Erdosteine, its salts, enantiomers or metabolites for use in the treatment of nociceptive and neuropathic pain conditions

Abstract

Erdosteine, its salts, enantiomers or metabolites for use in the treatment of non-plastic pain and neuropathic pain. Erdosteine, its salts, enantiomers and the active metabolite Met-1 inhibit the activation of TrkA by NGF and are therefore useful in the treatment of acute and chronic pain states, including neuropathic and neoplastic pain.

Description

[Technical Field]

[0001] The present invention relates to erdosteine, its salts, enantiomers or metabolites for use in the treatment of nociceptive and neuropathic pain states.

[0002] Several lines of evidence suggest that NGF and its signaling pathway via the TrkA receptor are important players in the mechanisms that maintain various types of activity, which, depending on the context, can be considered favorable or unfavorable, for example, promoting neuronal survival (Oo WM., Hunter JD. et al., 2021).

[0003] It has also been found that mutations in the NGF gene cause a painless disorder called Hereditary Sensory and Autonomic Neuropathy type V (Testa GM. et al., 2021; Denk F. et al., 2021, Khan N., Smith TM., 2015). Therefore, NGF / TrkA signaling is currently considered a potential target for pain treatment (Hirose M. et al., 2016). Furthermore, TrKA inhibition may be highly effective not only for neuropathic pain but also for nociceptive pain (as defined by the International Association for the Study of Pain (https: / / www.iasp-pain.org / resources / terminology / ?ItemNumber=1698, Aydede M., Shriver A et al., 2018) in which central mechanisms exist to maintain / develop this type of pain disorder. Indeed, algesic-modulated colic is a new clinical condition, and specific drugs designed to treat this new disease with potentially new targets could be the driving force (Pergolizzi JV Jr. et al., 2021).

[0004] The potential clinical application of TrkA inhibition for the treatment of pain has been well established (Chang DS et al., 2016; Yan W et al., 2019). Previous studies have confirmed that the interaction between NGF and TrkA is impaired by the reduction of thiol compounds (Kamata H. et al.). Furthermore, the target of this inhibition is not nociceptive pain, but other clinical conditions such as nociceptive and neuropathic pain.

[0005] Anti-NGF antibodies capable of inhibiting the binding of NGF to TrkA have been proposed for the treatment or prevention of chronic pain (WO2006 / 131951), but unfortunately, complete inhibition of NGF can cause severe side effects, suggesting that reversible modulation would be more effective.

[0006] Systemically administered small molecules with the same target as erdosteine and a better safety profile are clearly highly desirable for the treatment of nociceptive colic and neuropathic pain, according to current clinical use. Summary of the Invention

[0007] · Description of the Invention We have now discovered that erdosteine, its salts, enantiomers and its active metabolite Met-1 inhibit the activation of TrkA by NGF and are therefore useful in the treatment of nociceptive or neuropathic pain.

[0008] formula [ka] Erdosteine, (2-[N-3-(2-oxotetrahydrothienyl)]acetamido)-thioglycolic acid), was first disclosed in FR 2,502,153 and US 4,411,909. Racemic R,S-erdosteine is used to treat chronic and acute respiratory diseases. It undergoes first-pass metabolism and is formula [ka] is converted to the pharmacologically active metabolite Met-1 (Pharmacology 2006;77(3):150-4,2006 Jul 3).

[0009] Erdosteine is currently registered as an oral mucolytic agent, but its antioxidant and anti-inflammatory properties have also been established. Erdosteine exerts a protective role against lipid peroxidation (smokers, COPD patients) by increasing the availability of endogenous antioxidants such as glutathione in plasma and bronchoalveolar lavage fluid (BALF). Furthermore, erdosteine inhibits bacterial adhesion and enhances antibiotic activity.

[0010] The term "erdosteine" as used herein refers to any of its forms, including the racemate, single enantiomers, polymorphs, and salts.

[0011] The present invention builds on experiments demonstrating that erdosteine and its metabolite Met-1 can inhibit NGF-induced TrkA activation, suggesting that this effect is both time- and concentration-dependent, with longer exposure (within the time frame investigated) resulting in increased inhibition. This pharmacological property is therefore important given the increasing relevance of TrkA inhibition to clinical applications in pain relief (Chang D.S. et al., 2016; Yan W. et al., 2019).

[0012] Erdosteine can be used according to the present invention in the form of its racemate (R,S) or in any of its enantiomeric R or S forms as disclosed in EP 2060568, or in any polymorphic form.

[0013] In particular, the metabolite Met-1 of erdosteine disclosed in EP 974 353 has the following formula: [ka]

[0014] For the therapeutic applications contemplated, erdosteine or its metabolites will be administered by any suitable route at doses similar to those already used clinically for the treatment of respiratory diseases, typically 100-300 mg per day. The oral route is preferred, but other routes traditionally used for analgesics, such as parenteral, transdermal, and transmucosal routes, are also contemplated. Examples of suitable dosage forms include capsules, tablets, controlled-release formulations, solutions, and suspensions.

[0015] Neuropathic and nociceptive pain, both acute and chronic, may be successfully treated by administration of erdosteine or its metabolites.

[0016] The invention will now be explained in more detail in the following experimental section and figures. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1: MTT assay after exposure of SH-SY5Y cells to erdosteine and its metabolite Met-1 for 90 minutes. [Figure 2] Figure 2: MTT assay after 24 hours of exposure of SH-SY5Y cells to erdosteine and its metabolite Met-1. [Figure 3] Figure 3: Activation of the TrkA receptor by NGF after exposure of SH-SY5Y cells to Met-1, a metabolite of erdosteine, for 90 minutes. [Figure 4] Figure 4: Activation of the TrkA receptor by NGF after exposure of SH-SY5Y cells to erdosteine or its metabolite Met-1 for 90 min. [Figure 5] Figure 5: Activation of the TrkA receptor by NGF after exposure of SH-SY5Y cells to Met-1, a metabolite of erdosteine, for 90 minutes. [Figure 6] Figure 6: Activation of TrkA receptor by NGF after exposure of SH-SY5Y cells to 10 mM erdosteine for 90 min or 24 h. [Figure 7]Figure 7: Activation of TrkA receptor by NGF after exposure of SH-SY5Y cells to erdosteine at concentrations of 10 mM or less for 24 hours. [Figure 8] Figure 8: Effect of 24 h preincubation with 10 mM erdosteine and its R and S enantiomers on TrkA activation by NGF. [Figure 9] Figure 9: Effect of 24 h preincubation with different concentrations of erdosteine S enantiomers on TrkA activation by NGF. DETAILED DESCRIPTION OF THE INVENTION

[0018] · Detailed Description of the Invention We analyzed the effects of erdosteine and its metabolite Met-1 on NGF-induced TrkA receptor activation in SH-SY5Y cell cultures. Specifically, TrkA activation was induced with NGF (100 nM) for 10 min. The time and concentration of NGF were chosen based on previous experience and preliminary experiments. Indeed, NGF has been shown to increase TrkA autophosphorylation several-fold in SH-SY5Y cells. This is the first step in a series of events triggered when NGF binds to the TrkA receptor (Marsh HN et al., 2003) and is therefore an indicator of TrkA activation.

[0019] The experiment was based on a commercially available ELISA assay to assess the amount of phosphorylated TrkA (Pandre M.K. et al., 2018) and was used as directed in the manufacturer's protocol. Cell culture conditions were as described in Marchesi N. et al., 2020.

[0020] In preliminary experiments, we evaluated the in vitro toxicity of erdosteine and its metabolites (expected at concentrations around 10 mM and higher) and investigated whether exposure time affected this parameter. Therefore, two time points were considered: a short (90 min) and a long (24 h) exposure time. Longer exposure times can generally affect selected responses to test conditions / substances. Therefore, it is important to explore appropriate time intervals when starting an exploratory protocol.

[0021] SH-SY5Y cells were pre-exposed to the test substances for the indicated times, after which the medium was replaced and medium containing NGF was added. The exposure time to NGF was 10 minutes, as previously shown to be optimal for TrkA activation. Cytotoxicity was assessed by measuring mitochondrial activity using the MTT assay (Marchesi N. et al., 2020).

[0022] The methods employed and described below are standard methods adapted according to the published literature.

[0023] · Materials and Methods The following materials / substances were obtained from the indicated manufacturers and the methods described were applied.

[0024] · cultured cells Human neuroblastoma SH-SY5Y cells were obtained from ATCC (Manassas, VA) and cultured in T75 flasks at 37°C in a humidified incubator with 5% CO. SH-SY5Y cells were cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, L-glutamine (2 mM), non-essential amino acids (1 mM), and sodium pyruvate (1 mM). For MTT experiments, cells were exposed to 10, 20, and 30 mM erdosteine (Erdo; powder from Edmond Pharma) and 10, 20, and 50 mM Metabolite-1 (Met-1; powder from Edmond Pharma). For ELISA experiments, cells were exposed to various concentrations of erdosteine and Met-1 for 90 minutes and 24 hours, followed by 100 ng / ml NGF (rh β-NGF; SRP3015, Sigma-Aldrich) for 10 minutes. The NGF concentration and duration selected were the best combinations obtained from preliminary experiments using NGF concentrations ranging from 0.5 to 1000 ng / ml and durations ranging from 5 to 60 minutes, resulting in TrkA autophosphorylation rates ranging from 2.5-fold to several times higher than the basal rate. All experiments were performed under a laminar flow hood.

[0025] · MTT assay Mitochondrial enzyme activity was estimated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Sigma-Aldrich). 4 A cell suspension of SH-SY5Y cell line at 1000 cells / mL was seeded into a 96-well plate. After 90 minutes and 24 hours of treatment, 50 μL of MTT (concentration equivalent to 2.5 mg / mL) was added to each well. After 3 hours of incubation at 37°C, purple formazan crystals formed. The formed crystals were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich). Specifically, after removing the MTT from the wells, 100 μL of DMSO was added to dissolve the cellular and mitochondrial membranes and solubilize the formazan crystals. After 10 minutes, absorbance was measured at 595 nm using a Synergy HT microplate reader (BioTek Instruments), and the results were expressed as a percentage of the control.

[0026] · ELISA method The phosphorylated TrkA (Tyr674 / 675) protein level in SH-SY5Y cells was estimated by a specific ELISA kit (Cell Signaling, #7212C) according to the manufacturer's instructions. The PathScan® Phosphorylated TrkA (Tyr674 / 675) Sandwich ELISA Kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects transfection levels of phosphorylated TrkA (Tyr674 / 675) protein. TrkA Mouse mAb #4614 is coated onto microwells. Upon incubation with cell lysate, both phosphorylated and non-phosphorylated TrkA protein are captured by the coated antibody. After extensive washing to remove unbound antibody / reagents, phosphorylated TrkA (Tyr674 / 675) antibody #4610 is added to detect phosphorylated TrkA protein. Anti-rabbit IgG HRP-conjugated antibody #7074 is then used to recognize the bound detection antibody. Color development is achieved by adding the HRP substrate, TMB (3,3',5,5'-tetramethylbenzidine). The magnitude of the optical density is proportional to the amount of phospho-TrkA (Tyr674 / 675) protein. The color development was stopped, and the color intensity was measured at 450 nm using a Synergy HT microplate reader (BioTek Instruments). The antibodies used in this kit were custom-prepared specifically for this kit.

[0027] 1. Tolerance of test substances by SH-SY5Y cells after 90 min or 24 h exposure (Figures 1 and 2) The first assay performed was aimed at analyzing the tolerance of SH-SY5Y cells to erdosteine and Met-1 using the MTT assay. Mitochondrial activity below 70% of control (CTR) levels is considered an indicator of significant cytotoxicity (Srivastava GK et al., 2018). Each test substance was tested at various concentrations ranging from 10 to 50 mM. For erdosteine, solubility issues limited the maximum concentration to 30 mM, while Met-1 was tested up to 50 mM. As can be seen in Figure 1, exposure of cells to 30 mM erdosteine resulted in severe toxicity. 20 mM erdosteine resulted in a 16.3% decrease in mitochondrial activity, which, while within the acceptable range, indicates that cells begin to be affected by exposure to the substance. On the other hand, 10 mM erdosteine was indistinguishable from the control. Data on Met-1, a metabolite of erdosteine, show that all concentrations used were fully tolerated at this exposure time (90 minutes). The bars at the top of the columns represent standard errors for this and all other figures.

[0028] The next experiment aimed to observe the tolerance of cells to erdosteine and Met-1 after 24 hours of exposure at the same concentrations. Met-1 was dissolved in water (164 mg / ml) and erdosteine was dissolved in MEM (11 mg / ml), and after 20 minutes of incubation at 37°C, cells were exposed to the specified final concentrations of the compounds. As can be seen from the graph in Figure 2, at this time point (24 hours), erdosteine at both 20 and 30 mM concentrations caused a decrease in mitochondrial activity of approximately 80% or more. Also, Met-1 at concentrations of 20 and 50 mM caused significant changes in mitochondrial activity after 24 hours. Met-1 at 10 mM reduced mitochondrial activity by 17.5%, indicating the presence of an effect, but still exceeding the tolerance threshold. Overall, these data suggest that in TrkA activation experiments, erdosteine and Met-1 concentrations should be limited to 20 mM and 50 mM, respectively, for a 90-minute exposure of cells, and to 10 mM for both for a 24-hour exposure.

[0029] 2. Effects of Met-1 and erdosteine on NGF-induced TrkA activation (Figures 3–6) First, cells were exposed to Met-1 at concentrations of 20 mM and 40 mM for 90 minutes, and the effect on the increase in phosphorylated TrkA (pTrkA) was examined using an ELISA kit. In this experiment, the Met-1 concentration was kept below the limit of 50 mM. Cells were exposed to unsupplemented medium (first two columns in Figure 3) or medium containing 20 mM or 40 mM Met-1 (next four columns in Figure 3) for 90 minutes. The medium was then replaced, and control or medium containing 100 ng / ml NGF was added. Data are expressed as % of basal activity in the absence of added substance (CTR column). Bars above the columns represent standard errors of independent samples (n = 3 or more). TrkA activation was significantly inhibited by Met-1 at all concentrations.

[0030] [Table 1]

[0031] As can be seen in Figure 3, NGF (100 ng / ml) increased the amount of pTrkA several-fold. Met-1 alone, at these concentrations and for this time, did not alter basal pTrkA levels. Met-1 at both 20 mM and 40 mM concentrations blocked NGF-induced TrkA activation, as indicated by the decrease in pTrkA levels upon addition of fresh NGF-containing medium to cells exposed to Met-1 for 90 min. The inhibition at 20 mM and 40 mM Met-1 was 34% and 52%, respectively, in a concentration-dependent manner.

[0032] The bar graphs in Figure 4 report the effects of 10 mM erdosteine and 10 and 20 mM Met-1 on NGF-induced TrkA activation. Cells were exposed for 90 min to medium containing no substance (first two columns), 10 mM erdosteine (next two columns), 10 mM Met-1 (next two columns), or 20 mM Met-1 (last column). The medium was then replaced, and control medium or medium containing 100 ng / ml NGF was added. Data are expressed as % of basal activity in the absence of added substance (CTR column). Bars above the columns represent standard errors of independent samples (n = 3 or more).

[0033] [Table 2]

[0034] The inhibitory effect of Met-1 confirmed and expanded previous experiments: Inhibition by 10 mM and 20 mM NGF was 23% and 24.5%, respectively. Erdosteine at 10 mM had a slight inhibitory effect (8.2% reduction in response to NGF), but this did not reach statistical significance in this experiment. In the absence of NGF, both compounds did not alter basal activity.

[0035] Figure 5 reports the values observed in an ad hoc experiment using several Met-1 concentrations, and combines data from previous experiments. Cells were exposed to medium containing either no added substance or various concentrations of Met-1 for 90 min. The medium was then replaced, and medium containing 100 ng / ml NGF was added. Data are expressed as % of the activity relative to NGF in the absence of added substance (NGF alone or time point 0). Vertical bars represent standard errors of independent samples (n = 3 or more); unless otherwise indicated, the bars are within the symbol dimensions. Inhibition of TrkA activation by NGF in the presence of increasing concentrations of Met-1 within the examined interval (90 min) was linear (right inset).

[0036] The data were calculated and expressed as a percentage of the response to NGF in the absence of added compound. A more refined curve based on the percentage inhibition of phosphorylated TrkA by NGF (i.e., separating the inhibition relative to basal activity rather than calculating overall activity) makes the effects of the two compounds even clearer (not shown). Even though adequate statistical significance was observed starting from the 10 mM concentration, the inhibition was still evident, and the trend was clear.

[0037] Figure 6 shows data from an experiment in which cell viability and NGF-induced TrkA activation were assessed after 24 hours of exposure to 10 mM erdosteine. Cells were exposed to medium containing either no additives or 10 mM erdosteine for 90 minutes or 24 hours. The medium was then replaced with medium containing 100 ng / ml NGF. Data are expressed as % of activity without added substance. Vertical bars represent standard errors of independent samples (n = 3 or more). The two photographs on the right confirm that erdosteine has no toxic effect on SH-SY5Y cells after 24 hours, as evidenced by the MTT data in Figure 2.

[0038] [Table 3]

[0039] This concentration of erdosteine did not significantly affect cell viability, as shown in Figure 2 and the photograph on the right (20x magnification), demonstrating the absence of obvious cytotoxicity after treatment. In particular, the effect of 10 mM erdosteine on TrkA activation was tested in the same experiment after both 90 min and 24 h of exposure to the compound. After 90 min of exposure, a slight inhibitory effect of 10 mM erdosteine was confirmed (13.9% inhibition, reaching statistical significance, likely due to the particularly high response to NGF in this experiment; see also Figure 4). After 24 h of exposure, a strong decrease in TrkA phosphorylation (42.2% inhibition, p<0.0001) was observed. These findings highlight the ability of 10 mM erdosteine to counteract the effects of NGF and suggest that longer exposure to this compound increases its ability to inhibit NGF-induced TrkA activation.

[0040] Two additional experiments were performed to examine the effects of lower concentrations of erdosteine (5.0, 1.0, 0.5, and 0.1 mM). The results are summarized in Figure 7. Cells were exposed to medium containing either no added substance or erdosteine (0.1–10 mM) for 24 hours. The medium was then replaced, and medium containing 100 ng / ml NGF was added. Data are expressed as % of activity without added substance. Vertical bars represent standard errors from independent samples (n = 3 or more). Two experiments were performed combining exposure to erdosteine at 0.1, 0.5, and 1.0 mM and 1.0, 5.0, and 10.0 mM, respectively. Over the 24-hour interval investigated, inhibition of TrkA activation by NGF in the presence of increasing concentrations of erdosteine was linear between 0.1 and 1.0 mM, then flattening out (curves drawn by hand).

[0041] [Table 4]

[0042] As can be seen from the figure, at this exposure time, erdosteine already showed significant inhibitory activity at a concentration of 1.0 mM, and at a concentration of 0.5 mM, the inhibitory activity was evaluable but not statistically significant. At a concentration of 0.1 mM, the inhibitory activity had already plateaued at a concentration of 1 mM, and further increases in the inhibitory activity did not increase the inhibitory activity, remaining at around 40%. The presented data confirm that erdosteine and Met-1 can inhibit NGF-induced TrkA activation. Experiments also suggest that the effect is both time- and concentration-dependent, with longer exposure times (within the time frame investigated) resulting in higher rates of inhibition. This pharmacological property is considered important, as TrkA inhibition is increasingly relevant for potential clinical applications (Chang D.S. et al., 2016; Yan W. et al., 2019).

[0043] 3. Effects of erdosteine and erdosteine enantiomers on NGF-induced activation of TrkA. Cells were exposed to various concentrations of the test substance for 24 hours to examine the effects of the test compounds. Figure 8 reports the effect of erdosteine, its R and S isomers, on NGF-induced TrkA activation after 24 hours of exposure to the test substance. The R and S isomers did not affect basal activity. The reference substance, erdosteine, inhibited TrkA activation by 36%, the S enantiomer by 41%, and the R isomer by 30%.

[0044] [Table 5]

[0045] Then, to find the inactive concentration of isomer S, we finally performed one experiment in which cells were treated with the S-erdosteine isomer from 1 mM to 1 nM compared with 10- and 1-mM concentrations of racemic erdosteine (green dots in Figure 9). In Figure 9, all experiments with isomer S and racemic erdosteine were pooled.

[0046] [Table 6]

[0047] From the reported data, it can be concluded that the isomers of erdosteine, similar to erdosteine, are able to inhibit NGF-induced TrkA activation. In particular, the enantiomer of S-erdosteine showed an inhibitory effect comparable to that observed with erdosteine. Although it is not yet possible to directly compare the potencies of the S and R enantiomers, experiments reported in Figure 8 show that the S enantiomer exerts a greater inhibitory effect when the two enantiomers are directly compared at the same concentration (10 mM). In any case, both enantiomers are capable of inhibiting NGF-induced TrkA activation without exhibiting additive activity. Furthermore, there is a limit to the maximum range of inhibition. For the S-erdosteine isomer, the effect is already maximal at 100 nM S-erdosteine, and increasing the concentration to 10 mM does not change the effect. However, 1 nM S-erdosteine has no effect. This suggests that the IC50 can be estimated in the range of 1–100 nM. Interestingly, the IC50 of racemic erdosteine can be estimated between 0.5 and 1 mM from the experiments reported in Figure 9. The action of S-erdosteine to inhibit the activation of TrkA by NGF is quite intriguing and occurs at concentrations that allow us to hypothesize that this compound represents a new class of NGF inhibitors.

[0048] · References ·Aydede M.,Shriver A.,Pain.2018 Jun;159(6):1176-1177. ·Chang DS.et al.,J Pain Res.2016 Jun 8;9:373-83. ·Denk F.et al.,Annual Rev Neurosci.2017 Jul 25;40:307-325. ·Hirose M.et al.,Pain Pract.2016 Feb;16(2):175-82. ·Kamata H.et al.,Arch Biochem Biophys.2005 Feb 1;434(1):16-25. ·Khan N., Smith TM., Molecules.2015 Jun 9;20(6):10657-88. ·Marchesi N.et al.,PLoS One.2020 Nov 30;15(11):e0242627. ·Marsh HN.et al.,J Cell Biol.2003 Dec 8;163(5):999-1010. ·Oo WM.,Hunter JD.,BioDrugs.2021 Nov;35(6):611-641. ·Pandre MK.et al.,Anal Biochem.2018 Mar 15;545:78-83. ·Pergolizzi JV Jr.et al.,Expert Opin Pharmacother.2021 Aug 30;23:1,105-116 ·Srivastava GK.et al.,Sci Rep.2018 Jan 23;8(1):1425. ·Testa GM.et al.,Pharmacol Res.2021 Jul;169:105662. ·Yan W.et al.,J Med Chem.2020 Sep 10;63(17):10089.

Claims

1. Eldosteine, its salts, enantiomers, or metabolites thereof for use in the treatment of neuropathic and dysalgesic pain disorders.

2. The S-enantiomer of eldostein for use according to claim 1.

3. The R-enantiomer of eldosteine ​​for use according to Claim 1.

4. A metabolite of erdostein of the following formula for use according to claim 1. 【Chemistry 1】

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