Erdosteine, salts, enantiomers or metabolites thereof for treatment of nociceptive plastic pain and neuropathic pain states

Abstract

Erdosteine, its salts, enantiomers and their active metabolite Met-1 inhibit the activation of TrkA by NGF and are thus useful in the treatment of acute and chronic pain conditions, including neuropathic pain and nociceptive plastic pain.

Description

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

[0003] Several lines of evidence suggest that NGF and its signaling pathway through the TrkA receptor are important players in mechanisms that maintain various types of activities that can be considered beneficial (e.g., promoting neuronal survival) or adverse depending on the context (Oo WM., Hunter JD. et al., 2021).

[0004] It has also been found that mutations in the NGF gene cause the analgesic disease 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). In addition, TrKA inhibition may in fact contribute not only to neuropathic pain, but also to nociceptive plasticity pain (defined from the International Association for the Study of Pain, https: / / www.iasp- pain.org / resources / terminology / ?ItemNumber=1698 , Aydede M., Shriver A et al., 2018), where the central mechanisms that maintain / cause such pain diseases occur. In fact, nociceptive plasticity pain is a new clinical entity that can be driven by special drugs designed to modulate this new disease with new potential targets (Pergolizzi JV Jr. et al., 2021).

[0005] The potential clinical application of TrkA inhibition for the treatment of pain is well established (Chang DS. et al., 2016; Yan W. et al., 2019). Previous studies have identified that the interaction between NGF and TrkA can be impaired by the reduction of thiol compounds (Kamata H. et al., 2005). In addition, the target of this inhibition is not nociceptive pain, but other clinical entities such as nociceptive plasticity pain or neuropathic pain.

[0006] Anti-NGF antibodies capable of inhibiting NGF binding to TrkA have been suggested for use in treating or preventing chronic pain (WO2006 / 131951), but unfortunately, complete inhibition of NGF causes severe side effects, suggesting that reversible modulation may be more effective.

[0007] Based on its current clinical use, a small molecule that is systemically administered, has the same target as erdosteine, and has a superior safety profile would obviously be highly desirable for the treatment of nociceptive plasticity pain and neuropathic pain.

[0008] Description of the invention

[0009] It has now been found 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 plasticity pain or neuropathic pain.

[0010] Erdosteine ​​(2-[N-3-(2-oxotetrahydrothienyl)]acetylamino)-thioglycolic acid) of the following formula

[0011]

[0012] First disclosed in FR 2,502,153 and US 4,411,909. Racemic R,S-erdosteine ​​is used in the treatment of chronic and acute respiratory disorders. It is converted into its pharmacologically active metabolite Met-1 after first-pass metabolism (Pharmacology 2006; 77(3):150-4, July 3, 2006) of the following formula:

[0013]

[0014] Erdosteine ​​is currently registered as an oral mucolytic drug, but is also endowed with well-established antioxidant and anti-inflammatory activities. Erdosteine ​​exerts a protective effect against lipid peroxidation (smokers, COPD patients) by increasing the availability of endogenous antioxidants (e.g. glutathione) in plasma and bronchoalveolar lavage fluid (BALF). In addition, erdosteine ​​inhibits bacterial adhesion and enhances antibacterial activity.

[0015] The term "erdosteine" in the present specification refers to any form thereof, including racemic forms, single enantiomers, polymorphs and salts.

[0016] The present invention is based on experiments showing that erdosteine ​​and its metabolite Met-1 are able to inhibit the activation of TrkA by NGF, which also showed that the effect is time- and concentration-dependent, with longer exposure times (within the time frame explored) driving higher inhibition rates. Therefore, this pharmacological property is very important in view of the increasing relevance of clinical applications attributed to TrkA inhibition and pain relief (Chang DS. et al., 2016; Yan W. et al., 2019).

[0017] According to the invention, erdosteine ​​can be used in the racemic (R,S) form or in the form of one of its enantiomers R or S as disclosed in EP 2 060 568, in any polymorphic form.

[0018] The metabolite Met-1 of erdosteine ​​disclosed in EP 974 353, among others, has the following formula:

[0019]

[0020] For the therapeutic use contemplated, erdosteine ​​or its metabolites will be administered by any suitable route at doses equivalent to those already used in clinical practice for the treatment of respiratory diseases, typically 100 to 300 mg multiple times a day. The oral route is preferred, but routes commonly used for analgesics, such as parenteral, transdermal, transmucosal routes, may be considered. Examples of suitable dosage administration forms include capsules, tablets, controlled release formulations, solutions, suspensions, and the like.

[0021] Neuropathic pain and nociceptive pain can be successfully treated in both the acute and chronic stages by administering erdosteine ​​or its metabolites.

[0022] The invention will now be described in further detail in the following experimental section and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : MTT analysis of SH-SY5Y cells after exposure to erdosteine ​​and its metabolite Met-1 for 90 min.

[0024] Figure 2 : MTT analysis of SH-SY5Y cells exposed to erdosteine ​​and its metabolite Met-1 for 24 h.

[0025] Figure 3 : TrkA receptor is activated by NGF after SH-SY5Y cells were exposed to the metabolite of erdosteine, Met-1, for 90 minutes.

[0026] Figure 4 : Activation of TrkA receptor by NGF occurred in SH-SY5Y cells after exposure to erdosteine ​​or its metabolite Met-1 for 90 min.

[0027] Figure 5 : TrkA receptor is activated by NGF after SH-SY5Y cells were exposed to the metabolite of erdosteine, Met-1, for 90 minutes.

[0028] Figure 6 : Activation of TrkA receptor by NGF after SH-SY5Y cells were exposed to 10 mM erdosteine ​​for 90 min or 24 h.

[0029] Figure 7: Activation of TrkA receptor by NGF after SH-SY5Y cells were exposed to erdosteine ​​concentrations equal to or lower than 10 mM for 24 hours.

[0030] Figure 8 : Effect of 24 h preincubation with 10 mM erdosteine ​​and its R and S enantiomers on the activation of TrkA by NGF.

[0031] Fig. 9 : Effect of 24 h preincubation with different concentrations of the S enantiomer of erdosteine ​​on the activation of TrkA by NGF. DETAILED DESCRIPTION OF THE INVENTION

[0033] The effects of erdosteine ​​and its metabolite Met-1 on the activation of TrkA receptor by NGF were analyzed using SH-SY5Y cell cultures.

[0034] Specifically, NGF 100 ng / mL was used Induce the activation of TrkA for 10 minutes. The selection of time and NGF concentration is based on previous experience and preliminary experiments, which have actually shown the ability of NGF to cause a several-fold increase in TrkA autophosphorylation in SH-SY5Y cells. When NGF binds to the TrkA receptor, this is the first step in a series of events caused by it (Marsh HN. et al., 2003), so it is an indicator of TrkA activation.

[0035] The assay was based on a commercially available ELISA assay that assesses the amount of phosphorylated TrkA (Pandre MK. et al., 2018) and was used as indicated in the manufacturer's protocol. Cell culture conditions are described in Marchesi N. et al., 2020.

[0036] Initial studies were aimed at evaluating the in vitro toxicity of erdosteine ​​and its metabolites (which can be expected at concentrations of about or above 10 mM) and investigating whether exposure time affects this parameter. Therefore, two time points were explored: short (90 minutes) and long (24 hours). In general, longer exposure times can sometimes affect the selected response to the test conditions / substances; therefore, in the initial exploratory protocol, it is important to explore sufficient time intervals.

[0037] SH-SY5Y cells were pre-exposed to the test substances for the indicated times, then the medium was changed and medium containing NGF was added; the exposure time for NGF was 10 minutes, as indicated by previous tests showing that this time was optimal for TrkA activation.

[0038] Cytotoxicity was assessed by measuring mitochondrial activity using the MTT assay (Marchesi N. et al., 2020).

[0039] The methods used and described below are based on standard methods used in reported papers.

[0040] Materials and methods

[0041] The following materials / substances were obtained from the indicated manufacturers and the methods used are described.

[0042] Cultured cells

[0043] Human neuroblastoma SH-SY5Y cells were obtained from ATCC (Manassas, VA) and cultured in T75 flasks in a humidified incubator at 37°C and 5% CO. 2 SH-SY5Y cells were grown 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).

[0044] In the MTT assay, cells were exposed to 10, 20 and 30 mM erdosteine ​​(Erdo; powder from Edmond Pharma); 10, 20 and 50 mM metabolite-1 (Met-1; powder from Edmond Pharma).

[0045] For ELISA assays, cells were exposed to various concentrations of erdosteine ​​and Met-1 for 90 minutes and 24 hours, followed by exposure to 100 ng / mL NGF (rhβ-NGF; SRP3015, Sigma-Aldrich) for 10 minutes. The concentration and time selected for NGF were derived from the best combination of preliminary experiments, using NGF concentrations ranging from 0.5 to 1000 ng / mL and times from 5 to 60 minutes, causing TrkA autophosphorylation reactions in the range of 2.5 to several times the basal rate. All experiments were performed in a laminar flow cabinet.

[0046] MTT assay

[0047] Mitochondrial enzyme activity was estimated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Sigma-Aldrich). 4 Cell suspension of SH-SY5Y cell line at 10 cells / mL was seeded in a 96-well plate.

[0048] After each treatment at 90 minutes and 24 hours, 50 μL of MTT (concentration equal to 2.5 mg / mL) was added to each well. After incubation at 37°C for 3 hours, purple formazan was formed. The formed crystals were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich). Specifically, after removing MTT from the wells, 100 μL of DMSO was added to lyse the cell and mitochondrial membranes and dissolve the methyl sulfoxide. After 10 minutes, the absorbance value was measured at 595 nm using a Synergy HT microplate reader (BioTek Instruments) and the results were expressed as % relative to the control.

[0049] ELISA analysis

[0050] Phospho-TrkA (Tyr674 / 675) protein levels in SH-SY5Y cells were estimated using a specific ELISA kit (Cell Signaling, #7212C.) according to the manufacturer's instructions.

[0051] The Phospho-TrkA (Tyr674 / 675) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay that detects the transfection level of phospho-TrkA (Tyr674 / 675) protein. TrkA mouse mAb #4614 has been coated on the microwells. After incubation with cell lysate, phospho-TrkA and non-phospho-TrkA proteins are captured by the coated antibodies. After extensive washing to remove any unbound antibody / reagent, phospho-TrkA (Tyr674 / 675) antibody #4610 is added to detect phospho-TrkA protein. Anti-rabbit IgG, HRP-conjugated antibody #7074 is then used to recognize the bound detection antibody. HRP substrate TMB (3,3',5,5'-tetramethylbenzidine) is added for color development. The magnitude of the optical density is proportional to the amount of phospho-TrkA (Tyr674 / 675) protein.

[0052] Color development was stopped and color intensity was measured at 450 nm using a Synergy HT microplate reader (BioTek Instruments).The antibodies used in the kit were custom formulations specific for the kit.

[0053] 1. Tolerance of SH-SY5Y cells to the test substances after 90 minutes or 24 hours of exposure ( Figure 1 and 2 ).

[0054] The first analysis 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 the control (CTR) level was considered an indicator of significant cytotoxicity (Srivastava GK. et al., 2018). For each test substance, multiple concentrations were tested: 10 to 50 mM. In the case of erdosteine, the maximum concentration had to be limited to 30 mM due to solubility issues, while in the case of Met-1, concentrations up to 50 mM were tested. As described above, the mitochondrial activity was below 70% of the control (CTR) level as an indicator of significant cytotoxicity (Srivastava GK. et al., 2018). Figure 1 As can be appreciated, exposure of cells to 30 mM erdosteine ​​produced severe toxicity. 20 mM erdosteine ​​produced a 16.3% decrease in mitochondrial activity, which is acceptable but indicates that cells are beginning to suffer from exposure to this substance, while 10 mM erdosteine ​​was indistinguishable from the control. The data for Met-1 (a metabolite of erdosteine) indicate that at this exposure time (90 minutes), all concentrations used were well tolerated. In this figure and all other figures, the bars at the top of the columns are standard errors.

[0055] Subsequent experiments were designed to observe tolerance to erdosteine ​​and Met-1 at the same concentrations but after 24 hours of cell exposure. Met-1 was dissolved in water (164 mg / mL); erdosteine ​​was dissolved in MEM (11 mg / mL) and warmed at 37°C for 20 minutes to expose cells to the indicated final concentrations of the substances of interest.

[0056] As Figure 2 As can be seen in the graph, at this time (24 hours), 20 and 30 mM erdosteine ​​both produced approximately and more than 80% reduction in mitochondrial activity. Moreover, Met-1 produced significant changes in mitochondrial activity at 20 and 50 mM concentrations at 24 hours. 10 mM Met-1 produced a 17.5% reduction in mitochondrial activity, indicating the presence of an effect, but still above the tolerance threshold.

[0057] Taken together, these data suggest that for TrkA activation assays, the concentrations of erdosteine ​​and Met-1 should be limited to 20 and 50 mM or less, respectively, when cells are exposed for 90 min, and both to 10 mM when the exposure time is 24 h.

[0058] 2. Effects of Met-1 and erdosteine ​​on TrkA activation by NGF ( Figures 3 to 6 ).

[0059] First, the effect of Met-1 exposure for 90 minutes on the increase of phospho-TrkA (pTrkA) was tested by ELISA kit at concentrations of 20 and 40 mM. In this assay, the Met-1 concentration was kept below the 50 mM limit.

[0060] Cells were exposed to the presence of no added substances ( Figure 3 The first two columns contained 20 and 40 mM Met-1 ( Figure 3 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 substances (CTR column). The bars on the columns represent the standard error of independent samples (n=3 or more). The activation of TrkA was significantly inhibited by both concentrations of Met-1.

[0061] Statistics: Tukey's multiple comparison test

[0062]

[0063] As Figure 3 It can be seen that NGF (100ng / mL) increases the amount of pTrkA several fold. For this exposure time, Met-1 alone at these concentrations does not change the basal pTrkA level. When fresh medium containing NGF is added to cells pre-exposed to Met-1 for 90 minutes, both 20 and 40mM Met-1 prevent TrkA from being activated by NGF, as indicated by the decreased values ​​of pTrkA. The inhibition in the case of 20 and 40mM Met-1 was 34 and 52%, respectively, in a concentration-dependent manner.

[0064] Figure 4 The bar graph in reports the effect of 10 mM erdosteine ​​and 10 and 20 mM Met-1 on the activation of TrkA by NGF. Cells were exposed to medium containing no added substances (first two columns), 10 mM erdosteine ​​(next two columns), 10 mM Met-1 (next two columns), 20 mM Met-1 (last column) for 90 minutes. 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 substances (CTR column). The bars on the columns represent the standard error of independent samples (n=3 or more).

[0065] Statistics: Tukey's multiple comparison test

[0066]

[0067] The inhibition of Met-1 confirmed and extended previous experiments: NGF inhibition at 10 and 20 mM was 23 and 24.5%, respectively, and erdosteine ​​had a small inhibitory effect at 10 mM (8.2% reduction in response to NGF), however, it did not reach statistical significance in this assay. In the absence of NGF, neither compound altered basal activity.

[0068] Figure 5 The Met-1 values ​​observed in specific (ad hoc) experiments using several Met-1 concentrations are reported and data from previous experiments are summarized. Cells were exposed to culture medium containing no added substances or various concentrations of Met-1 for 90 minutes. The culture medium was then replaced and culture medium containing 100ng / mL NGF was added. The data are expressed as % of the activity in response to NGF in the absence of added substances (NGF alone or 0 points). The vertical bars represent the standard error of independent samples (n=3 or more) and are located within the symbol size if no indication is given. At this time (90 minutes), the inhibition of TrkA activation by NGF in the presence of increasing concentrations of Met-1 within the explored interval is linear (inset on the right).

[0069] The data were calculated and expressed as a percentage of the response to NGF in the absence of the added compound. A more refined curve based on the percentage inhibition of phosphorylated TrkA activation due to NGF addition (i.e., isolating activation above basal rather than calculating overall activity) made the effects of the two compounds more apparent (not shown). Even though modest statistical significance was observed starting from 10 mM concentration, inhibition was evident in all cases and the trend was clear.

[0070] Figure 6 Experimental data for exposure to 10 mM erdosteine ​​for 24 hours are reported for evaluation of cell viability and activation of TrkA by NGF. Cells were exposed to medium without additives or containing 10 mM erdosteine ​​for 90 minutes or 24 hours. The medium was then replaced and medium containing 100 ng / mL NGF was added. Data are expressed as % of activity in the absence of additives. Vertical bars represent standard errors of independent samples (n=3 or more). The two figures on the right confirm that erdosteine ​​does not produce toxic effects on SH-SY5Y cells at 24 hours, as shown by Figure 2 The MTT data are shown in Figure 2.

[0071] Statistics: Tukey's multiple comparison test

[0072]

[0073] At this concentration, erdosteine ​​did not show a significant effect on cell viability, e.g. Figure 2and the photograph on the right (20x magnification), which demonstrates the absence of overt cytotoxicity following treatment. In particular, the effect of 10 mM erdosteine ​​on TrkA activation was tested in the same assay after 90 min and 24 h of exposure to the compound. A small inhibitory effect of 10 mM erdosteine ​​after 90 min exposure (inhibition, 13.9%, reached statistical significance), was demonstrated, probably due to the particularly high response to NGF in this assay, see also Figure 4 ), in addition, a strong decrease in TrkA phosphorylation was observed after 24 hours of exposure (42.2% inhibition, p<0.0001). These findings highlight the ability of erdosteine ​​to counteract the effects of NGF at 10 mM and suggest that longer exposure to the compound increases its ability to attenuate TrkA activation by NGF.

[0074] Two follow-up experiments were conducted to explore the effects of lower erdosteine ​​concentrations (5.0, 1.0, 0.5, 0.1 mM). The results are summarized in Figure 7 Cells were exposed to medium without additives or containing 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 in the absence of additives. Vertical bars represent standard errors of independent samples (n=3 or more). Two experimental combinations were exposed to 0.1, 0.5, 1.0 and 1.0, 5.0, 10.0 mM erdosteine, respectively. At this time (24 hours), in the presence of increasing concentrations of erdosteine ​​within the explored interval, the inhibition of TrkA activation by NGF was linear between 0.1 and 1.0 mM, and then reached a plateau (hand-drawn curve).

[0075] Statistics: Tukey's multiple comparison test

[0076]

[0077] As can be understood from the figure, at this exposure time, erdosteine ​​already has a significant inhibitory effect at a concentration of 1.0 mM and has a clear but not strongly statistically significant trend at a concentration of 0.5 mM. A concentration of 0.1 mM is ineffective. At a concentration of 1 mM, the effect has reached a plateau, and further increasing the concentration does not increase the inhibition, which is at a level of about 40%.

[0078] The data presented demonstrate that erdosteine ​​and Met-1 are able to inhibit the activation of TrkA by NGF. These experiments also show that the effects are time- and concentration-dependent, with longer exposure times (within the explored timeframe) driving higher inhibition rates. This pharmacological property is very important in view of the increasing relevance of clinical applications attributed to TrkA inhibition (Chang DS. et al., 2016; Yan W. et al., 2019).

[0079] 3. Effects of erdosteine ​​and erdosteine ​​enantiomers on the activation of TrkA by NGF

[0080] We have examined the effects of test compounds by exposing cells to various concentrations of the test substances for 24 hours.

[0081] Figure 8 The effects of erdosteine, its R and S isomers on the activation of TrkA by NGF after 24 hours of exposure to the test substances are reported. The R and S isomers had no effect on the basal activity. The reference substance erdosteine ​​inhibited TrkA activation by 36%, the S enantiomer by 41% and the R isomer by 30%.

[0082] Statistics: Dunnett's multiple comparison test

[0083]

[0084] Subsequently, to find the inactive concentration of isomer S, we finally performed an experiment in which cells were treated with S erdosteine ​​isomer from 1 μM down to 1 nM and with 10 and 1 mM concentrations ( Fig. 9 The green dot in Figure 2 shows the comparison of racemic erdosteine. Fig. 9 In this study, we pooled all experiments with isomer S and racemic erdosteine.

[0085] Statistics: Dunnett's multiple comparison test

[0086]

[0087]

[0088] The reported data allow to conclude that the erdosteine ​​isomers are able to counteract the activation of TrkA by NGF as well as erdosteine. In particular, the S-erdosteine ​​enantiomer has an effect comparable to that observed with erdosteine ​​in terms of the degree of inhibition. A direct comparison of the efficacy of the S and R enantiomers is not possible at present, even in Figure 8In the activity test directly comparing the two enantiomers at the same concentration (10 mM) reported in , the S enantiomer appears to exert greater inhibition. In any case, both enantiomers were able to inhibit the activation of TrkA by NGF and did not show additive activity. It also appears that the maximum degree of inhibition has limits, and in the case of the S-erdosteine ​​isomer, there is already a maximum with 100 nM S-erdosteine ​​and no change even by increasing the amount up to 10 mM, while with 1 nM S-erdosteine ​​there is zero effect, which initially indicates that the IC50 can be estimated in the range of 1-100 nM.

[0089] Interestingly, the IC50 of racemic erdosteine ​​can be Fig. 9 The experiments reported in were evaluated between 0.5 and 1 mM. The effect of S-erdosteine ​​in inhibiting the activation of TrkA by NGF is quite interesting and occurs at concentrations that allow to assume that the compound may be representative of a new class of NGF inhibitors.

[0090] References

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[0093] Denk F. et al., Annual Rev Neurosci. 2017 Jul 25;40:307-325.

[0094] Hirose M. et al. Pain Pract. 2016 Feb;16(2):175-82.

[0095] • Kamata H. et al., Arch Biochem Biophys. 2005 Feb 1;434(1):16-25.

[0096] Khan N., Smith TM., Molecules. 2015 Jun 9;20(6):10657-88.

[0097] Marchesi N. et al. PLoS One. 2020 Nov 30;15(11):e0242627.

[0098] • Marsh HN. et al., J Cell Biol. 2003 Dec 8;163(5):999-1010.

[0099] ·Oo WM., Hunter JD., BioDrugs. 2021 Nov; 35(6): 611-641.

[0100] Pandre MK. et al., Anal Biochem. 2018 Mar 15;545:78-83.

[0101] Pergolizzi JV Jr. et al., Expert Opin Pharmacother. 2021 Aug 30;23:1,105-116.

[0102] 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. Erdosteine, its salts, enantiomers or metabolites for use in the treatment of neuropathic pain and nociceptive plasticity pain disorders.

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

3. The R-enantiomer of erdosteine ​​for use according to claim 1.

4. The metabolite of erdosteine ​​of the formula for use according to claim 1

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