[(2S)-1,4-dioxan-2-yl]methyl}-8- (trifluoromethyl)-4,5-dihydro-2H-furo[2,3-g]indazole-7-carboxamide (Compound 1) between 5 and 30 mg, for use in a GPR84 depending disease in which said pharmaceutical form is administered in a daily dose between 5 and 30 mg, with positive target engagement as defined as reduction of mean CD11b expression by at least 10 % compared to pre-dosing level and a total CSF trough concentration of compound 1 higher than IC60. Compound 1 can be prepared according to the schemes 1, 2, 3, and 4 according to WO2021122415 page 40, 43, 45, and 47 which are incorporated herein as a reference and are part of the present application. Furthermore, the specific preparation of Compound 1 reported on page 39, line 21 to page 47, line 11 of WO2021122415 is also incorporated herein as a reference and is part of the present application. Compound 1 according to the present invention can be utilized to inhibit, antagonize, block, reduce, decrease GPR84 signal transduction, activity, and cellular function. This method comprises orally administering an amount of a Compound 1 according to the invention, or a pharmaceutically acceptable salt, isomer, polymorph, metabolite, hydrate, solvate or ester thereof; which is effective to treat the GPR84 depending disease. Compound 1 of the present invention demonstrate a valuable pharmacological spectrum of action being an effective antagonist of GPR84. Compound 1 can be effectively used for the treatment or prophylaxis of GPR84 diseases, in particular of autoimmune diseases such as multiple sclerosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, primary and secondary autoimmune uveitis, inflammatory disorders like endometriosis, inflammatory eye diseases, inflammatory kidney diseases, inflammatory liver diseases like non- alcoholic, alcoholic- and toxic fatty liver diseases, lung diseases like asthma, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and metabolic and metabolic-endocrine disorders like metabolic syndrome, insulin resistance, diabetes mellitus type I and type II, and polycystic ovary syndrome (PCOS) disorders, neuropathic and inflammatory pain disorders. Compound 1 demonstrates a valuable pharmacological spectrum of action. Compound 1 has been found to be effective antagonist of GPR84 and it is possible therefore that said compound to be used for the treatment or prophylaxis of diseases, in particular of inflammatory-driven pain diseases such as neuropathic pain diseases like diabetic neuropathic pain, and chemotherapy induced pain, and post-breast surgery pain, cancer bone pain, trigeminal neuralgia, post-mastectomy pain, fibromyalgia, multiple sclerosis pain, post-herpetic neuralgia, Fabry disease, gout, and bladder pain syndrome. The term “treating”, or “treatment” as used in the present text is used conventionally, e.g., the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of a disease or disorder, such as PCOS or IPF. For pharmaceutical form for oral administration, it is possible to formulate Compound 1 intended for the use according to the invention to pharmaceutical forms known in the art that deliver the compounds rapidly and / or in a modified manner, such as, for example, tablets (uncoated or coated tablets, for example with enteric or controlled release coatings that dissolve with a delay or are insoluble), orally-disintegrating tablets, films / wafers, films / lyophilizates, capsules (for example hard or soft gelatine capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. It is possible to incorporate the compounds intended for the use according to the invention in crystalline and / or amorphized and / or dissolved form into said dosage forms. According to a particular form of embodiment the pharmaceutical form for oral administration is an immediate release (IR) tablet. An IR tablet is a pharmaceutical form known in the art, said IR tablet dissolve without delaying or prolonging dissolution or absorption of the drug. Generally, an IR tablet or capsule is swallowed whole and instantaneously disintegrates to make the drug available for absorption and subsequent pharmacologic action. More particularly, Compound 1 for use according to the present invention can be administered in a daily dose is between 8 and 20 mg. Furthermore, the oral daily dose of Compound 1 is 10, 11, 12, 13, 14, 15, 16, 17 or 18 mg. By administering Compound 1 for use according to the invention in an oral daily dose of 10, 11, 12, 13, 14, 15, 16, 17 or 18 mg, the resulting ratio between CSF trough concentration and unbound plasma trough concentration is of around 0.3. Compound 1 for use in a GPR84 depending disease orally administered in a daily dose of 15 mg, achieved an unbound plasma trough concentration and a total CSF trough concentration higher than the IC80 of 17.9 μg / L, and a positive target engagement defined as reduction of mean CD11b expression by at least 10 % compared to pre- dosing level. The present invention covers the use of Compound 1 in the dosages described supra, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, for the prophylaxis or treatment of a GPR84 depending disease, in particular of inflammatory- driven pain diseases such as neuropathic pain diseases like diabetic neuropathic pain, and chemotherapy induced pain, and post-breast surgery pain, cancer bone pain, trigeminal neuralgia, post-mastectomy pain, fibromyalgia, multiple sclerosis pain, post- herpetic neuralgia, Fabry disease, gout, and bladder pain syndrome in humans and animals. DEFINITIONS CSF: cerebrospinal fluid HV: healthy volunteers IC80: inhibitory concentration with 80% of maximum effect PD: pharmacodynamic effect QD: once daily Trough concentration: the concentration of the compound 1 in measured matrix (e.g. plasma or CSF) at the end of the dosing interval (i.e.24 h for once daily dosing). Total concentration in the measured matrix is the sum of bound and unbound drug concentration. Unbound concentration was calculated by the total concentration multiplied by fraction unbound determined via in vitro assay (i.e.61.7% in human). FIGURES Figure 1 describes the geometric mean total plasma concentration-time course of Compound 1 after 10, 25 and 50 mg Compound 1 single and multiple once daily (QD) dosing. The IC80 marked in the figure (29 μg / L) was referring to the total concentration in plasma, which was corresponding to an unbound concentration of 17.9 μg / L. The trough concentrations at steady state after QD dosing (216 h) were above IC80 for all dose groups. Figure 2a and 2b describes Ex vivo induced CD11b activated epitope expression in neutrophils. Inhibitory effect of GPR84 ant. on ex vivo induced CD11b activated epitope expression in neutrophils tested in a novel whole blood FACS assay. Surface expression of CD11b activated epitope as a pharmacodynamic marker (à target engagement) of neutrophil activation measured in HV before and after (at 2 timepoints – Day0 and Day9) GPR84 antagonist treatment as well as in placebo treated HVs. Figure 3 describes the individual total concentrations of Compound 1 in plasma and CSF at different time points on Day5 after 15 mg once daily dosing of Compound 1 (A) and the correlation between total plasma and CSF concentrations (B). There was strong correlation between total plasma and CSF concentrations, resulting in the ratio (CSF / plasma) of around 0.3, which allows for the estimation of total CSF concentrations based on plasma concentrations. EXPERIMENTAL SECTION cAMP HTRF® Assay for identification of cellular GPR84 antagonists By using a Homogenous Time-Resolved Fluorescence (HTRF®) based assay (#62AM5PEJ, Cisbio, Condolet, France) the inhibition of the Gi-coupled GPR84 receptor can be detected. CHO-K1 cells stably expressing human GPR84 receptor (purchased from DiscoveRx, now Eurofins) were used and treated with Forskolin (F6886, Sigma, Germany) to stimulate membrane adenylyl cyclases and thereby unspecific cAMP formation. Activation of the Gi-coupled GPR84 by a natural or small molecule agonist (e.g.6-n-octyl aminouracile, inhouse) results in inhibition of cellular cAMP formation which can be released again by antagonists to this receptor. Detection and quantification of cellular cAMP levels in this HTRF assay is achieved by interaction between a fluorescent cAMP tracer (cAMP-d2) and an Eu-cryptate labelled anti-cAMP antibody. Following excitation at 337 nm this pairing allows for the generation of a fluorescence resonance energy transfer (FRET) between the partners and results in FRET induced emissions at 665 nm and 620 nm, the latter representing background signal by Eu-cryptate labelled anti-cAMP antibody. Maximal signal is obtained in the absence of any cellular cAMP (no competition for the binding of the tracer to the antibody). Given the combination of the Gi coupling properties of GPR84 and the competitive nature of the detection system agonist treatment should result in an increase in the HTRF signal due to lowered cAMP levels. Any signal decrease in the presence of Forskolin, agonist and compound is indicative of antagonist mediated abrogation of GPR84 signaling. For the assay, frozen aliquots of CHO-K1 cells expressing hGPR84 (prepared by acCELLerate, Hamburg, Germany) were thawed and a cell suspension (1.67E+06 cells / mL) in assay media (Ham’s F12 Nutrient Mix, Thermo Fisher Scientific, Waltham, USA; 5% fetal calf serum, Biomol, Hamburg, Germany) containing cAMP-d2 (dilution 1:20, supplied with the kit #62AM5PEJ, Cisbio, Condolet, France) was prepared. After recovery of cells for 20 minutes at 37 °C, 3 μL / well cell suspension including cAMP-d2 were added to a pre-dispensed assay plate (Greiner Bio-One, Kremsmuenster, Austria) containing 50nl / well test compound in 100% DMSO or 100% DMSO as control. This was followed by a 30 minutes incubation step at room temperature. The stimulation time was started by addition of 2 μL / well assay media containing 2.5xEC80agonist 6-OAU and 2.5xEC90Forskolin (negative control: 2.5xEC90Forskolin in assay media) and was continued for 30 minutes at room temperature. The reaction was stopped by addition of 3 μL / well lysis buffer containing cAMP Eu-Cryptate antibody (dilution 1:20) (both supplied with the kit #62AM5PEJ, Cisbio, Condolet, France). To enable complete lysis, plates were incubated for 60 minutes at room temperature before measurement in an HTRF reader, e.g. a PHERAstar (BMG Labtech, Ortenberg, Germany). From the fluorescence emissions at 665 nm (FRET) and at 620 nm (background signal of Eu-cryptate) the ratio (emission at 665 nm divided by emission at 620 nm x 10000) was calculated and the data were normalized (reaction without test compound, only 100% DMSO = 0% inhibition; all other assay components except agonist = 100% inhibition). For dose response testing on the same microtiter plate, compounds were tested at 11 different concentrations in the range of 20 μM to 0.07 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.89 nM, 0.25 and 0.07 nM; dilution series prepared before the assay at the level of the 100-fold conc. stock solutions by serial 1:3.5 dilutions in 100% DMSO) in duplicate values for each concentration. The potency in GPR84 cAMP HTRF® assay of Compound 1 given as IC50 [μM] was of 0.012. In vitro assay: Calculation of the inhibitory concentrations The concentration response relation was modelled with an Imax model of the form: EI0 ,where I0 denotes baseline response, Imax the maximum effect (both in %), logIC50the natural logarithm of the total concentration where 50% of the maximum effect is evoked (I0+Imax) and the slope factor hill describes the steepness of the concentration- response relation. The experiments were repeated in quadruplicate as indicated by experiment number EXP. The estimation was performed in R (4.1.1) with non- informative priors, and the results are compiled in Table 1 While the precision of the parameter estimates is high – as indicated by narrow credibility intervals. As protein binding is negligible in the medium, total concentrations are equal to unbound concentrations and the estimated potencies have to be compared with the unbound in vivo concentrations. HumanName Value 5% 95% UnitValue inValue in μg / l μg / L (total plasma) I0 2.6 0.79 5.7 %Imax 109 105 112 %Estimated logIC50 2.5 2.4 2.6 log(nM)hill 1.2 1.1 1.4 1IC10 1.9 1.5 2.4 nM0.9 1.5IC20 3.7 3.2 4.5 nM1.7 2.8IC30 5.9 5.2 6.7 nM2.8 4.5DerivedIC40 8.5 7.6 9.5 nM4.0 6.5fromIC50 12 11 13 nM5.7 9.2estimatesIC60 17 15 19 nM8.0 13IC70 24 21 28 nM11.3 18IC80 38 31 46 nM17.9 29IC90 76 57 96 nM35.8 58Table 1: Cellular in vitro potency. Total concentration (C) versus response (E) based on change in intracellular cAMP in presence of the stimulator forskolin. Reg FACS – Target engagement assay method Whole blood samples are collected in sodium citrate blood collection tubes. From each donor, 3 x 100 L whole blood are transferred into tubes and either 100 L RPMI medium + 0.2% DMSO (unstimulated sample) or 50 L TNFalpha working solution (8 ng / mL) and 50 L cytochalasin B (80 g / mL) (both stimulated samples) are added. Subsequently, all samples are incubated for 15 min± 1 min at 37°C in a water bath. To the unstimulated sample as well as to one of the two stimulated samples, 50 L medium + 0.5% DMSO are added. To the second stimulated sample 50 L 6-OAU working solution (50 M) are added. All samples are incubated for another 2h ± 5 min at 37°C in a water bath. The blood cells are then washed twice and incubated for 15 ± 2 min with normal mouse lgG to saturate Fe receptors. The samples are washed again and are then stained with the respective fluorochrome-conjugated antibodies (CD11b AE PE, CD45 Horizon V450, CD16 PE-Cy7) in the refrigerator for 30 ± 5 min. For lysis of the erythrocytes, lysis buffer is added right after the staining and the samples are incubated at 37°C for 15 ± 1 min in a water bath. Finally, the cells are washed twice, and each sample is divided onto two tubes - all samples will determined in duplicates. Positive target engagement is defined as reduction of mean CD11b expression by at least 10 % compared to pre-dosing level. The results of the ex vivo induced CD11b activated epitope expression in neutrophils tested in the whole blood FACS assay described above are presented in Fig.2a and 2b. The surface expression of CD11b activated epitope as a pharmacodynamic marker (à target engagement) of neutrophil activation was measured in HV before and after (at 2 timepoints – Day0 and Day9) GPR84 antagonist treatment as well as in placebo treated HVs. The positive target engagement as defined as reduction of mean CD11b expression by at least 10 % compared to pre-dosing level and a CSF trough concentration of compound 1 higher than IC80 was successfully achieved. Determination of total concentration of Compound 1 in human plasma Quantitative analysis of total Compound 1 concentration in human plasma was performed after addition of stable isotope labelled internal standards and protein precipitation with methanol followed by separation employing high-performance liquid chromatography and tandem mass spectrometric detection (LC-MS / MS). The method validation and analysis of the study samples were performed in compliance with the pertinent guidelines on Bioanalytical Method Validation. The calibration range of the procedure was from 0.5 (lower limit of quantification [LLOQ]) to 1000 g / L (upper limit of quantification [ULOQ]). QC samples in the concentration range from 1.5 to 750 g / L were determined with an accuracy of 97% to 101%. The trough concentration is defined as the concentration in measured matrix (e.g. plasma or CSF) at the end of the dosing interval (i.e.24 h for once daily dosing). In vitro assay: determination of fraction unbound of Compound 1 in human plasma The extent of binding to plasma proteins of different species was investigated with radiolabeled [14C]BAY 3178275 in the concentration range of 134 g / L to 10652 g / L (nominal concentrations ranging from 100 g / L to 10000 g / L) by the method of equilibrium dialysis. The human plasma was obtained from blood samples, which were drawn from healthy Caucasian volunteers at the Clinical Research Service Center in Wuppertal (Germany). The human plasma was prepared by centrifugation of the K- EDTA blood samples and plasma from at least three individuals was pooled and stored at -15 °C until use. In addition, plasma from 5 healthy male volunteers was individually investigated. The separation of protein bound and free (unbound) test substance was performed by dialysis across a semipermeable membrane with a pore size of 12-14 kDa according to Scholtan (Scholtan W., “The binding of long-acting sulfonamides on serum proteins”, Arzneimittelforschung, 1961; (11) 707-20). The free drug diffuses through the membrane into the buffer side until equilibrium is reached. The concentration of unbound drug can then be derived by measuring its concentration in the plasma and the buffer after a sufficient dialysis time. Material Dialysis apparatus: 96-well Dialysis apparatus, Teflon (Ht-Dialysis, Gales Ferry, USA). Dialysis membrane: regenerated cellulose membrane, pore size 12-14 kDa, (Ht- Dialysis, Gales Ferry, USA). Dialysis buffer: Dulbeccos 20 mM PBS-buffer, pH 7.4 (Sigma D 8537). Incubator: Heraeus BB6060 with 7% carbon dioxide aeration (Kendro, Langenselbold, Germany). Laboratory-shaker: IKA Vibrax-VXR (Janke & Kunkel, IKA Labortechnik, Staufen, Germany). Liquid scintillation counting: LS6500 Liquid Scintillation Counter (Beckman, USA). Samples were mixed with Ultima Gold Scintillation Liquid (Perkin Elmer, USA) in 20 mL Polyvials (Greiner, Germany). Study Procedure Plasma or protein containing buffer solutions adjusted to pH 7.4 (either by thawing the plasma in an incubator with 7% CO2 aeration or by exposing the plasma to a carbogen stream for 2 min) were dialyzed against buffer under the following conditions: Temperature: 37 °C Dialysis time: 5 h Exclusion size: 12-14 kDa Sample volume: 150 L Buffer volume: 150 L The total radioactivity in the plasma (C) and the dialysis buffer (Cu) were determined via LS-counting. These values were used for the calculation of the unbound fraction. The amount of organic solvent added to the plasma may not exceed 2% of the total incubation volume. Before starting the study, the recovery of a low drug concentration added to PBS from the dialysis cells (including membrane) was investigated. Generally, a recovery 90% of the actual concentration in the assay was accepted. Evaluation Results The protein binding of Compound 1 was low in all tested species with mean fu´s of 60.3% in male human plasma, of 63.1% in female human plasma (mean of male and female = 61.7%) and of 62.8% in male and female Wistar rat plasma. There was no concentration dependent plasma protein binding in the tested concentration range observed. Determination of total concentration of Compound 1 in human CSF Quantitative analysis of total Compound 1 concentration in human CSF was performed after addition of stable-labeled internal standard and protein precipitation with methanol followed by separation employing high-performance liquid chromatography and tandem mass spectrometric detection (LC-MS / MS). The method validation and analysis of the study samples were performed in compliance with the pertinent guidelines on Bioanalytical Method Validation. The calibration range of the procedure was from 0.5 (LLOQ) to 1000 g / L (ULOQ). QC samples in the concentration range from 1.5 to 750 g / L were determined with an accuracy of 98.3% to 100%. Clinical study First-in-human study of Compound 1 was conducted in healthy volunteers to investigate the safety, tolerability, and pharmacokinetics of single ascending dose: 0.5 mg, 2 mg, 5 mg, 15 mg, 25 mg, 50 mg, 100 mg and multiple ascending dose: 10 mg, 25 mg, 50 mg. Pharmacodynamics of Compound 1 was also investigated in multiple ascending dose part. Additionally, CSF exposure of Compound 1 after 15 mg multiple dosing was also investigated. Dose proportional increase of exposure was observed from 5 mg to 50 mg. Geometric mean total trough concentration in plasma after 10 mg QD dosing was 49.5 μg / L, corresponding to a mean unbound trough concentration of 30.5 μg / L, which was higher than IC80. It is expected coverage of IC80 with mean trough concentration in plasma could already be reached with 5 mg once daily dosing. There was strong correlation between total CSF and plasma concentrations at the same timepoint after 15 mg QD, resulting in a CSF / plasma ratio of 0.3. The ratio is not expected to change substantially from 5 mg to 50 mg based on the physiology and the pharmacology of Compound 1, so a CSF / plasma exposure ratio of 0.3 applies for 5 mg dose as well. Compound 1 was well tolerated, with very positive safety profile at all dosages tested. Multiple dose analysis comprised the ex-vivo neutrophil activation assay. A clear pharmacodynamic effect / target engagement was observed via ex vivo induced CD11b activated epitope expression in neutrophils. It has been shown that Compound 1 administered between the dose of 10 and 15 mg has an effective dose together with target engagement (already shown at 10 mg) and CNS penetration according to CSF exposure of over 30% shown in particular for the dosage of 15 mg. Furthermore, Compound 1 showed target engagement starting already at 10 mg and of course at higher dosage as well (25 and 50 mg).

Claims

CLAIMS 1. N-{[(2R)-1,4-dioxan-2-yl]methyl}-2-{[(2S)-1,4-dioxan-2-yl]methyl}-8- (trifluoromethyl)-4,5-dihydro-2H-furo[2,3-g]indazole-7-carboxamide (Compound 1) for use in a GPR84 depending disease characterized in that the daily dose is between 5 and 30 mg, and the unbound plasma trough concentration of Compound 1 is higher than the IC80 of 17.9 μg / L.

2. N-{[(2R)-1,4-dioxan-2-yl]methyl}-2-{[(2S)-1,4-dioxan-2-yl]methyl}-8- (trifluoromethyl)-4,5-dihydro-2H-furo[2,3-g]indazole-7-carboxamide (Compound 1) for use in a GPR84 depending disease characterized in that the daily dose is between 5 and 30 mg, the total cerebrospinal fluid (CSF) trough concentration of Compound 1 is higher than IC60 of 8 μg / L.

3. Compound 1 for use according to claim 1 or 2, characterized in that the daily dose is with a positive target engagement defined as reduction of mean CD11b expression by at least 10 % compared to pre-dosing level.

4. Compound 1 for use according to any one of the preceding claims, characterized in that the daily dose is between 8 and 20 mg.

5. Compound 1 for use according to any one of the preceding claims, characterized in that the oral daily dose is 10, 11, 12, 13, 14, 15, 16, 17 or 18 mg. 6 Compound 1 for use according to any one of the preceding claims, characterized in that the oral daily dose is 10, 11, 12, 13, 14, 15, 16, 17 or 18 mg and the ratio between total CSF trough concentration and total plasma trough concentration is of around 0.

3.

7. Compound 1 for use in a GPR84 depending disease characterized in that the daily dose is 15 mg, and the unbound plasma trough concentration and the total CSF trough concentration of Compound 1 is higher than the IC80 of 17.9 μg / L, and positive target engagement defined as reduction of mean CD11b expression by at least 10 % compared to pre-dosing level.

8. A pharmaceutical form for oral administration comprising N-{[(2R)-1,4-dioxan-2- yl]methyl}-2-{[(2S)-1,4-dioxan-2-yl]methyl}-8-(trifluoromethyl)-4,5-dihydro-2H- furo[2,3-g]indazole-7-carboxamide (Compound 1) between 3 and 25 mg, for use in a GPR84 depending disease characterized in that it is administered in a daily dose between 5 and 30 mg, with positive target engagement as defined as reduction of mean CD11b expression by at least 10 % compared to pre-dosing level and a total CSF though concentration of compound 1 higher than IC60 of 8 μg / L.

9. A pharmaceutical form for oral administration according to claim 8, characterized in that the oral formulation is an instant release (IR) oral formulation.

10. Compound 1 for use according to claims 8 or 9, characterized in that the daily dose is between 8 and 20 mg.

11. Compound 1 for use according to claims 8 to10, characterized in that the oral daily dose is 10, 11, 12, 13, 14, 15, 16, 17 or 18 mg.

12. Compound 1 or a pharmaceutical form for oral administration comprising Compound 1 for use in a GPR84 depending disease according to claims 1 to 11 for the treatment or prophylaxis of chemotherapy induced neuropathic pain.

13. Compound 1 or a pharmaceutical form for oral administration comprising Compound 1 for use in a GPR84 depending disease according to claims 1 to 12 for the treatment or prophylaxis of post-breast surgery pain, cancer bone pain, trigeminal neuralgia, post-mastectomy pain, fibromyalgia, multiple sclerosis pain diseases, post-herpetic neuralgia, Fabry, gout, or bladder pain syndrome.

14. Compound 1 or a pharmaceutical form for oral administration comprising Compound 1 for use in a GPR84 depending disease according to claims 1 to13 for the treatment or prophylaxis of multiple sclerosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, primary and secondary autoimmune uveitis, inflammatory disorders like endometriosis, inflammatory eye diseases, inflammatory kidney diseases, inflammatory liver diseases like non-alcoholic, alcoholic- and toxic fatty liver diseases, lung diseases like asthma, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and metabolic and metabolic-endocrine disorders like metabolic syndrome, insulin resistance, diabetes mellitus type I and type II, and polycystic ovary syndrome (PCOS) disorders.

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