Isolithocholic acid derivatives and use thereof

Abstract

The subject matter of the invention is a compound of the formula (I), or a salt, prodrug, solvate, hydrate, or stereoisomer thereof, wherein Rn represents groups for n=1, 2, or 3, as follows R1 represents -NHCH(CH3)2, R2 represents -CONH2, R3 represents -CH2NH2. Another subject matter of the invention is a compound according to the present invention for use in a prevention and / or treatment of inflammatory diseases of the respiratory system and / or prevention and / or treatment of cytokine storm-associated diseases.

Description

[0001] 67936 / 25 1

[0002] Isolithocholic acid derivatives and use thereof

[0003] Field of the invention

[0004] The present invention relates to isolithocholic acid derivatives and use thereof for prevention or treatment of inflammatory diseases of the respiratory system.

[0005] Background

[0006] European patent EP3668879B1 discloses steroid compounds that have a formula represented by the following: (I) and wherein Rl, R2, R3a, R3b, R4a, R4b, R5, R6a, R6b, R7, R8, and n are as described herein. The compounds may be prepared as pharmaceutical compositions, and may be used for promoting metabolic reprogramming of macrophages, and for the prevention and treatment of a variety of conditions in mammals including humans, including by way of nonlimiting example, inflammatory conditions, autoimmune disorders, and graft-versus-host disease.

[0007] International publication W02020260558A1 discloses isolithocholic acid (3|3-hydroxy-5|3- cholan-24-oic acid) and isoallolithocholic acid (3|3-hydroxy-5a-cholan-24-oic acid) together with the respective 22-homo-analogs or the deuterated analogs, which are modified in 3- position, for preventing or treating Clostridioides difficile-associated disease in a mammalian subject.

[0008] European patent application EP3004132A1 discloses compositions and methods for treating diseases or disorders associated with the metabolic syndrome using conjugates of bile acids with basic amino acids or a decarboxylated amino acid such as agmatine are provided. Further provided are bile acid- basic amino acid conjugates comprising chenodeoxycholic acid with an amino acid selected from arginine, lysine, histidine, ornithine or a decarboxylated amino acid such as agmatine.

[0009] Secondary bile acids are microbial products converted from primary bile acids. They have been shown to exert anti-inflammatory properties in the intestines through nuclear receptors and / or G protein-coupled receptors [1, 2], Several bile acids were shown to be potent modulators of adaptive immunity. Lithocholic acid (LCA) inhibited the activation of Thl cells in vitro via Vitamin D Receptor (VDR) signalling [3], 3-oxo-lithocholic acid and isolithocholic acids inhibited Thl7 differentiation in vitro and in vivo by binding RORyt, a master regulator of the Thl7 subset, and inhibiting its transcriptional activity [4, 5], Finally, isoallolithocholic acid 67936 / 25 2

[0010] (isoalloLCA) promoted the generation of mitochondrial reactive oxygen species, leading to enhanced expression of FOXP3 and differentiation of regulatory ? cells in vitro [4, 6], However, the potential impact of secondary bile acids on respiratory immunity has not been described. It is widely accepted that the gut microbiota profoundly shapes our immunity [7-9], Dysbiosis in patients suffering from conditions characterized by airway inflammation has been described [10-14], which triggered interest in reducing symptoms of these diseases via probiotic administration [15, 16], However, this approach raises three major concerns. First, probiotics fail to colonize the host, which prevents the long-lasting effect

[0017] , Second, as live organisms, probiotics adjust their metabolic functions according to new environments they are introduced to (different in terms of the microbiota / immune-metabolic states in each person), and thus, may lose their beneficial potential

[0018] , Finally, the concentrations of microbial products become diluted along the transit from the gut to the lungs (as demonstrated in the case of short-chain fatty acids [19, 20]), which might hinder their protective effect. In light of the above, the approach to administer a microbial metabolite or its derivatives directly to the site of the interest overcomes problems with probiotic regimens in the following ways: i) administration of purified microbial compounds does not rely on microbial colonization; ii) the function of administered compounds is not a subject of adaptation to a new environment; iii) their safety is easier to ensure, and iv) the route along the gut-lung axis is bypassed via direct intranasal administration, maximizing bioavailability of the compound in the airways.

[0011] A recent SARS-Cov-2 pandemic illustrated an emerging medical need to develop therapeutic interventions to reduce airway pathology in patients experiencing airway inflammation. One strategy to achieve this outcome is the use of anti-inflammatory drugs. Despite extensive efforts, many clinical trials failed to prove the efficacy of candidate compounds. These include immunomodulators such as anti-interleukin-6 or anti-interleukin-1 monoclonal antibodies

[0021] , hydrocortisone

[0022] , azithromycin

[0023] , doxycycline

[0024] , interferon beta-la

[0025] , or the Janus kinase (JAK)1 / JAK2 inhibitor ruxolitinib

[0026] ,

[0012] Several therapeutics did show a clinical benefit. These include dexamethasone

[0027] , anti-l L-6R monoclonal antibody Tocilizumab

[0028] , or Janus-associated tyrosine kinase (JAK) 1 and JAK 2 inhibitor, Baricitinib

[0029] , However, their efficacy is still limited (e.g. the improved mortality rate in the case of dexamethasone treatment is 22.9% vs 25.7% in the control group)

[0027] , In addition, those drugs are delivered orally or intravenously, which increases the likelihood of off-target effects. The primary mode of action behind compounds described in this application 67936 / 25 3 relies on the effective inhibition of neutrophil influx, which is a hallmark of several respiratory diseases, including bacterial or viral infection-induced ARDS

[0030] , neutrophilic severe asthma

[0031] , chronic obstructive pulmonary disease (COPD)

[0032] , bronchiectasis

[0033] , or idiopathic pulmonary fibrosis (IPF)

[0034] , Of note, most mediators that drive recruitment of neutrophils and other inflammatory cells, such as CXCL1, CCL2, CCL5, CXCL10, and IL-6 have been implicated in the pathogenesis of ARDS, neutrophilic asthma, COPD or IPF [31, 35-43] have been effectively inhibited by the compounds described in this application

[0013] Influx of neutrophils into the lung tissue is responsible for immunopathology in ARDS [30, 44, 45], Moreover, major cause of the immunopathology of ARDS are carried by influx of dendritic cells and pro-inflammatory (Ly6C+) monocytes [46-50], It is worth to mention that compounds described in this application do not induce cytotoxicity markers, such as Cystatin C and clusterin, which have been recommended for use as toxicity markers in preclinical development by the Predictive Safety Testing Consortium in collaboration with the US Food and Drug Administration (FDA), the European Medicines Agency and the Japanese Pharmaceuticals and Medical Devices Agency (PMDA) and the number of pharmaceutical companies

[0052] ,

[0014] Bile acids are known to be recognized by two classes of receptors, nuclear receptors (farnesoid X receptor, pregnane X receptor, vitamin D receptor, RAR-related orphan receptor gamma, the liver X receptor, and Nuclear Receptor Subfamily 4 Group A Member 1 and constitutive androstane receptor) and G protein-coupled receptors (sphingosine-l-phosphate receptor 2, Takeda G protein-coupled receptor 5, and muscarinic acetylcholine receptor M3) [53, 54], Activation of a nuclear receptor, farnesoid X receptor (FXR) was shown to induce expression of a transcriptional repressor, SHP. In turn, SHP was demonstrated to interact with a transcription factor API, preventing API-driven expression of inflammatory genes

[0055] , or directly bind to regulatory elements of inflammatory genes

[0056] , Activation of a G protein-coupled receptor, Takeda G protein-coupled receptor 5 (TGR5, also known as the G protein-coupled bile acid receptor 1, GPBAR1), was reported to activate protein kinase A (PKA) and cAMP-responsive element-binding protein (CREB), thereby reducing NF-KB activity in a STAT-l-dependent fashion [57-59], Finally, both FXR and TGR5 were demonstrated to inhibit NLRP3 inflammasome activation. TGR5 signaling led to PKA-dependent phosphorylation of NLRP3, which resulted in NLRP3 ubiquitination and prevention of inflammasome assembly. FXR, on 67936 / 25 4 the other hand, physically interacted with caspase 1 and NLRP3, which prevented inflammasome assembly

[0053] , Summary of the invention

[0015] The first aspect of the invention is a compound of the formula (I):

[0016] Formula (I) or a salt, prodrug, solvate, hydrate, or stereoisomer thereof, wherein Rnrepresents groups for n=l, 2, or 3, as follows:

[0017] Ri represents -NHCHfCHsh,

[0018] R2 represents -CONH2,

[0019] R3 represents -CH2NH2.

[0020] In some embodiments the compound is selected from the group consisting of: 67936 / 25 5

[0021] The second aspect of the invention is a composition comprising at least one compound or salt, prodrug, solvate, hydrate, or stereoisomer thereof.

[0022] In some embodiments a composition further comprises at least one carrier. The third aspect of the invention is a compound according to the present invention for use in a prevention and / or treatment of inflammatory diseases of the respiratory system and / or prevention and / or treatment of cytokine storm-associated diseases.

[0023] In some embodiments a compound for use in the prevention and / or treatment of inflammatory diseases of the respiratory system by inhibition of neutrophil influx. In some embodiments a compound for use in the prevention and / or treatment of inflammatory diseases of the respiratory system is based on inhibition of the production of one or more of the following cytokine: CXCL1, CCL2, CCL5, CXCL10, IL-6.

[0024] In some embodiments a compound is used in the inflammatory diseases of the respiratory system such as: ARDS, viral respiratory infections, bacterial respiratory infections, neutrophilic asthma, chronic obstructive pulmonary disease, bronchiectasis, idiopathic pulmonary fibrosis. 67936 / 25 6

[0025] The particular advantage of our invention is a localized delivery in a form of a nasal spray and the capacity to inhibit a broad spectrum of inflammatory genes, which increases the efficacy of the treatment.

[0026] According to the invention, compounds disclosed in the invention may be used in many technical fields, especially in medicine, veterinary, pharmaceutical field mainly but not limited to the prevention or treatment of diseases, particularly inflammatory diseases of the respiratory system or cytokine storm-associated diseases.

[0027] According to the invention we propose isolithocholic (isoLCA) derivatives to be formulated as prophylactic and / or therapeutic formulations against respiratory conditions characterized by neutrophilia, including but not limited to the following disorders: ARDS, viral or bacterial respiratory infections, neutrophilic asthma, chronic obstructive pulmonary disease, bronchiectasis or idiopathic pulmonary fibrosis.

[0028] Brief description of drawings

[0029] Fig. 1 IsoLCA profoundly inhibits the pro-inflammatory response in lung cells. A) Experimental setup. Primary murine lung cells were isolated using dispase II and 1% agarose solution and MACS-sorted into CD45+ and CD45- fractions. Cells were then stimulated with LPS (10 ng / ml) in the presence of tested metabolites, and the pro-inflammatory response was assessed using cytometric bead arrays. B) Lung Immune cells (CD45+) and C) Lung structural cells (CD45-) were stimulated with LPS (10 ng / ml) in the presence of tested metabolites. The pro- inflammatory response was assessed by cytometric bead arrays and compared to control conditions (cells stimulated with LPS in the presence of a vehicle). D), E), F) Select analytes (<0.5 fold change) from the immune cell compartment. G, H) Select analytes (<0.5 fold change) from the structural cell compartment.

[0030] Fig. 2 presents the activity of LCA, bearing the opposite orientation of the C3-hydroxyl group (at the concentration of 50 pM), being a base to synthesise 21 derivatives of isoLCA.

[0031] Fig. 3 presents compounds Bl-3, B1-X2, and B1-X4 (Table 1) retain the anti-inflammatory potential of isoLCA. A) Cellular toxicity assay of 3 isoLCA derivatives (at 50 pM). Equal volumes of PBS or medium and CellTiter-Glo reagent were added per well with immune lung cells or structural lung cells. After 2 minutes of incubation in a thermomixer at room temperature, 800 rpm luminescence was measured using Tecan M1000PRO. B) Activity of these derivatives of 67936 / 25 7 isoLCA (below 80% of live control) (at 50 pM). Data points represent biological replicates pooled from 3 independent experiments. Error bars represent the SEM. *p<0.05, **p<0.01.

[0032] Fig. 4 presents the therapeutic potential of isoLCA, Bl-3, and B1-X2 (Table 1) in a mouse model of ARDS; A) Experimental setup. C57BL / 6J mice were administered with 20 pg of LPS intranasally. isoLCA, Bl-3, B1-X2, or B1-X4 (Table 1) were administered intranasally 6 hours, 24 hours, and 48 hours later at the dose of 0,5 pM / kg body weight. Lungs were collected on day 3; B) Numbers of neutrophils, dendritic cells, Ly6C+, and Ly6C_monocytes were obtained with the use of flow cytometry and calculated as fold change in comparison to the control group (administration of LPS followed by the vehicle treatment); C) Concentrations of Cystatin C and Clusterin were measured by ELISA and calculated as fold change to the control (DMSO). Data points represent 8 biological replicates pooled from 2 independent experiments. Error bars represent the SEM. *p<0.05, **p<0.01, ****p<0.0001.

[0033] Fig. 5 presents H1NMR spectrum of B1-X2 compound.

[0034] Fig. 6 presents H1NMR spectrum of Bl-3 compound.

[0035] Fig. 7 presents H1NMR spectrum of B1-X4 compound.

[0036] Fig. 8 presents the therapeutic potential of isoLCA and Bl-3 in a mouse model of allergic airway inflammation; A) Experimental setup for allergic airway inflammation; B) Eosinophil infiltration; C) DC infiltration; D) plasma levels of total IgE; E) plasma levels of total IgGl; F) Ly6C+monocyte infiltration; G) neutrophil infiltration. Numbers of neutrophils, dendritic cells, Ly6C+ monocytes were obtained with the use of flow cytometry and calculated as fold change in comparison to the control group. Plasma levels of total IgE and IgGl were obtained with ELISA assay using goat anti-mouse IgE or IgGl and alkaline phosphatase-conjugated goat antimouse IgE or IgGl (SoutherBiotech).

[0037] Figure 9 shows a mechanism of action of isoLCA in mouse and human alveolar macrophages A) Experimental setup. Mice were administered with LPS intranasally on day 0, followed by intranasal instillation of isoLCA 6h, 24h, and 48h later. Inflammatory cell influx into the lungs was analysed 72 hours after LPS administration. B-D) Fold change in neutrophil numbers in the lungs (B) and concentrations of CXCL1 (C) and CXCL10 (D) in the bronchoalveolar lavage fluid of mice treated with isoLCA. E, Representative hematoxylin and eosin (H&E) staining of lung sections from mice in A-D. Scale bar = 100 pm. F, Dot plot depicting changes in gene expression across clusters caused by isoLCA treatment in LPS-treated mice, as per A. G) Top five pathways induced by isoLCA treatment in alveolar macrophages from mice treated as 67936 / 25 8 described in A. H) List of genes related to the citric acid (TCA) cycle and respiratory electron transport upregulated by isoLCA treatment to a statistically significant degree (p<0.05). I) Pie charts depicting TCA cycle substrate utilization of AML cells treated as per Fig. IL. J-M, AML cells were treated as described in Fig. IL. J) Metabolic activity was measured 2 hours posttreatment by resazurin conversion to resorufin. K) Concentration of lactate in culture medium was assessed 18 hours post-treatment. L) Intracellular ROS / RNS level was assessed 2 hours and 18 hours post-treatment by the conversion of 2',7'-Dichlorodihydrofluorescein diacetate (DCFH2-DA) to the fluorescent form 2',7'-dichlorofluorescein (DCF). M) Mitochondrial membrane potential was evaluated 18 hours post-treatment by accumulation of tetramethylrhodamine methyl ester perchlorate (TMRM). N-Q, Data pooled from three independent experiments, each performed in technical duplicates or triplicates. CXCL10 concentration in the culture media of AML cells (N, O) and primary mouse lung CD45+ cells (P, Q) was assessed under hypoxic conditions (1% 02) or in the presence of a TCA cycle inhibitor (UK-5099 for human cells, Roxadustat for mouse cells), respectively.

[0038] Embodiments

[0039] Compound of lithocholic acid in amount of 1 eq and thionyl chloride in amount of 2 eq were dissolved in anhydrous methanol (10 V; 10 x volume of reagents) and stirred for 2 hours in 70°C. Protected lithocholic acid was separated and purified. Reaction of compound (3) in amount of 1 eq with chloromesylate in amount of 2 eq and triethyloamine in amount of 3 eq was carried out in dichloromethane (30 V) at 0°C for 2 hours in quantitative yield. Procedure was carried out before all synthesis of the invention.

[0040] Embodiment 1 67936 / 25 9

[0041] Isolated and purified compound (3) underwent substitution with NaCN in THF at 65°C for 24 hours, resulting in compound (4). Sodium hydroxide and methanol in water solution were added to the mixture, yielding compound Cpd B1-X2.

[0042] Cpd B1-X2

[0043] Embodiment 2

[0044] Isolated and purified compound (3) underwent reaction with isopropylamine in N-methyl-2- pyrrolidone (NMP) at 120°C for 12 hours in sealed tube. Sodium hydroxide and methanol in water solution were added to the mixture, yielding compound Cpd Bl-3.

[0045] Cpd Bl-3

[0046] Embodiment 3

[0047] Isolated and purified compound (3) underwent substitution with NaCN in DMF at 80°C for 20 hours, resulting in compound (4). Isolated compound (4) was subjected to hydrogenation process using catalyst Pd / C in EtOH, AcOH at 80°C for 12 hours. Ester hydrolysis was done using sodium hydroxide, resulting in compound Cpd B1-X4. 67936 / 25 10

[0048] The obtained compounds namely Cpd B1-X2, Cpd Bl-3, Cpd B1-X4 were in the form of white solids with a purity of >97%.

[0049] Embodiment 4 - Inhibition properties of the pro-inflammatory response in the lungs

[0050] We screened the library of metabolites for their capacity to inhibit a panel of inflammatory mediators that are typically observed during acute respiratory distress syndrome. For this, we used lungs isolated from naive mice C57BL / 6J bred in the animal house at our Institute, and after using MACS sorting (Miltenyi Biotec), we obtained CD45 positive cells. We stimulated them with LPS (1 pg / ml), which mimics the inflammation in the presence or absence of metabolites in 50 pM concentration, established as non-toxic. After 24h, we collected the supernatant, and the production of inflammatory mediators was analysed by cytometric bead arrays (Legend Plex) or ELISA (R&D or Invitrogen). By stimulating lung immune cells with lipopolysaccharide (a compound that mimics a bacterial infection), we found isoLCA to be the most effective among tested compounds at inhibiting the proinflammatory response. It inhibits the production of CXCL1, CCL2, CCL5, CXCL10 and IL-6 to a statistically significant degree (fold change <0,5 versus LPS alone) (Fig. 1). Based on this, we decided to synthesise 21 derivatives of isoLCA, since we noted that its natural isomer, lithocholic acid (LCA), which differs in the three-dimensional orientation of the C3-hydroxyl group, lost the immunosuppressive potential (Figure 2). This data indicated that the C3-hydroxyl group is important for the activity of isoLCA and suggested that its modification can alter the magnitude of the effect. We screened those compounds; 8 of them were non-toxic for lung cells ( Fig.3 A), and 3 of them showed immunosuppressive potential on lung cells even stronger than isoLCA (Fig.3B). 67936 / 25 11

[0051] Embodiment 5 - Protection properties against ARDS

[0052] To test whether identified compounds hold the potential to be developed as drugs against ARDS, we validated our findings in a mouse model of ARDS. C57BL / 6J mice bred in the animal house at our Institute were administered with lipopolysaccharide from Escherichia coli O127:B8 (SigmaAldrich) intranasally (20 pg), and isoLCA (Cayman) and its derivatives were administered intranasally 6 hours, 24 hours, and 48 hours later at a dose of 0.5 pM / kg body weight (Fig. 4). After 3 days, mice were sacrificed, and cell infiltration in the lung and cytokine levels in BALF fluid were measured. (Fig. 4A). Bl-3, and B1-X2 (Table 1) potently inhibited the influx of neutrophils into the lung tissue. Notably, Bl-3 (Table 1) also inhibited the influx of dendritic cells and pro-inflammatory (Ly6C+) monocytes (Fig. 4B).

[0053] None of the compounds affected the population of patrolling monocytes, which are involved in removing damaged cells and wound healing (defined as Ly6C—

[0051] ).

[0054] Finally, none of the compounds elevated the levels of kidney toxicity markers, cystatin C and clusterin (Fig. 4C). Together, these data indicate the therapeutic potential of Bl-3, and B1-X2 (Table 1) in the context of ARDS.

[0055] Embodiment 6 - Bl-3 attenuated allergic airway inflammation in a mouse model

[0056] Mice were anesthetized by inhalation of 3.5% isoflurane in oxygen for 6 min. House dust mite (HDM) (Citeq Biologies) was reconstituted at a concentration of 20 pg protein content in 30 pL of sterile PBS and administered intranasally on days 0, 1, 11, 12, and 13 (Fig. 8A). Mice were euthanized on day 14, and cell infiltration in the lung and plasma levels of total IgE and IgGl were measured. Bl-3 inhibited the influx of eosinophils, dendritic cells (Fig. 8B-C), neutrophils as well as pro-inflammatory (Ly6C+) monocytes (Fig. 8F-G) and lowered the levels of allergically significant immunoglobulins IgE and IgGl (Fig. 8D-E).

[0057] Embodiment 7 - Mechanism of action

[0058] Using single-cell RNA sequencing of mouse lung cells, we have indicated an additional, previously undescribed mechanism by which isolithocholic acid boosts mitochondrial function in alveolar macrophages, by inducingtranscriptional changes in those cells, including upregulation of tricarboxylic acid (TCA) cycle genes such as SdhcL ml-Co and mt-Nd4 (Fig.

[0059] 9a-h). We also confirmed this mechanism of action in human alveolar-like macrophages 67936 / 25 12 differentiated from monocytes isolated from peripheral blood from healthy donors - cells treated with isoLCA exhibited enhanced mitochondrial activity, decreased lactate production, reduced reactive oxygen / nitrogen species, and preserved mitochondrial membrane potential upon LPS stimulation (Fig. 9j-m). Their anti-inflammatory phenotype was abrogated under hypoxia or pharmacological TCA cycle inhibition (UK 5099), indicating a direct mechanistic link between metabolic rewiring and isoLCA’s immunomodulation (Fig. 9n, o). These results were further validated in primary mouse lung immune cells (Fig. 9p, q).

[0060] In the context of isoLCA derivatives, we discovered that they can suppress the production of chemokines recruiting neutrophils, reducing inflammation across diverse models of lung disease, including ARDS and allergic airway inflammation.

[0061] 67936 / 25 13

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Claims

67936 / 25 18CLAIMS1. A compound of the formula (I):Formula (I) or a salt, prodrug, solvate, hydrate, or stereoisomer thereof, wherein Rnrepresents groups for n=l, 2, or 3, as follows:Ri represents -NHCHfCHsh,R2 represents -CONH2,R3 represents -CH2NH2.

2. The compound according to claim 1, characterized in that the compound is selected from the group consisting of:67936 / 25 193. A composition comprising at least one compound or salt, prodrug, solvate, hydrate, or stereoisomer thereof as defined in any one of claims 1-2.

4. A composition according to claim 3, further comprising at least one carrier.

5. A compound as defined in any one of claims 1-2 for use in the prevention and / or treatment of inflammatory diseases of the respiratory system and / or prevention and / or treatment of cytokine storm-associated diseases.

6. A compound for use according to claim 5, wherein prevention and / or treatment of inflammatory diseases of the respiratory system by inhibition of neutrophil influx.

7. A compound for use according to any one of claims 5-6, characterised in that, the prevention and / or treatment of inflammatory diseases of the respiratory system is based on inhibition of the production of one or more of the following cytokine: CXCL1, CCL2, CCL5, CXCL10, IL-6.

8. A compound for use according to any one of claims 5-7, wherein inflammatory disease of the respiratory system belongs to such group of instances as: ARDS, viral respiratory infections, bacterial respiratory infections, neutrophilic asthma, chronic obstructive pulmonary disease, bronchiectasis, idiopathic pulmonary fibrosis.

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