Agents for the treatment of the peripheral nervous system

Abstract

A compound for use in the treatment of CIDP, an immune-mediated or autoimmune disease of the peripheral nervous system, wherein the compound increases the resistance of Schwann cells and spinal ganglia to oxidative stress, containing propionic acid, its physiologically acceptable salts and / or esters with C1-C8 alkyl alcohols.

Description

[0001] The invention relates to an agent containing short-chain C1 to C6 carboxylic acids for use in the treatment of the peripheral nervous system in immune-mediated diseases or autoimmune diseases and in particular for use in the treatment of chronic inflammatory demyelinating polyneuropathy (CIDP).

[0002] Short-chain fatty acids (SCFAs) such as acetate, propionate (PA), and butyrate are produced endogenously through the fermentation of dietary fiber by the gut microbiome. Short-chain fatty acids are known to modulate cellular function, among other things, by binding to G protein-coupled free fatty acid receptors (FFARs) 2 and 3 and by inhibiting histone acetylation by histone deacetylases (HDACs).

[0003] Oral PA has demonstrated anti-inflammatory potential in multiple sclerosis patients by increasing the frequency and activity of regulatory T cells, leading, among other things, to reduced relapse frequency and reduced brain atrophy. Furthermore, it has been demonstrated that PA concentrations in stool and serum are reduced in MS patients.

[0004] The publication by Duscha, Alexander, et al., "Propionic acid shapes the multiple sclerosis disease course by an immunomodulatory mechanism" in Cell, 2020, Vol. 180, No. 6, pp. 1067-1080, describes the use of propionic acid for the treatment of multiple sclerosis, an autoimmune, chronic inflammatory disease of the central nervous system. Propionic acid is thought to exert a neuroprotective function.

[0005] The publication by YOON, Min-Suk, et al., "Treatment of an acute motor and sensory axonal neuropathy with propionate in a 33-year-old male" in Therapeutic Advances in Neurological Disorders, 2018, Vol. 11, pp. 1756286418809580, discloses the use of propionic acid to treat autoimmune inflammatory neuropathies. The subject was treated with 500 mg of sodium propionate capsules.

[0006] In chronic inflammatory demyelinating polyneuropathy (CIDP), neuronal degeneration, including apoptosis of myelinating Schwann cells (SCs), is primarily caused by autoimmune inflammation in the peripheral nervous system (PNS).

[0007] The mechanisms of degeneration resulting from neuroinflammation also include oxidative stress triggered by reactive oxygen species. Oxidative stress is of great importance for the pathophysiology of immune-mediated diseases of the PNS, as myelin is a sensitive tissue due to its high lipid content.

[0008] Since Schwann cells are involved in immune regulation and crucial for the nutrition and repair of neurons, secondary neurodegeneration also plays a crucial role in the impairments caused by such diseases. However, none of the currently approved drugs target oxidative stress or directly promote neuroregeneration. This could represent a promising new treatment approach.

[0009] It is therefore an object of the invention to provide an agent for use in the treatment of immune-mediated diseases or autoimmune diseases of the PNS which exhibits an antioxidant effect.

[0010] This object is achieved by an invention having the features of claim 1. Advantageous embodiments are the subject of the dependent claims. It should be noted that the features listed individually in the claims can also be combined with one another in any desired and technologically expedient manner, thus revealing further embodiments of the invention.

[0011] The agent according to the invention for use in the treatment of immune-mediated diseases or autoimmune diseases of the peripheral nervous system contains propionic acid, its physiologically acceptable salts and / or esters with C1-C8 alkyl alcohols and increases the resistance of Schwann cells and spinal ganglia to oxidative stress.

[0012] Preferably, the physiologically acceptable salts are sodium, potassium, magnesium, calcium, zinc and / or iron salts.

[0013] The agent according to the invention preferably contains sodium propionate.

[0014] Preferably, the physiologically acceptable esters are the methyl esters or ethyl esters.

[0015] The agent according to the invention is preferably administered in a daily dose of up to 10 g, more preferably up to 7 g, and especially up to 3 to 5 g. Such a dose promises the best possible therapeutic effect.

[0016] The agent according to the invention is preferably offered in capsule or tablet form, which allows for easy intake and dosage.

[0017] The agent according to the invention is preferably offered in capsules or tablets with a dose of 0.1 to 5 g, in particular 1 to 3 g of the carboxylic acids, their salts or esters.

[0018] The agent is preferably intended for use in the treatment of CIDP.

[0019] Preferably, the agent contains another short-chain C1 to C6 carboxylic acid, in particular butyrate.

[0020] Preferably, the product contains butyrate, its physiologically acceptable salts and / or esters with C1-C8 alkyl alcohols in a daily dose of up to 10 g.

[0021] Surprisingly, experiments have demonstrated a direct neuroprotective effect of PA in the PNS through increased catalase expression following elevated histone 3 acetylation. Catalase plays a central role in oxidative protection in autoimmune diseases and age-related degeneration, and a dependence on histone 3 acetylation is known. Oxidative stress induces an Nrf2-dependent antioxidant response in the DRG. Since PA induces another antioxidant defense mechanism (catalase), a complementary effect of both mechanisms can be assumed.

[0022] Inflammatory neuropathies are characterized by an inflammatory process and reduced antioxidant resistance and reduced neuroregeneration, leading to demyelination and axonal damage.

[0023] It is known that CIDP patients already exhibit severe axonal damage at the time of initial diagnosis, suggesting oxidative stress and the destruction of axonal integrity early in the disease course.

[0024] The experiments conducted within the scope of the invention surprisingly showed that the viability of PA-treated cells was increased after H2O2 or SNAP treatment. Increased expression of gap-43, a marker for neuroregeneration and plasticity, was also demonstrated in vitro. Accordingly, PA treatment may be beneficial in addition to first-line immunotherapy due to its anti-inflammatory, antioxidant, and neuroregenerative properties. The experimental results demonstrate a neuroprotective and neuroregenerative treatment option and its mechanisms of action for immune-mediated neuropathies.

[0025] In vitro experiments, PA demonstrated neuroprotective and neuroregenerative effects of the FFAR3 agonist in the PNS. PA mediated an antioxidant effect in SCs and DRGs by downregulating HDAC 2 and upregulating HDAC 8 expression, leading to hyperacetylation of histone 3 and upregulation of the important antioxidant enzyme catalase. Furthermore, PA treatment leads to enhanced neuroregeneration after oxidative stress through induction of Gap-43. The FFAR3 agonist β-hydroxybutyric acid can mimic this effect, while the FFAR3 antagonist pertussis toxin abolishes it.

[0026] Propionate increases the resistance of Schwann cells and dorsal root ganglia to oxidative stress through catalase expression.

[0027] Flow cytometric analysis of propidium iodide (PI)-stained SCs was used to investigate the potential of PA to protect against H2O2-induced oxidative stress. Treatment of SCs with 0.03 mM H2O2 resulted in submaximal cell death, as evidenced by a 47.0±9.1% PI-positive signal. Strikingly, concomitant treatment with PA for 24 hours significantly increased cell death to an average of 33.6±9.6% compared to H2O2-treated SCs (p<0.0001, n=28). Fig. 2A) reduced.

[0028] The results could be reproduced with the FFAR3 ligand β-hydroxybutyric acid (BHB): SCs treated simultaneously with H2O2 and BHB showed significantly restored cell viability (cell death: 28.7±9.8%, p<0.0001, n=9, Fig. 2A). A combination of PA and BHB had the same effect as the separate administration of the two substances (cell death: 37.2±12.3%, p=0.0036 compared to H2O2 alone, n=9, Fig. 2A). Neither PA nor BHB showed any toxic effect on naive SCs when a concentration of 10 mM was used ( Fig. 2A).

[0029] Under naive conditions, a mean axonal growth of 1859±362 µm was observed in cultured DRGs after 24 hours. After induction of oxidative stress by S-nitroso-N-acetylpenicillamine (SNAP, p=0.0002, n=30, Fig. 2B), axonal growth was significantly reduced by approximately 25.3%. Twenty-four-hour PA pretreatment resulted in improved growth despite oxidative stress, indicating a neuroprotective effect (p=0.03, n=16, Fig. 2B). The neuroprotective effect of PA could be enhanced by the simultaneous administration of pertussis toxin (PTX, p=0.0394, n=13, Fig. 2B) are repealed.

[0030] To translate the in vitro results into an ex vivo approach, rats were treated orally with 150 mM propionate dissolved in 400 µl of water or with a control gavage for 5 days. After extraction, half of the DRGs in each group were subjected to oxidative stress. Only the DRGs of the control group showed a significant reduction in axonal outgrowth after SNAP treatment (p=0.04, n=4). Fig. 2C), which shows a neuroprotective effect of PA even after prior oral administration to live rats.

[0031] Since PA increased resistance to oxidative stress in SCs and DRGs, real-time PCR was performed to analyze the expression levels of antioxidants such as catalase, NAD(P)H quinone dehydrogenase 1 (NQO1), and heme oxygenase 1 (HO-1). A 72-hour treatment with PA increased the relative expression of catalase mRNA by approximately 21.5% compared to naive SCs (p=0.0009, n=9). Fig. 3D), while catalase expression was also increased after 24 hours of treatment, but did not reach significance. Similarly, catalase mRNA expression in DRGs was significantly increased after 24 hours of PA treatment (p=0.007, n=6, Fig. 3E). No significant changes in the relative mRNA expression of HO-1 and NQO1 were observed after PA treatment ( Fig. 3F-G).

[0032] Propionate induces neuroregeneration through the expression of growth-associated protein 43.

[0033] The 24-hour application of propionate to DRG after induction of oxidative stress by SNAP resulted in enhanced outgrowth, indicating induction of neuroregeneration (p=0.0141, n=17, Fig. 4A). PA treatment in naive DRG (p<0.0001, n=6, Fig. 4B) and in DRG after SNAP treatment (p=0.0026, n=5, Fig. 4C) leads to increased expression of the plasticity protein growth-associated protein 43 (gap-43).

[0034] Treatment with β-hydroxybutyrate (BHB) leads to hyperacetylation of histone 3 and 4 in Schwann cells, whereas propionate only leads to hyperacetylation of histone 3.

[0035] Histone 3 acetylation was increased 3.9-fold after 72 hours of PA treatment compared to that in naive SC (p=0.0002, n=6, Fig. 5A). After 24 hours of PA treatment, only a non-significant increase of 1.5-fold was observed compared to the control. In contrast to BHB treatment, PA treatment did not lead to a significant change in the acetylation of histone 4. The acetylation of histone 3 lysine 9 (5.6-fold, p<0.0001, n=6, Fig. 5A) and histone 4 lysine 5 (16.9-fold, p<0.0001, n=6, Fig. 5B), lysine 8 (20.1-fold, p<0.0001, n=6, Fig. 5C) and lysine 16 (6.1-fold, p<0.0001, n=6, Fig. 5E), but not lysine 12, were significantly increased after 24 hours of BHB treatment.

[0036] Propionate increases histone acetylation by altering the expression of histone deacetylase 2 and 8.

[0037] The relative expression of HDAC 2 (p=0.02, n=9, Fig. 5G) was significantly reduced by approximately 11.6% after 72 hours of PA treatment and the expression of HDAC 8 (p=0.02, n=9, Fig. 5l) by approximately 19.4%. A 24-hour treatment resulted in a significant upregulation of HDAC 8 expression (p=0.048, n=9, Fig. 5l). The relative expression of HDAC 1 and HDAC 3 did not change after PA treatment for 24 or 72 hours ( Fig. 5F, Fig. 5H).

[0038] In summary, a surprising neuroprotective and neuroregenerative effect of the FFAR3 agonist PA was demonstrated in vitro in the PNS. PA mediated an antioxidant effect in SCs and DRGs by downregulating HDAC 2 and upregulating HDAC 8 expression, leading to hyperacetylation of histone 3 and upregulation of the important antioxidant enzyme catalase. Furthermore, PA treatment enhanced neuroregeneration after oxidative stress by inducing Gap-43. The FFAR3 agonist BHB was able to mimic this effect, whereas the FFAR3 antagonist PTX abolished it.

[0039] The increasing prevalence of autoimmune diseases in Western countries is associated with alterations in the gut microbiome and the daily intake of a low-fiber, refined carbohydrate-enriched diet. Since SCFAs such as acetate, propionate, and butyrate are produced by the fermentation of fiber by the gut microbiome, the presence of FFAR3 in the PNS may have an important physiological function in detecting nutritional status.

[0040] Evidence suggests that the response of the extraintestinal system to available food intake to maintain homeostasis is mediated by a neural circuit between the gut and brain via FFA 3 receptors on peripheral nerves. Therefore, SCFAs regulate not only the body's energy balance but also inflammatory processes and antioxidant defense cascades. Similar to previous findings demonstrating FFAR3 in the sympathetic ganglia and enteric nervous system, we provide evidence for the existence of FFAR3, but not FFAR2, in SCs and DRGs.

[0041] Histone modification in SC has implications for development, PNS integrity, and injury resistance. In this study and previous reports, PA was able to increase histone 3 acetylation through HDAC inhibition. The demonstration that both FFAR3 ligands, BHB and PA, led to a comparable increase in resistance to oxidative stress through similar mechanisms and that the G protein inhibitor PTX reversed these effects is highly convincing for an FFAR3-mediated process. However, there is evidence that both PA and BHB are capable of inducing free fatty acid receptor-independent HDAC inhibition.

[0042] Regardless of the mechanisms triggering HDAC inhibition, the resulting histone acetylation is a central step in PA-mediated signaling pathways. PA affects class 1 HDACs most potently. Contrary to previous assumptions, recent findings have demonstrated distinct roles of HDAC subtypes in preventing or promoting neurodegeneration. In the present study, we demonstrated significant downregulation of HDAC 2 by PA treatment. Previous studies have shown that overexpression of HDAC 2 leads to reduced dendritic spine density, synapse number, synaptic plasticity, and memory formation in mice. However, HDAC 2 also plays a special role in SC differentiation by regulating the important transcription factor Oct6: SCs occur in various forms, e.g., as myelinating and repairing SCs.HDAC 2 activation increases myelination but impairs differentiation into repair SCs, which is particularly important after cellular damage, e.g., after injury or inflammation. Although a balance between both conditions is likely essential, PA treatment could promote differentiation into repair SCs and thereby increase resistance to oxidative stress.

[0043] Furthermore, PA administration led to increased expression of HDAC 8. Overexpression of HDAC 8 is known in various cancer types and may regulate neural differentiation.

[0044] The invention and the technical environment are explained in more detail below with reference to the figures. It should be noted that the figures show a particularly preferred embodiment of the invention. However, the invention is not limited to the embodiment shown. In particular, the invention encompasses, to the extent technically feasible, any combination of the technical features listed in the claims or described in the description as relevant to the invention.

[0045] They show: Fig. 1 the detection of the free fatty acid receptor 3 (FFAR 3) on Schwann cells and spinal ganglia. Fig. 2 the neuroprotective effect of the free fatty acid receptor 3 agonist propionate on Schwann cells and dorsal root ganglia in vitro and ex vivo. Fig. 3 Downstream mechanisms of the neuroprotective effect exerted by the free fatty acid receptor 3 agonist propionate on Schwann cells and dorsal root ganglia through increased catalase expression Fig. 4 neuroregenerative effects exerted by propionate through increased Gap-43 expression Fig. 5 Propionate treatment leads to histone hyperacetylation through HDAC modification.

[0046] Fig. Figure 1 shows the detection of the free fatty acid receptor 3 on Schwann cells and dorsal root ganglia. Free fatty acid receptor 3 was detected immunohistochemically and at the RNA level in Schwann cells (A.3) and dorsal root ganglia (DRG; B.2).

[0047] Fig. Figure 2 shows that the free fatty acid receptor 3 agonist propionate exerts neuroprotective effects on Schwann cells and dorsal root ganglia. Treatment of Schwann cells with the two free fatty acid receptor 3 ligands, propionate (PA) or ß-hydroxybutyrate (BHB), significantly reduced H2O2-induced cell death compared to the control in flow cytometry ( Fig. 2A).

[0048] Similarly, pretreatment with propionate increased axonal growth after SNAP application compared to control ( Fig. 2B). Conversely, the addition of the G-protein receptor inhibitor pertussis toxin abolished the neuroprotective effect of propionate in SNAP-treated dorsal root ganglia (Figure B). Similarly, DRGs from control rats, but not from propionate-treated rats, showed a significant reduction in axonal outgrowth after SNAP application ( Fig. 2C) as an indication of a neuroprotective effect of propionate.

[0049] Fig. 3 presents the results Fig. 2. A 72-hour propionate treatment of Schwann cells resulted in a significant increase in relative catalase expression compared to the control group ( Fig. 3D), while expression was also increased after 24 hours of treatment, but did not reach the significance level. In the dorsal root ganglia, catalase expression was increased 24 hours after propionate treatment ( Fig. 3E). No significant changes in the relative mRNA expression of HO-1 and NQO1 were observed after 24 hours of PA treatment in DRG ( Fig. 3F, Fig. 3G). Data are expressed as mean ± standard deviation (SD). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0050] Fig. 4 shows in Fig. 4A shows that propionate induces neuroregenerative effects through increased Gap-43 expression. Treatment with propionate leads to enhanced axonal growth when applied after SNAP administration. In the same vein, propionate treatment leads to increased expression of the neuroregeneration marker growth-associated protein 43 under naive conditions ( Fig. 4B) and under SNAP-induced oxidative stress ( Fig. 4C). Data are expressed as mean ± standard deviation (SD). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0051] Fig. Figure 5 shows that propionate treatment leads to histone hyperacetylation. Treatment with propionate leads to increased acetylation of histone 3, but not of histone 4. The acetylation of histone 3 lysine 9 ( Fig. 5A) and histone 4 lysine 5 ( Fig. 5B), Lysine 8 ( Fig. 5C) and lysine 16 ( Fig. 5E), but not lysine 12 ( Fig. 5D), is significantly increased after 24 hours of β-hydroxybutyrate treatment. After propionate treatment, the relative expression of histone deacetylase 2 is significantly reduced ( Fig. 5G) and the expression of histone deacetylase 8 was significantly increased ( Fig. 5l) in Schwann cells. A 24-hour treatment only leads to a non-significant trend towards a reduction of HDAC 2 and 8. The relative expression of HDAC 1 and HDAC 3 does not change after PA treatment for 24 hours or 72 hours ( Fig. 5F, Fig. 5H). Data are expressed as mean ± standard deviation (SD). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Claims

[1] An agent for use in the treatment of CIDP, an immune-mediated or autoimmune disease of the peripheral nervous system, wherein the agent increases the resistance of Schwann cells and dorsal root ganglia to oxidative stress, containing propionic acid, its physiologically acceptable salts and / or esters with C1-C8 alkyl alcohols. [2] An agent for use in the treatment of CIDP according to claim 1 in a daily dose of up to 10g. [3] Agent for use in the treatment of CIDP according to any one of claims 1 or 2, characterized by that it contains sodium propionate. [4] Agent for use in the treatment of CIDP according to any one of the preceding claims, characterized by that the physiologically acceptable esters are the methyl esters or ethyl esters. [5] An agent for use in the treatment of CIDP according to any one of the preceding claims in capsule or tablet form. [6] Agent for use in the treatment of CIDP according to claim 5, characterized by that the capsules or tablets contain a dose of 0.1 to 5 g, in particular 1 to 3 g, of the carboxylic acids, their salts or esters. [7] Agent for use in the treatment of CIDP according to any one of the preceding claims, characterized by that the agent contains at least one other short-chain C1 to C6 carboxylic acid, in particular butyrate. [8] Agent for use in the treatment of CIDP according to claim 7, characterized by that butyrate, its physiologically acceptable salts and / or esters with C1-C8 alkyl alcohols are intended for a daily dose of up to 10 g.

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