WO2019211783A1

WO2019211783A1 - Indoleamine 2,3-dioxygenase signalling modulator and therapeutic use thereof

Info

Publication number: WO2019211783A1
Application number: PCT/IB2019/053588
Authority: WO
Inventors: Antonio Macchiarulo; Ursula Grohmann
Original assignee: Universita' Degli Studi Di Perugia
Priority date: 2018-05-04
Filing date: 2019-05-02
Publication date: 2019-11-07
Also published as: IT201800005062A1

Abstract

The invention refers to the use of the compound 335984-DIDO in the therapeutic treatment of multiple sclerosis, a pharmaceutical composition comprising the compound 335984- DIDO and a method for formula (I).

Description

Indoleamine 23-dioxygenase signalling modulator and therapeutic use thereof

The present invention falls within the field of therapeutic treatments of autoimmune diseases. More specifically, the invention relates to the use of an indoleamine 2,3- dioxygenase (IDOl) signalling modulator for the therapeutic treatment of multiple sclerosis (MS).

Multiple sclerosis (MS) is a chronic and progressive neurodegenerative disease currently affecting around 2.3 million people worldwide. MS incidence rates are significantly higher in Europe and other regions of the northern hemisphere. In Europe, the number of patients currently suffering from MS is estimated at around 700,000, with incidence rates ranging between 2.3-12.2/100,000 per year.

From a pathophysiological point of view, multiple sclerosis is characterised by the loss of the insulating layer surrounding the axons of neurons, i.e. the myelin sheath. This loss causes neuronal damage and the formation of plaques in the central nervous system.

This disease can be distinguished into two main types, namely relapsing-remitting multiple sclerosis (RRMS), which affects about 80% of MS patients, and primary-progressive multiple sclerosis (PPMS), which affects the remaining 20%. The two types differ in the disease progression patterns, and so far most of the available therapeutic treatments have only been approved for RRMS.

MS symptoms manifest themselves as cognitive impairment, muscle weakness, loss of muscle coordination, speech and vision disorders. The high prevalence of MS (2.3 million people worldwide) leads to a substantial economic burden (on average,€ 50,000 per year per patient), since MS mainly affects relatively young people (the disease is often diagnosed between 20 and 40 years of age). The effects of neurodegeneration accumulate over time and within 15 years of diagnosis 80% of patients become unable to work.

Research carried out in the field of MS has established that the causes of the disease are immunological, in particular autoimmunity-related, and for this reason MS is also considered as the autoimmune disease with the highest prevalence.

Currently there is no cure for MS and the available therapeutic options are mainly intended to suppress the activity of pro-inflammatory mediators. Moreover, in addition to their substantial ineffectiveness, most of the treatments available today require frequent administrations (from once a day to once a month) because the pathways in which they act only show short-term effects. Examples of such drugs include Ocrelizumab (mAb for B lymphocyte depletion), Zinbryta (mAb to prevent T lymphocyte activation), Interferon beta la/lb (recombinant anti-inflammatory cytokines), Natalizumab (mAb to block effector T lymphocyte uptake through the blood-brain barrier), Fingolimod (a small molecule that sequesters leukocytes), dimethyl fumarate (which promotes regulatory T lymphocytes or Tregs), corticosteroids and adrenocorticotropic hormones (ACTH). Although various therapeutic options are available, neurodegeneration progressively persists in MS patients, leading to a deterioration in the quality of life and a reduction in life expectancy (5-10 years less than the average).

Therefore, there is an urgent need to identify new drugs for MS treatment, which are more effective in modulating inflammatory events in the central nervous system. These new drugs should also be targeted to new molecular mechanisms, so as to provide long-term immunoregulatory effects, in view of the chronic nature of the disease.

Patients with juvenile diabetes and multiple sclerosis are known to exhibit a deficiency of the indoleamine-2, 3-dioxygenase 1 (IDOl) enzyme. This enzyme produces kynurenines, i.e. substances that prevent over-activation of the immune defences. In contrast, IDOl is too active in cancer patients, where it acts by turning off the immune defence response to the growth of cancer cells.

Previous studies have shown that IDOl not only produces kynurenines to inhibit lymphocyte activation, but can also send signals to immune cells, thereby reprogramming the immune defences towards long-term immune tolerance functions, a fundamental effect for the treatment of chronic diseases such as autoimmune diseases [Pallotta MT, et al. Nat. Immunol. 201 1 , 12(9), 870-8]. While the production of kynurenines is linked to the catalytic activity of the enzyme, the sending of signals to immune cells is associated with post-transcriptional modifications of two amino acid motifs in the primary sequence of IDOl, designated as ITIM or Immune Tyrosine Inhibitory Motif [Albini E, et al. J. Cell Mol. Med. 2017, 1, 165-176]

To date, the importance of the production of kynurenines through the enzyme’s catalytic activity has generated the development of several strategies aimed at blocking this function with chemical compounds capable of inhibiting the enzyme. For instance, patent applications W02010005958, WO2016181349 and WO2012142237 are known, which are related to 1D01 inhibitors, i.e. chemical compounds aimed at reducing kynurenine production and stopping tumour growth, and currently at an advanced clinical trial stage.

According to patent application EP0021806A1, 2,3-dihydro-imidazo[2,l-b]benzothiazole analogues would appear to exert antidepressant and anti-Parkinson pharmacological activities, acting as inhibitors of the monoamine oxidase (MAO) enzyme. The scientific publication by Smith et al, Bioorganic & Medicinal Chemistry, 20 (2012), 1354-1363, describes the identification and biological characterization of some 2,3-dihydro- imidazo[2,l-b]benzothiazole analogues as weak inhibitors of IDO’s catalytic activity (IDOl IC50 = 50 mM). In this publication, 2,3-dihydro-imidazo[2,l-b]benzothiazole analogues are investigated as possible inhibitors of tumour growth.

The present inventors have now surprisingly found that certain 2,3-dihydro-imidazo[2,l- bjbenzothiazole analogues are capable of binding the IDOl enzyme and modulating the signalling thereof that is specifically mediated by ITIM amino acid motifs, thereby reprogramming the immune defences towards immune tolerance effects.

This result is surprising since neither patent application EP0021806A1, nor the scientific publication by Smith et al, Bioorganic & Medicinal Chemistry, 20 (2012), 1354-1363, nor other publications report experimental data concerning the possible in vitro and/or in vivo activity of these compounds in modulating the signalling mediated by IDOl ITIM motifs. Therefore, their effectiveness as pharmacological agents against autoimmune diseases in which IDOl plays an important role was not predictable in the light of the prior art. Furthermore, US patent application US 2009/253706 reports that IDOl inhibition through administration of 1 -methyl-tryptophan is effective in a rheumatoid arthritis model, while the article E.KWIDZINSKI, THE FASEB JOURNAL, "Indolamine 2,3 -dioxygenase is expressed in the CNS and down regulates autoimmune inflammation "(2005-06-13) reports that administration of 1 -methyl-tryptophan worsens ' experimental autoimmune encephalomyelitis. In the light of the prior art there was no reasonable expectation of success in using an IDO l inhibitor, such as 1 -methyl-tryptophan, in the therapeutic treatment of multiple sclerosis.

One aspect of the present invention is therefore the compound as shown below for use in the therapeutic treatment of multiple sclerosis. The compound used in the invention is designated as 338954-DIDO and represents an advantageous alternative to the few therapeutic approaches known to date for the treatment of this chronic disease.

338954-DIDO

A second aspect of the invention is a pharmaceutical composition comprising or consisting of at least the compound 338954-DIDO as the active ingredient, in association with one or more pharmaceutically acceptable adjuvants and/or excipients, for use in the therapeutic treatment of multiple sclerosis.

A third aspect of the invention is a method for modulating the activity of the IDOl enzyme in a subject, comprising administering to said subject the compound 338954-DIDO or a pharmaceutical composition comprising or consisting of at least the compound 338954- DIDO as the active ingredient. In a preferred embodiment, the subject is affected by multiple sclerosis. In another preferred embodiment, the subject is a human being. In a further preferred embodiment, the subject is administered with a pharmaceutically effective amount of the compound 338954-DIDO or the pharmaceutical composition comprising or consisting of at least the compound 338954-DIDO as the active ingredient. A pharmaceutically effective amount of the compound 338954-DIDO, for example, ranges between 0.1 - 100 mg/kg, preferably 0.1 - 1 mg/kg, or 1 - 10 mg/kg, or 10 - 20 mg/kg, or 20 - 30 mg/kg, or 30 - 40 mg/kg, or 40 - 50 mg/kg, or 50 - 60 mg/kg, or 60 - 70 mg/kg, or 70 - 80 mg/kg, or 80 - 90 mg/kg, or 90 - 100 mg/kg.

The present invention is hereinafter described by way of illustration, but not by way of limitation, according to preferred embodiments thereof, with particular reference to the attached figures and tables, in which:

Figure 1 shows the structural formula of 338954-DIDO;

Figure 2 shows the computational analysis of molecular dynamics trajectories of the IDOl structure, which led to the identification of two enzyme conformations (Conformation A and Conformation B) specifically associated with the catalytic activity (Conformation A) and signalling (Conformation B) thereof;

Figure 3 shows the concentration-dependent interaction curve between the compound 338954-DIDO and the IDOl enzyme, as obtained from microscale thermophoresis studies;

Figure 4 shows the results from the administration of the compound 338954-DIDO in an induced skin hypersensitivity model. The compound 338954-DIDO is capable of exerting a tolerogenic immune effect through an IDOl -dependent mechanism;

Figure 5 shows the ability of the compound 338954-DIDO to improve the clinical score in a dose-dependent manner in a model of experimental autoimmune encephalopathy (EAE);

Table 1 shows the results from the inhibition of the compound 338954-DIDO on the enzymatic activity of wild type IDOl (adopting the conformation A) and IDOl with mutated ITIM residues (preferably adopting the conformation B).

The invention was achieved through a computational analysis carried out on crystallised structures of the IDOl enzyme, in which two enzyme conformations (Conformation A and Conformation B, Figure 2), specifically associated with the catalytic activity (Conformation A) and signalling (Conformation B) thereof, could be identified.

Site-directed mutagenesis experiments on amino acids of the primary sequence of IDO 1, which are important for the energy stability of these conformations, have therefore allowed the generation of two cell lines stably expressing the enzyme preferably adopting conformation A (wild type IDOl), or the enzyme preferably adopting conformation B (IDOl with mutated 1TIM residues). The aforementioned cell lines were then used in assays for in vitro screening of libraries containing low molecular weight chemical compounds, aimed at identifying chemical entities capable of binding the enzyme preferably adopting conformation B (with the signalling function). These studies showed that 338954-DIDO is able to selectively interact with the conformation B of IDOl .

The biological and pharmacological characterization of the compound 338954-DIDO is given by way of non-limiting example in the following experimental section.

338954-DIDO

Experimental Section

Results

Biophysical studies using the microscale thermophoresis methodology have shown direct interaction of the aforesaid compound with the IDO enzyme, revealing a dissociation constant Ko=20mM (Figure 3).

Studies inhibiting the kynurenine-producing catalytic activity with the compound 338954- DIDO on cell lines expressing the enzyme adopting conformation A (catalytic activity) showed no inhibitory activity (IC50 for IDOl Conformation A> IOOmM, Table 1).

On the other hand, inhibition studies with the compound 338954-DIDO on cell lines expressing the enzyme adopting conformation B (with the signalling function) showed inhibitory activity (IC50 for IDO Conformation B = 5.01 mM), thus highlighting the ability of the compound to interact specifically with the conformation B of IDOl associated with the signalling mediated by ITIM motifs. Based on these results, the compound 338954-DIDO was subsequently investigated in in vivo induced immune response pharmacological assays (delayed-type hypersensitivity test, DTH assay).

In detail, an inflammatory response is induced in two groups of female C57BL/6-strain mice through intravenous administration of CD8- dendritic cells and 5% IDOl -expressing plasmacytoid dendritic cells in the first group (ID01+/+ pDCs), or 5% non-IDOl- expressing plasmacytoid dendritic cells in the second group (IDOl-/- pDCs), both pulsed with the weakly immunogenic HY peptide. The plasmacytoid cells were also incubated with the compound 338954-DIDO or the carrier alone before administration. Two weeks later, the mice are inoculated into the plantar paw pad with the HY peptide in the absence of dendritic cells, whereas the left paw receives the carrier alone. After 24 hours, the animals are sacrificed and the immune response (DTH) is measured in the two groups (receiving plasmacytoid cells pre-treated or not pre-treated with the compound 338954- DIDO) through the increase in weight of the paw inoculated with the peptide alone compared to the administration carrier. Figure 4 shows that pre-treatment of plasmacytoid dendritic cells with the compound 338954-DIDO induces an immune tolerance response to the skin hypersensitivity induced by an IDOl -dependent mechanism, as this effect is absent in the second group of mice treated with IDOl-/- pDCs.

Based on the aforementioned data, the pharmacological effectiveness of the compound 338954-DIDO was then characterised in a multiple sclerosis animal model (experimental autoimmune encephalopathy model, EAE). The compound 338954-DIDO was administered intraperitoneally at doses of 10 mg/kg and 30 mg/kg. The results shown in Figure 5 show the effectiveness of the compound in improving the clinical score in a dose-dependent manner.

Materials and Methods

Computational Studies

Molecular dynamics studies were performed by using Desmond calculation program (version 3.8). First of all, an aqueous solvation grid was built around the crystallised structure of IDOl (pdb codes = 4PK5, pPK6, 2D0T, 2D0U, 4U72, 4U74, 5ETW, 5EK2, 5EK3, 5EK4) by using the SPC (Simple Point Charge) model as the solvent within an orthorhombic form of the grid. This was done to make the simulation in an environment reproducing that in which the enzyme acts in vivo. The constant volume, constant temperature (NVT) algorithm was then chosen, defining a temperature of 300.0 K. The duration of the simulation was set at a time of 100 nanoseconds (ns), sampling every ns, thus obtaining 100 conformational models for each trajectory. These models were then used to investigate eleven molecular descriptors of the binding site by means of the SiteMap calculation program. These descriptors include“SiteScore, size, Dscore, volume, exposure, enclosure, contact, phobic, philic, balance, don/acc”. The results were examined through a statistical approach for analysing the main components, which allowed two conformations representative of the IDOl enzyme (Conformation A and Conformation B, Figure 2) to be identified. Screening of chemical compound libraries

The screening of chemical compound libraries on the A and B conformations of IDOl was performed through molecular docking studies and the use of the Glide calculation program. In particular, the A and B conformations representative of IDOl were first treated with the Protein Preparation Wizard (PPW) procedure of the program; residual water molecules were removed; the oxidation state of heme with Fe³⁺ was considered; the geometric parameters of the protein structure were optimised. A grid was built on the protein conformations thus obtained, within which the binding settings of the molecules contained in the compound library were searched. The grid was generated by defining as the centre a point located on the ray with the endpoint in Fe³⁺ and perpendicular to the plane of the heme group, and placed at a distance of 2 A from the Fe³⁺ atom. The total grid dimensions were set at 20x20x20 A. Before carrying out the docking, the molecules contained in the chemical libraries were filtered to remove potential false positives or toxic chemical compounds (PAINS filters) and the rest were treated with the Ligprep procedure, where the choice was made to generate all the tautomeric and ionization forms in the pH range = 7.0±0.3.

The molecules capable of better interacting with the binding pocket of conformations A and B were identified by assessing an energy score with the Gscore-SP function (Standard Precision). Compounds having a Gscore-SP <-7.5 kcal/mol were selected, purchased and/or synthesized, and then tested in a biophysical microscale thermophoresis assay. This assay is based on the physical phenomenon of microscale thermophoresis.

Human recombinant IDO 1 protein was labelled by using a fluorophore that emits in the red range (Monolith NT Protein Labeling Kit RED, excitation 605-645 nm, emission 680-685 nm), supplied by NanoTemper Technologies; once labelled, the enzyme was solubilised in a buffer supplemented with 0.025% Tween20, and a diluted solution with a concentration of 100 nM was prepared. The selected chemical compounds were used at the same initial concentration of 100 mM, in accordance with the KD values expected from the relative energy scores obtained in the docking study. The percentage of DMSO solvent used was reduced to a minimum, based on the solubility of the individual molecules.

In order to carry out the analysis, 16 aliquots were prepared containing 10 mΐ of human IDOl (hIDOl; 50 nM final concentration) and 10 mΐ of the unlabelled chemical compound solution; the latter was placed in the first aliquot at the final concentration mentioned above, and then 1 :1 serial dilutions were prepared in the other fifteen aliquots. For the thermophoresis analysis, hydrophilic capillary kits sold by the Nanotemper company were used, in which the sixteen aliquots were loaded. The ligand/protein interaction analysis in the sixteen capillaries was then performed by using the Monolith NT.1 15 instrument, setting a LED Power of 20% and an MST Power of 80% (Figure 3).

Cell assays

The purification procedures for plasmacytoid dendritic cells (pDC) and CD8- dendritic cells (CD8- DC) from C57/BL6 mice have been reported previously in the literature (Grohmann U, et al. J. Exp. Med. 2003; 198: 153-60; Pallotta MT, et al. Nat. Immunol. 2011 ; 12: 870-8). Purified pDC cells were exposed for 24 hours at 37°C to the chemical compounds selected by the biophysical microscale thermophoresis studies, at a concentration of 0.3 mM and 30 mM. The carrier alone was used as a control. IDOl - knockout C57BL/6 mice were used to assess the IDO 1 -dependency of the effects of the compounds.

The functional activity of 1D01 in P815 murine mastocytoma tumour cells (Pl-HTR) was measured in terms of ability to metabolize tryptophan into L-kynurenine, whose concentration was measured by high performance liquid chromatography (HPLC) in culture supernatants at 16 hours after the addition of 100 mM tryptophan for the last 8 hours (Grohmann U., et al. Nat. Immunol. 2002; 3: 1097-101). Specifically, Pl-HTR cells were grown in Iscove’s modified Dulbecco’s culture medium supplemented with 10% FCS. Two Pl-HTR cell lines were respectively transfected by electroporation with plasmid constructs encoding wild type murine IDOl and the murine IDOl-Yl 11F/Y249F mutant. Two stable transfected cell lines were then obtained by puromycin selection. The two cell lines at a concentration of 0,1 x 10⁶ cells/ml were incubated with serial dilutions of the chemical compounds, starting from a concentration of 30 mM for 16 hours. After incubation, the cell culture supernatants were collected and the concentration of kynurenine was detected by HPLC. Dose-response curves were then constructed, from which the IC50 parameter was obtained for the chemical compounds towards wild type IDOl and the IDOl-Yl 1 1F/Y249F mutant (Table 1). All experiments were performed in triplicate and repeated twice.

Table 1

Induced skin hypersensitivity test (DTH)

Female C57BL/6 mice, between 8 and 12 weeks of age, were purchased from Charles River Breeding Laboratories (Calco, Milan, Italy). The animals were housed and fed under specific pathogen-free conditions. All studies in animal models were performed in compliance with the national guidelines (A-3143-01) and the Ethics Committee guidelines for animal use and care at the University of Perugia.

A skin test assay was used to measure the delayed-type hypersensitivity of the class I- restricted major histocompatibility complex in response to an immune response stimulus induced by sensitization with combinations of DCs pulsed with the synthetic HY peptide, as previously described (Pallotta MT, et al. Nat. Immunol. 2011; 12: 870-8), with the minority component of pDCs pre-treated or not pre-treated for 24 hours with the compound 338954-DIDO, and challenge after 2 weeks with the peptide alone in the plantar pad of the left hind paw, whereas the right paw only received the carrier. 20 ng/ml TGF-b cytokine, instead of the compound 338954-DIDO, was used as a positive control of the tolerogenic stimulus (Pallotta MT, et al. Nat. Immunol. 2011 ; 12: 870-8). The H-2D^b- restricted HY peptide (amino acid sequence WMHHNMDLI) contains the immunodominant epitope of the male-specific minor histocompatibility antigen and is therefore recognized by CD8+ T cells in C57BL/6 female mice. The response to the immune response stimulus in the paw of the peptide-treated mice compared to the carrier- treated mice was measured in terms of weight after 24 hours, and the results are shown as the weight of the peptide-treated paw compared to that of the counterpart injected with the carrier (Figure 4).

In the delayed-type hypersensitivity test assay, Student’s t-test was used for statistical analysis of the results by comparing the average weight of the experimental paws with that of the corresponding control injected with saline solution. At least six mice were used per experimental group, as calculated from the power analysis to obtain a power of at least 80% with a statistical significance value a = 0.05. Autoimmune encephalopathy (EAE) model

The EAE model was obtained by subcutaneously administering 200 pg of the murine Myelin OligoDendrocyte glycoprotein peptide (MOG; sequence MEVGWYRSPFSRVVHL YRNGK) emulsified in 100 mΐ of complete Freund's adjuvant to C57BL/6 mice, between 8 and 12 weeks of age, as previously described (Fallarino F. et al., Nat. Med. 2010; 16, 897-902; Mendel I. et al, Eur. J. Immunol. 1995, 25, 1951-1959. The animals were purchased from Charles River Breeding Laboratories (Calco, Milan, Italy), and housed and fed under specific pathogen-free conditions. All studies in EAE animal models were performed in compliance with the national guidelines (A-3143-01) and the Ethics Committee guidelines for animal use and care at the University of Perugia. A 200 ng dose of pertussis toxin in 100 ml of phosphate buffered saline (PBS) was intraperitoneally administered on the day of vaccination (day 0) and two days later. The neurological signs (ascending paralysis from the tail to the forelimbs) lasted at least 2 months and eventually resulted in the death of the animals. The compound 335984-DIDO was dissolved in water and administered daily at a dose of 10 mg/kg from day 1. To assess the dose-dependency of the effect, the compound 335984-DIDO was also administered at a dose of 30 mg/kg. Control mice only received the aqueous carrier in the absence of the compound 335984-DIDO.

Claims

1. A compound of formula

for use in the therapeutic treatment of multiple sclerosis in a subject.

2. The compound for use according to claim 1 , wherein the subject is a human being.

3. A pharmaceutical composition comprising at least a compound according to claim 1 as the active ingredient, in association with one or more pharmaceutically acceptable adjuvants and/or excipients, for use in the therapeutic treatment of multiple sclerosis in a subject.

4. The pharmaceutical composition for use according to claim 3, wherein the subject is a human being.

5. A method for modulating the activity of the IDOl enzyme in a subject, comprising administering to said subject a pharmaceutically effective amount of a compound of formula

or a pharmaceutical composition comprising it.

6. The method according to_. claim 5, wherein the activity of the IDOl enzyme that is modulated is the IDOl signalling mediated by the ITIM amino acid motifs.

7. The method according to claim 5, wherein the subject is a human being.

8. The method according to claim 5, wherein the subject is a human being suffering from multiple sclerosis.

9. The method according to claim 5, wherein the pharmaceutically effective amount of a compound of formula

is between 0.1-100 mg/kg.