Use of therapeutic agents having sodium hydrogen antitransporter 1 inhibitory activity for treatment and prevention of diseases associated with chronic fatigue, fatigue and / or fatigue intolerance

Abstract

The present invention relates to the use of a substance having sodium hydrogen antiporter 1 inhibitory activity (NHE1 inhibitor) as an active ingredient in a therapeutic agent having sodium hydrogen antiporter 1 inhibitory activity, using an NHE1 inhibitor for the treatment and prevention of various diseases, syndromes, disease states or conditions associated with chronic fatigue, fatigue and / or fatigue intolerance.

Description

Technical Field

[0001] The present invention relates to the use of substances having sodium-hydrogen antiporter 1 inhibitory activity (NHE1 inhibitors) as active ingredients in therapeutic agents having sodium-hydrogen antiporter 1 inhibitory activity, using NHE1 inhibitors for the treatment and prevention of various diseases, syndromes, disease states or conditions associated with chronic fatigue, exhaustion and / or exertion intolerance.

[0002] These diseases, syndromes, disease states or conditions will be encompassed by the term "diseases associated with chronic fatigue" and abbreviated as (DACF), which includes diseases diagnosed as: myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS), generalized exertion intolerance, exertion intolerance, post-vac-syndrome following vaccination against viruses and pathogenic agents, post-infectious fatigue following viral, bacterial, or fungal infections, cancer-related fatigue (where chronic fatigue and exhaustion are symptoms of or associated with cancer), and fatigue associated with fibromyalgia, Ehlers-Danlos syndrome, Marfan syndrome, Gulf War illness, and the autoimmune diseases rheumatoid arthritis, ANCA vasculitis (antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis) and Sjögren's syndrome ( syndrome, and other autoimmune diseases with debilitating symptoms of fatigue and exhaustion. Background Art

[0003] Sodium proton transporter 1 is also called sodium / proton exchanger 1 or Na + / H + Exchanger type 1 (abbreviated as NHE1) or SLC9A1 (Soluble carrier family 9A1), encoded by the SLC9A1 human gene (NFliegel et al. doi:10.1007 / BF00936442). The sodium proton transporter (SLC9A1) is a ubiquitous membrane-bound transporter involved in volume regulation and pH regulation of vertebrate cells. It is activated by a variety of signals including growth factors, mitogens, neurotransmitters, tumor promoters and others (Cardone et al. doi:10.3390 / ijms20153694). Under normal circumstances, NHE1 activates the sodium proton transporter by removing one intracellular proton (H + ) ions to exchange for a single extracellular sodium (Na + ) ions to maintain intracellular pH (pHi) and volume (Fliegel.doi:10.1016 / j.biocel.2004.02.006).

[0004] In certain pathological states [e.g., heart failure, cardiovascular disease, diabetes], NHE1 is activated, leading to rapid accumulation of sodium in cells (Fliegel.doi:10.3390 / ijms20102378) and acidification of the extracellular space. High sodium concentration drives Na + / Ca 2+ exchanger (NCX), because the sodium gradient is the NCX export calcium (Ca2 + ) provides the driving force. In its (physiological) forward mode, NCX exports one calcium ion in exchange for the uptake of three sodium ions that control cellular calcium. Under high sodium concentrations, NCX changes its transport direction (so-called reverse mode NCX) to import calcium and export sodium, resulting in increased intracellular calcium and calcium load. The resulting calcium accumulation triggers cellular damage. NHE1 is critically responsible for the cellular sodium load, which leads to a deleterious calcium load. NHE1 inhibitors generally protect organs from damage in ischemia-reperfusion situations, which are due to sodium overload, which then leads to calcium overload via reverse mode NCX. The involvement of NHE1 in cardiac pathology has been studied for decades and is supported by a large number of experimental studies showing that effective NHE1 inhibition can not only protect the myocardium from ischemia and reperfusion injury, but also attenuate myocardial remodeling and heart failure (Evans et al. doi:10.1016 / j.pmrj.2009.04.010). The cardioprotective effects of NHE-1 inhibitors, including cariporide and rimeporide, have been extensively studied in various animal models of myocardial infarction and malnutrition-induced cardiomyopathy, including DMD (Ghaleh et al. doi: 10.1016 / j.ijcard.2020.03.031). Other preclinical experiments (Chahine et al. doi: 10.1016 / j.yjmcc.2005.01.003; Bkaily and Jacques, doi: 10.1139 / cjpp-2017-0265) have highlighted the importance of myocardial necrosis due to sodium and calcium imbalance in the pathophysiology of heart failure and demonstrated the role of NHE1 inhibition using cariporide and rimeporide (CAS187870-78-6) in preventing Ca2+. + and Na + Beneficial effects amidst the deleterious effects of overload.

[0005] In experimental models, NHE1 inhibition is cardioprotective, reducing cardiac pathology including fibrosis, left ventricular function, and improving survival.

[0006] NHE1 inhibition can normalize intracellular sodium and calcium, thereby improving cell function; preventing progressive congestive heart failure; regulating inflammatory processes; preventing fibrosis. Known NHE1 inhibitors are, for example, cariporide, ramipril, eneporide, all of which belong to the class of benzoyl-guanidine derivatives (based on the benzene ring), or amiloride, EIPA (5-(N-ethyl-N-isopropyl) amiloride), DMA (5-(N,N-dimethyl) amiloride), MIBA (5-(N-methyl-N-isobutyl) amiloride) and HMA5-N,N-(hexamethylene) amiloride, all of which belong to the class of pyrazinoyl-guanidine derivatives (based on the pyrazine ring).

[0007] NHE1 is also expressed in skeletal muscle as an important proton-extruder. As in many organs, especially the heart, NHE1 inhibitors have been shown in animal experiments to inhibit ischemia-reperfusion injury in skeletal muscle. This suggests that it is related to calcium overload-induced cell damage caused by high intracellular sodium in skeletal muscle. NHE1 is strongly involved in cellular sodium loading, especially during exercise, by eliminating the increase in intracellular protons through a 1:1 exchange with sodium ions. Therefore, NHE1 enhances intramuscular sodium influx and sodium loading. Intracellular sodium is replaced by Na + / K + ATPase removes and exports it from the cell. If the latter does not work properly, intracellular sodium rises, with negative consequences for intracellular calcium. The latter can cause harmful changes in skeletal muscle.

[0008] The pathophysiology of disorders associated with chronic fatigue (DACF) has been elusive, and thus no rational pharmacological or therapeutic strategies have been derived or developed. Therefore, due to the lack of understanding of these diseases and their pathophysiology, no specific effective treatments are currently available. The present invention has been established through a unified and comprehensive disease hypothesis elaborated by the inventors, who have discovered that DACF can be treated by preventing the increase in sodium ions in skeletal muscle. The pathological increase in sodium in skeletal muscle has two causes: increased sodium influx caused by NHE1, and Na + / K + The increased influx is due to the need to export excess H produced in skeletal muscle due to poor metabolic conditions leading to anaerobic metabolism. + ions. In patients with chronic fatigue, blood lactate levels are elevated. Cellular energy and mitochondrial dysfunction are present in chronic fatigue, especially in skeletal muscle. Since the sodium-proton exchanger NHE1 imports 1 sodium ion to export 1 H + , so intracellular sodium tends to increase when there is an increased demand for cellular proton export due to poor metabolic conditions (in anaerobic metabolism).+ / K + ATPase activity is unable to cope with the increased sodium influx caused by NHE1, and the sodium in the myocytes of skeletal muscle increases. The latter leads to an increase in intracellular calcium (Ca) in skeletal muscle by changing the activity of the sodium-driven sodium-calcium exchanger (NCX). 2+ ) and mitochondrial calcium (Ca 2+ ) are adversely altered. These pathological ionic changes in skeletal muscle play an important pathophysiological role in the development and persistence of chronic fatigue, exhaustion and / or exertion intolerance. The inventors have found that the harmful increase in sodium in skeletal muscle cells in DACF can be prevented by inhibiting NHE1, which inhibits sodium influx into skeletal muscle.

[0009] In summary, inhibition of NHE1 would inhibit sodium overload in myocytes of skeletal muscle, thereby preventing the deleterious calcium changes that deteriorate the energetic function of skeletal muscle leading to muscle fatigue and many of the symptoms of ME / CFS and chronic fatigue.

[0010] Among the diseases associated with fatigue, exhaustion and / or exertion intolerance, myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS) is the most debilitating condition. Although ME / CFS also frequently affects about 0.3% of the population, most physicians are not familiar with it. ME / CFS is characterized by extreme fatigue, post-exertional malaise (PEM), exertion intolerance, cognitive dysfunction, orthostatic intolerance, including postural tachycardia (POTS) and is associated with a variety of different symptoms (including muscle weakness and pain). Despite the fact that unrefreshing sleep is also a typical symptom and unrefreshing sleep may worsen the patient's condition, sleep disorders are not the initial cause of extreme fatigue and exhaustion, but rather a consequence of ME / CFS that may further worsen the condition.

[0011] Prior to the present invention, no specific treatment was available, and current symptomatic treatments can only provide some symptom relief, not a cure. Many patients with ME / CFS are bedridden or in wheelchairs; most are unable to work. ME / CFS is often, but not always, triggered by various viral infections such as Epstein–Barr virus (EBV), enterovirus, influenza virus, dengue fever.

[0012] Therefore, there is a strong need for drugs or therapeutic agents that can be used to treat and prevent chronic fatigue, exhaustion and / or exertion intolerance associated with various diseases, syndromes, disease states (DACF).

[0013] Clearly, there is a strong need in the art for new approaches to treat and prevent chronic fatigue, exhaustion and / or exertion intolerance associated with various diseases, syndromes, condition (DACF). Summary of the invention

[0014] The present invention provides a novel approach to treat and prevent DACF by using a substance having sodium-hydrogen antiporter 1 inhibitory activity (NHE1 inhibitor). DETAILED DESCRIPTION

[0015] The term "NHE1 inhibitor" as used herein includes chemical compounds, proteins or polypeptides, nucleic acids, ribozymes, DNA enzymes, protein degraders, gene therapy, or other such agents that directly or indirectly inhibit or block the transport activity of NHE1 proteins in a selective or non-selective manner. In some cases, the agent can directly bind to or directly interact with the NHE1 protein. Agents bound to NHE1 can be used to inhibit or block NHE1 activity by any suitable means (e.g., by blocking ion binding sites). In other cases, NHE1 inhibitors can indirectly inhibit NHE1 activity, for example, by reducing the expression of NHE1 proteins. In some cases, NHE1 inhibitors can inhibit NHE1 activity by changing the cellular distribution of NHE1 (e.g., by interfering with the binding between NHE1 and intracellular anchoring proteins).

[0016] The term disease associated with chronic fatigue used in this application includes different diseases, syndromes, disease states, or conditions associated with chronic fatigue, exhaustion, and / or intolerance to fatigue. It includes myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS), systemic intolerance to fatigue, intolerance to fatigue, post-vaccination syndrome (post-vac-syndrome) after vaccination against viruses and pathogenic factors, post-infection fatigue and exhaustion after viral, bacterial, or fungal infections. In addition, the term includes such diseases, wherein chronic fatigue and exhaustion are symptoms of cancer or are associated with cancer (cancer-related fatigue), fibromyalgia, Ehlers-Danlos syndrome, Marfan syndrome, Gulf War disease, autoimmune disease rheumatoid arthritis, ANCA vasculitis (vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA)-) and Sjögren's syndrome, and other autoimmune diseases with fatigue and exhaustion as debilitating symptoms. Hereinafter, these diseases, syndromes, disease states, or conditions are covered by the disease associated with chronic fatigue (DACF). However, in the present invention, the term chronic fatigue-related diseases does not include Covid-19 sequelae, long Covid and post-Covid-19 syndrome.

[0017] In a preferred embodiment of the present invention, the NHE1 inhibitor is a compound that is potent enough to be effective at IC 50<50 μM, preferably less than or about 1 μM, inhibits the transport activity of NHE1. Even more preferably, the NHE1 inhibitor is potent enough to inhibit the transport activity of NHE1 with an IC 50 Inhibits the activity of NHE1 at a concentration of about 0.1 to about 600 nM. In particular, the NHE1 inhibitor is potent and has an IC 50 From about 0.2 to about 100 nM, preferably with IC 50 The enzyme activity of NHE1 is inhibited at about 1 to about 100 nM.

[0018] In the present invention, NHE1 inhibitors prevent sodium overload and subsequent calcium overload in skeletal muscle to prevent adverse ionic changes that cause skeletal muscle mitochondrial and energy dysfunction. Inhibition of NHE1 can prevent sodium (Na + ) and subsequent harmful increases in calcium (Ca 2+ ) changes. Inhibition of NHE1 prevents sodium (Na + )-induced disturbances in key ions, metabolism, and energy in skeletal muscle and improve energy status in skeletal muscle by preventing subsequent adverse changes in cellular and mitochondrial calcium.

[0019] The improvement in skeletal muscle energy status caused by NHE1 inhibition prevents the release of vasoactive nociceptive (pain) mediators in skeletal muscle. Normally, these mediators are released to compensate for poor metabolic status (increase blood flow to skeletal muscle) and physiologically act only locally in skeletal muscle. Since they are overproduced in disease states due to very poor metabolic status of the body's large muscle groups, they overflow into the systemic circulation, whereby these mediators with pain properties can reach and act on every organ in the body. These mediators cause many different symptoms. In addition to fatigue and exhaustion, related symptoms include brain fog, impaired cognition, disturbed sleep, orthostatic intolerance, muscle weakness, pain, post-exertion malaise (PEM), which is an aggravation of typical symptoms after low-intensity or moderate-intensity exercise, and many other diseases.

[0020] Through these beneficial effects, NHE1 inhibition improves the symptoms of DACF and may cure DACF disease, prevent relapses, and prevent disease in a prophylactic manner.

[0021] Specific examples of the NHE1 inhibitors used in the present invention are substances described below, which are selected from the following compounds: ramipril, cariporide, eniporide, amiloride, EIPA (5-(N-ethyl-N-isopropyl) amiloride), DMA (5-(N,N-dimethyl) amiloride), MIBA (5-(N-methyl-N-isobutyl) amiloride), HMA (5-(N,N-(hexamethylene) amiloride), 2-aminophenoxazine-3-one, 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one, zoniporide, compound 9t, and SL-591227.

[0022] Thus, the present invention includes methods of treating or preventing DACF comprising administering one or more NHE1 inhibitors to a subject having or at risk of developing DACF.

[0023] As used herein, unless the context clearly indicates otherwise, "treat" and similar words such as "treatment" or "treating" are methods of obtaining beneficial or desired results, including and preferably clinical results. Treatment may optionally include reducing or ameliorating a disease or condition such as DACF, or slowing the progression of a disease or condition such as DACF.

[0024] As used herein, unless the context clearly indicates otherwise, "prevent" and similar words such as "prevention", "preventing", etc., are methods of preventing the onset or recurrence of a disease or condition (e.g., DACF) or preventing the recurrence of a disease or condition, for example, after a viral infection such as Epstein-Barr virus infection, or preventing the recurrence of symptoms of a disease or condition, or optionally a method of delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition.

[0025] As used herein, an "effective amount" or "therapeutically effective amount" of a substance, such as an NHE1 inhibitor, is an amount sufficient to affect a desired biological or psychological effect (e.g., a beneficial outcome, including a clinical outcome). For example, in the context of treating DACF using the methods of the invention, an effective amount of an NHE1 inhibitor is an amount sufficient to treat and prevent DACF. Typically, an effective amount of an NHE1 inhibitor is provided to a subject.

[0026] Pharmaceutical composition, route of administration, unit dosage form, kit:

[0027] The composition of the present invention can be applied to a subject as a pharmaceutical composition or preparation. In a particular embodiment, the pharmaceutical composition of the present invention can be any form that allows the composition to be administered to a subject. For example, the composition can be in the form of a solid, a liquid, or a gas (aerosol). Typical routes of administration include, but are not limited to, oral administration, topical administration, parenteral administration, sublingual administration, rectal administration, vaginal administration, and nasal administration. The term parenteral as used herein includes subcutaneous injection, intravenous injection, intramuscular injection, epidural injection, intrasternal injection or infusion technology.

[0028] The pharmaceutical composition used according to the present invention comprises: an NHE1 inhibitor, and a pharmaceutically acceptable diluent, excipient, or carrier.

[0029] "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical art. For example, sterile saline and phosphate buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes, and even flavoring agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid can be added as preservatives. In addition, antioxidants and suspending agents can be used.

[0030] The pharmaceutical compositions of the present invention are typically formulated so as to allow the active ingredients contained therein to be bioavailable when the composition is administered to a subject. The composition to be administered to a subject can take the form of one or more dosage units, wherein, for example, a tablet, capsule, or cachet can be a single dosage unit, and a container containing a combination of agents according to the present invention in aerosol form can accommodate multiple dosage units. In a particular embodiment, the composition comprising an NHE1 inhibitor and ultimately another therapeutic agent is administered in one or more doses of a tablet formulation, typically orally. The tablet formulation can be, for example, an immediate release formulation, a controlled release formulation, or a sustained release formulation. In one embodiment, the tablet formulation comprises an effective amount of a composition comprising an NHE1 inhibitor and ultimately another therapeutic agent. In a particular embodiment, the tablet comprises about 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, or 600 mg of the NHE1 inhibitor and, if used in combination, a final amount of about 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, or 600 mg of another therapeutic agent.

[0031] As an example, a unit administration form of a compound according to the invention in the form of a tablet may contain the following components: ramipril 300 mg, mannitol 223.75 mg, croscarmellose sodium 6 mg, corn starch 15 mg, hydroxypropylmethylcellulose 2.25 mg, and magnesium stearate 3 mg.

[0032] Alternatively, according to the present invention, the capsule formulation may contain, for example, 300 mg of ramipril and a pharmaceutically acceptable excipient, wherein the excipient may contain one or more selected from the following substances: a disintegrant, a filler and a lubricant, and may contain an effective amount of a binder.

[0033] Preferably, the NHE1 inhibitor is provided to the subject in an amount ranging from 0.1-1000 mg / day, 1-1000 mg / day, 10-100 mg / day, or 25-50 mg / day.

[0034] NHE1 proteins and inhibitors

[0035] In the practice of the methods of the present invention, representative NHE1 inhibitors that inhibit the activity of NHE1 include: molecules that bind to NHE1 and inhibit the transport activity of NHE1 (e.g., small molecule inhibitors or blocking peptides or proteins / protein fragments / fusion proteins that bind to NHE1 and reduce enzyme activity), molecules that reduce the expression of NHE1 at the transcriptional and / or translational levels (e.g., NHE1 antisense nucleic acid molecules, NHE1-specific RNAi molecules and NHE1 ribozymes, DNA enzymes), and NHE1-directed gene therapy, thereby preventing ion transport caused by NHE1. NHE1 inhibitors can be used alone as primary therapy, or as adjunctive therapy in combination with other therapies to enhance the therapeutic benefits as discussed in this application.

[0036] Inhibition of NHE1 is characterized by at least one of the following changes occurring as a result of administration of the NHE1 inhibitor according to the methods of the invention: inhibition of NHE1 transport activity, reduction in gene or protein expression levels of NHE1 as measured, for example, by gene expression analysis (e.g., RT-PCR analysis) or protein analysis (e.g., Western blot).

[0037] In some embodiments, the NHE1 inhibitor is a molecule or composition that inhibits the expression of NHE1, such as an antisense or small inhibitory nucleotide (e.g., siRNA) that specifically hybridizes with cellular mRNA and / or gene DNA corresponding to the target NHE1 gene, thereby inhibiting their transcription and / or translation, or a ribozyme that specifically cleaves the target NHE1 mRNA.

[0038] Potency of NHE1 Inhibitors:

[0039] In one embodiment, the NHE1 inhibitor used in the methods of the invention is a compound that is sufficiently potent to be effective at an IC 50 <50 μM, preferably less than or about 1 μM, inhibits the transport activity of NHE1. In one embodiment, the NHE1 inhibitor is potent enough to inhibit the transport activity of NHE1 with an IC 50 In one embodiment, the NHE1 inhibitor is potent so that the IC 50 From about 0.2 to about 100 nM, preferably with IC 50 The activity of NHE1 is inhibited at about 1 to about 100 nM.

[0040] Determination of IC of NHE1 inhibitors 50 Representative methods are well known in the art.

[0041] NHE1 selective inhibitors:

[0042] Types of NHE1 inhibitors

[0043] The NHE1 inhibitor can be any type of agent, including, but not limited to, chemical compounds, proteins or polypeptides, peptidomimetics, nucleic acid molecules, ribozymes, DNA enzymes, protein degraders, or gene therapy. In some embodiments, the NHE1 inhibitor is a small molecule inhibitor, including natural and synthetic substances with low molecular weight, for example, peptides, peptidomimetics and non-peptide inhibitors such as chemical compounds.

[0044] Chemical compounds:

[0045] NHE1 inhibitors used in the methods of the present invention include agents that are administered into the bloodstream by conventional routes (eg, oral, intramuscular, subcutaneous, transdermal, oral, intravenous, etc.) and ultimately transported through the vascular system to inhibit NHE1 in skeletal muscle and vasculature.

[0046] The following is a description of exemplary NHE1 inhibitors for use in the methods of the invention. They can be orthosteric, allosteric, or other inhibitors. If not explicitly defined later, the following terms are used:

[0047] i) Aryl: a functional group derived from an aromatic ring or a ring system having or not having heteroatoms when one hydrogen is removed from the ring structure,

[0048] ii) Alkyl: In a broad sense, a hydrocarbon group formed when a hydrogen atom is removed from an alkane, alkene, or alkyne group,

[0049] iii) Alkoxy: a functional group comprising an alkyl group bonded to oxygen,

[0050] iv) Acyl group: a functional group having the formula RC=O, wherein R is an aryl group, an alkyl group or other structures.

[0051] The NHE1 inhibitor used in the method of the present invention is selected from known NHE1 inhibitors, for example, ramipril, cariporide, eniporide, all of which belong to the class of benzoyl-guanidine derivatives (based on the benzene ring), or amiloride, EIPA (5-(N-ethyl-N-isopropyl) amiloride), DMA (5-(N,N-dimethyl) amiloride), MIBA (5-(N-methyl-N-isobutyl) amiloride) and HMA5-N,N-(hexamethylene) amiloride, which belongs to the class of pyrazinoyl-guanidine derivatives (based on the pyrazine ring) or 2-aminophenoxazine-3-one, 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one, zoniporide, compound 9t, or SL-591227.

[0052] The chemical names and chemical structures of some selected NHE1 inhibitors are as follows:

[0053] Rimepril: N-(4,5-bis(methylsulfonyl-2-methylbenzoyl)guanidine (CAS 187870-78-6)

[0054]

[0055] Cariporide: N-(diaminomethylene)-3-methylsulfonyl-4-(propan-2-yl)benzamide

[0056]

[0057] Enipamide: N-(diaminomethylene)-2-methyl-5-methylsulfonyl-4-pyrrol-1-ylbenzamide

[0058]

[0059] Amiloride: 3,5-diamino-N-carbamimidoyl-6-chloropyrazine-2-carboxamide

[0060] HMA (5-(N,N-hexamethylene) amiloride)

[0061]

[0062] EIPA (5-(N-ethyl-N-isopropyl) amiloride)

[0063]

[0064] DMA (5-(N,N-dimethyl)amiloride)

[0065]

[0066] MIBA (5-(N-methyl-N-isobutyl) amiloride)

[0067]

[0068] Aminophenoxazine derivatives: 2-aminophenoxazine-3-one

[0069]

[0070] Another aminophenoxazine derivative: 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazin-3-one

[0071]

[0072] Zonipoli

[0073]

[0074] Compound 9t (CAS 335063-97-3)

[0075] 5-(4-Fluoro-3-methylphenyl)-2-methoxy-4-[4-(5-methyl-1H-imidazol-4-yl)piperidin-1-yl]pyrimidine

[0076]

[0077] SL-591227(CAS 326488-60-2)

[0078] 3-[(Cyclopropylcarbonyl)amino]-N-[2-(dimethylamino)ethyl]-4-[4-(4-methyl-1H-imidazol-5-yl)-1-piperidinyl]benzamide

[0079]

[0080] or as a pharmaceutically acceptable salt such as DMA (5-(N,N-dimethyl)amiloride) (hydrochloride)

[0081]

[0082] or zoniporol (hydrochloride)

[0083]

[0084] In another embodiment, the NHE1 inhibitor used in the method of the present invention is selected from the group consisting of pyrrolidinyl compounds and piperidinyl compounds generally or specifically disclosed in WO 2010 / 005783, which is expressly incorporated herein by reference in its entirety.

[0085] Protein, polypeptide or peptide inhibitors:

[0086] In some embodiments, the NHE1 inhibitor includes an isolated NHE1 polypeptide or peptide inhibitor, including isolated natural peptide inhibitors and synthetic peptide inhibitors that inhibit NHE1 activity. The term "isolated NHE1 polypeptide or peptide inhibitor" as used herein refers to a polypeptide or peptide that inhibits NHE1, competes with NHE1 to bind ions, and / or directly interacts with NHE1 to inhibit NHE1 transport activity, which is substantially pure and substantially free of other substances that may be present in nature to the extent practical and suitable for its intended use.

[0087] Peptide inhibitors have been successfully used in vivo to interfere with protein interactions and binding sites. For example, peptide inhibitors of adhesion molecules related to the LFA-1 structure have been approved for clinical use in coagulation disorders. Short linear peptides (<30 amino acids) that prevent or interfere with integrin-dependent adhesion have been described. Longer peptides with a length of 25 to 200 amino acid residues have also been successfully used to block integrin-dependent adhesion. In general, longer peptide inhibitors have higher affinity and / or slower off-rates than short peptides and may therefore be more potent inhibitors. Cyclic peptide inhibitors have also been shown to be effective inhibitors of integrin in vivo for the treatment of human inflammatory diseases. One method for producing cyclic peptides includes the synthesis of peptides, wherein the terminal amino acid of the peptide is cysteine, so that the peptide exists in a cyclic form through a disulfide bond between the terminal amino acids, which has been shown to increase the in vivo affinity and half-life for the treatment of hematopoietic tumors.

[0088] The following materials also inhibit NHE1: NHE1 binding proteins, antibodies, nanobodies or functionally related proteins or protein fragments or fusion proteins, such as single-chain variable fragments or DARPins (designed ankyrin repeat domain proteins) or lipocalins / anticalins or other such molecules, which enter cells or are modified with other parts / conjugated to other parts so that they can enter cells.

[0089] Synthetic NHE1 peptide inhibitors

[0090] Examples of NHE1 inhibitory peptides used in the methods of the present invention are amino acid sequences that mimic target regions important for NHE1 transport activity (e.g., the sodium binding domain of NHE1). Molecular modeling and rational molecular design can also be used to generate and screen molecular structures that mimic NHE1 binding regions and peptides that inhibit the transport activity of NHE1. Molecular structures used for modeling include CDR regions of anti-NHE1 monoclonal antibodies. Methods for identifying peptides that bind to specific targets are well known in the art. For example, molecular imprinting can be used to construct macromolecular structures such as peptides that bind to specific molecules from scratch. See, for example, Shea, KJ, "Molecular Imprinting of Synthetic Network Polymers: The De Novo Synthesis of Macromolecular Binding and Catalytic Sites," Trends in Polymer Science 2 (5): 166-173 (1994).

[0091] As an illustrative example, a method for preparing a mimetic of an NHE1 binding peptide is as follows. Functional monomers (templates) of the binding region of an anti-NHE1 antibody that exhibits NHE1 inhibition are polymerized. The template is then removed, and a second type of monomer is then polymerized in the void left by the template to provide a new molecule that exhibits one or more desired properties similar to the template. In addition to preparing peptides in this manner, other NHE1 binding molecules (which are NHE1 inhibitors) can also be prepared, such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active substances. This method can be used to design a variety of biomimetic substances that are more stable than their natural counterparts because they are typically prepared by free radical polymerization of functional monomers, resulting in compounds with non-biodegradable backbones.

[0092] NHE1 inhibitory peptides can be prepared using techniques known in the art, such as solid phase synthesis techniques originally described by Merrifield in J. Amer. Chem. Soc. 85: 2149-2154, 1963. Automated synthesis can be achieved, for example, using an Applied Biosystems 431A peptide synthesizer (Foster City, Calif.) according to the instructions provided by the manufacturer. Other techniques can be found in the following literature, for example, Bodanszky, M., et al., Peptide Synthesis, 2nd Edition, John Wiley & Sons, 1976, and other references known to those skilled in the art. Peptides can also be prepared using standard genetic engineering techniques known to those skilled in the art.

[0093] Candidate NHE1 inhibitory peptides can be tested for their ability to act as NHE1 inhibitors in one of several assays including, for example, an NHE1 cell assay.

[0094] NHE1 expression inhibitor, nucleic acid

[0095] In some embodiments of the methods of the present invention, the NHE1 inhibitor is an NHE1 expression inhibitor capable of inhibiting NHE1-dependent sodium and hydrogen transport. In the practice of this embodiment of the present invention, representative NHE1 expression inhibitors include NHE1 antisense nucleic acid molecules (e.g., antisense mRNA, antisense RNA, antisense DNA, or antisense oligonucleotides), NHE1 RNAi molecules, NHE1 ribozymes, and NHE1 DNA enzymes.

[0096] Antisense RNA and DNA molecules are used to directly block the translation of NHE1 mRNA by hybridizing with NHE1 mRNA and preventing the translation of NHE1 protein. Antisense nucleic acid molecules can be constructed in a variety of different ways, as long as they are able to interfere with the expression of NHE1. For example, antisense nucleic acid molecules can be constructed by reversing the coding region (or a portion thereof) of NHE1 cDNA relative to its normal transcription direction to allow transcription of its complement. Methods for designing and administering antisense oligonucleotides are well known in the art and are described in, for example, Mautino et al., Hum Gene Ther 13:1027-37, 2002; and Pachori et al., Hypertension 39:969-75, 2002, which are incorporated herein by reference.

[0097] Antisense nucleic acid molecules are generally substantially identical to at least a portion of one or more target genes. However, nucleic acids do not need to be identical to inhibit expression. Generally, higher homology can be used to compensate for the use of shorter antisense nucleic acid molecules. The minimum identity percentage is generally greater than about 65%, but a higher identity percentage may more effectively inhibit the expression of endogenous sequences. Generally, a significantly larger identity percentage greater than about 80% is preferred, although about 95% to absolute identity is generally the most preferred.

[0098] The antisense nucleic acid molecule need not have the same intron or exon pattern as the target gene, and non-coding segments of the target gene may be equally effective as coding segments in achieving antisense inhibition of target gene expression. DNA sequences having at least about 8 or so nucleotides can be used as antisense nucleic acid molecules, but longer sequences are preferred. In the present invention, a representative example of a useful inhibitor of NHE1 is an antisense NHE1 nucleic acid molecule having at least 90% identity to the complement of a portion of the NHE1 cDNA.

[0099] Targeting antisense oligonucleotides to bind to NHE1 mRNA is another mechanism that can be used at the level of NHE1 protein synthesis. For example, Paolo's U.S. Patent 7,579,455 or the synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptor was inhibited by antisense oligonucleotides directed against their respective mRNA sequences (Galli's U.S. Patent 5,739,119, and Shewmaker's U.S. Patent 5,759,829). In addition, examples of antisense inhibition have been demonstrated with nuclear protein cyclins, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA a The receptor is related to human EGF (see, e.g., US Pat. No. 5,801,154 to Baracchini; US Pat. No. 5,789,573 to Baker; US Pat. No. 5,718,709 to Considine; and US Pat. No. 5,610,288 to Rubenstein).

[0100] A system has been described that allows one of ordinary skill in the art to determine which oligonucleotides can be used in the present invention, which system includes the use of RNase H cleavage as an indicator of sequence accessibility within the transcript to probe for suitable sites in the target mRNA. Scherr, M. et al., Nucleic Acids Res. 26:5079-5085, 1998; Lloyd et al., Nucleic Acids Res. 29:3665-3673, 2001. A mixture of antisense oligonucleotides complementary to certain regions of the NHE1 transcript is added to an extract of cells expressing NHE1 and hybridized to create RNAseH vulnerable sites. This method can be combined with computer-assisted sequence selection, which can predict the optimal sequence selection for antisense compositions based on their relative ability to form dimers, hairpins, or other secondary structures that will reduce or prohibit specific binding to the target mRNA in the host cell. These secondary structure analysis and target site selection considerations can be performed using OLIGO primer analysis software (Rychlik, I., 1997) and BLASTN 2.0.5 algorithm software (Altschul, SF et al., Nucl. Acids Res. 25: 3389-3402, 1997). The length of the antisense compound for the target sequence preferably comprises about 8 to about 50 nucleotides. Antisense oligonucleotides comprising about 9 to about 35 nucleotides are particularly preferred. The inventors contemplate that all oligonucleotide compositions ranging from 9 to 35 nucleotides in length (i.e., those having a length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 or so bases) are highly preferred for the practice of the antisense oligonucleotide-based methods of the invention. Highly preferred target regions of NHE1 mRNA are those located at or near the AUG translation start codon, and those sequences that are substantially complementary to the 5' region of the mRNA (e.g., between regions 0 and +10 of the nucleotide sequence of the NHE1 gene).

[0101] The term "oligonucleotide" as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or its mimetics. The term also includes oligonucleotide bases consisting of naturally occurring nucleotides, sugars, and covalent nucleoside (backbone) bonds, as well as oligonucleotides with non-natural modifications. These modifications allow the introduction of certain desired properties that cannot be provided by naturally occurring oligonucleotides, such as reduced toxicity, improved stability against nuclease degradation, and enhanced cellular uptake.

[0102] Another alternative to antisense is to use "RNA interference" (RNAi). Double-stranded RNAs (dsRNAs) can induce gene silencing in mammals. The natural function of RNAi and co-inhibition seems to be to protect the genome from invasion by mobile genetic elements, such as retrotransposons and viruses that produce abnormal RNA or dsRNA in host cells when they become active (see, e.g., Jensen, J. et al., Nat. Genet. 21: 209-12, 1999). Double-stranded RNA molecules can be prepared by synthesizing two RNA chains that can form double-stranded RNA molecules, wherein the length of each RNA chain is about 19 to 25 (e.g., 19-23 nucleotides). For example, the dsRNA molecules used in the method of the present invention can include RNA corresponding to a portion of at least one of human NHE1 and its complement. Preferably, at least one chain of RNA has a 3' overhang of 1-5 nucleotides. The synthesized RNA chains are combined under conditions that form double-stranded molecules. The RNA sequence may comprise at least an 8 nucleotide portion of human NHE1 with a total length of 25 nucleotides or less. The design of siRNA sequences for a given target is within the knowledge of one of ordinary skill in the art. Commercial services are available for designing siRNA sequences and guaranteeing at least 70% knockdown of expression (Qiagen, Valencia, Calif.). Exemplary NHE1 shRNAs and siRNAs are commercially available from Sigma-Aldrich Company (product #SHDNA_-NM_002603; SASI_Hs01_00183420 to SASI_Hs01_00010490).

[0103] dsRNA can be administered as a pharmaceutical composition and by known methods, wherein the nucleic acid is introduced into the desired target cell. Conventionally used gene transfer methods include calcium phosphate, DEAE dextran, electroporation, microinjection and viral methods. Such methods are taught in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

[0104] Ribozymes and DNA enzymes

[0105] In some embodiments, the NHE1 inhibitor is a ribozyme or DNA enzyme that specifically cuts the mRNA of the target NHE1 or NHE1 itself. Ribozymes targeting NHE1 can be used as NHE1 inhibitors to reduce the amount and / or biological activity of NHE1. Ribozymes are catalytic RNA molecules that can cut nucleic acid molecules with sequences that are completely or partially homologous to the sequences of ribozymes. Ribozyme transgenes encoding RNA ribozymes can be designed, which specifically pair with the target RNA and cut the mitochondrial skeleton at a specific position, thereby functionally inactivating the target RNA. When performing this cutting, the ribozyme itself will not change, so it can recover and cut other molecules. Including ribozyme sequences in antisense RNA can give them RNA cutting activity, thereby increasing the activity of antisense constructs.

[0106] Ribozymes used in the practice of the present invention generally comprise a hybridization region of at least about 9 nucleotides that is complementary in nucleotide sequence to at least a portion of a target NHE1 mRNA and a catalytic region suitable for cleaving the target NHE1 mRNA (see generally, European Patent No. 0 321 201; WO 88 / 04300; Haseloff, J. et al., Nature 334:585-591, 1988; Fedor, MJ et al., Proc. Natl. Acad. Sci. USA 87:1668-1672, 1990; Cech, TR et al., Ann. Rev. Biochem. 55:599-629, 1986).

[0107] Ribozymes can be targeted directly to cells either in the form of RNA oligonucleotides incorporating the ribozyme sequence, or can be introduced into cells as expression vectors encoding the desired ribozyme RNA. Ribozymes can be used and applied in much the same manner as described for antisense polynucleotides.

[0108] Antisense RNA and DNA, RNAi molecules, ribozymes and DNA enzymes for the inventive method can be prepared by any method known in the art for the synthesis of DNA molecules and RNA molecules. These include the technology known in the art for chemical synthesis of oligodeoxyribonucleotides and oligoribonucleotides, such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be produced by in vitro and in vivo transcription of the DNA sequence encoding the antisense RNA molecule. Such DNA sequence can be incorporated into numerous vectors, and these vectors have incorporated suitable RNA polymerase promoters such as T7 or SP6 polymerase promoters. Alternatively, according to the promoter used, the antisense cDNA constructs of the synthetic antisense RNA constitutively or inductively can be stably introduced into the cell line.

[0109] Various well-known modifications of DNA molecules can be introduced as a means of improving stability and half-life. Useful modifications include, but are not limited to, adding flanking sequences of ribonucleotides and deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or using phosphorothioate or 2'O-methyl bonds rather than phosphodiester bonds within the oligodeoxyribonucleotide backbone.

[0110] Protein Degraders

[0111] Protein degradation technologies also reduce or inhibit NHE1 activity. Many technologies are in use, such as protein degradation targeting chimeras (PROTACs), which are bispecific molecules comprising a target protein binder (here NHE1) and a ubiquitin ligase binder, the two binders are connected by a linker or other degradation technology, and the molecules include PROTACs, SNIPERs, HaloPROTACs, HyTs, LYTACs, AUTACs, ATTECs, RIBOTECs, monomeric degraders, dual-mechanism degraders, SARDs, TF-PROTACs, dual PROTACs, SERDs, bispecific aptamer chimeras, AbTACs, GlueTACs, AUTOTACs, CMA-based degraders, MADTACs, ATACs, molecular glues, biodegraders, biodegradable protein mRNAs, biodegradable protein mRNAs in lipid nanoparticle formulations, all targeting NHE1 (e.g., Li, H. et al., J Hematol Oncol 14(1): 138, 2021).

[0112] Gene therapy

[0113] Modification, complementation, replacement or deletion of the NHE1 gene in the selected tissue will also result in inhibition of the corresponding NHE1 activity.

[0114] The present invention provides a method for treating a disease associated with chronic fatigue (DACF) by administering to a patient in need thereof an amount of an NHE1 inhibitor effective to inhibit the transport activity of NHE1, wherein such inhibition of NHE1 activity is the primary therapeutic mode of action of the NHE1 inhibitor in treating DACF.

[0115] For each of the NHE1 inhibitory chemical compounds used in the method of the present invention, all possible stereoisomers and geometric isomers are included. The compounds include not only racemic compounds, but also optically active isomers. When the NHE1 inhibitor is desired as a single enantiomer, it can be obtained by resolving the final product or by stereospecific synthesis from isomerically pure starting materials or using chiral auxiliary reagents, for example, see Ma, Z. et al., Tetrahedron: Asymmetry 8 (6): 883-888, 1997. The resolution of the final product, intermediate, or starting material can be completed by any suitable method known in the art. In addition, in the case where the tautomer of the compound can be used, the present invention is intended to include all tautomeric forms of the compound.

[0116] NHE1 inhibitors comprising an acidic moiety can form pharmaceutically acceptable salts with suitable cations. Suitable pharmaceutically acceptable cations include alkali metals (e.g., sodium or potassium) and alkaline earth metals (e.g., calcium or magnesium) cations. Pharmaceutically acceptable salts of NHE1 inhibitors (which contain a basic center) are acid addition salts formed with pharmaceutically acceptable acids. Examples include hydrochlorides, hydrobromides, sulfates or bisulfates, phosphates or hydrogen phosphates, acetates, benzoates, succinates, fumarates, maleates, lactates, citrates, tartrates, gluconates, methanethiocyanates (methanefulgonate), benzenesulfonates and p-toluenesulfonates. In view of the foregoing, any reference to compounds useful in the methods of the present invention that appears in this application is intended to include NHE1 inhibitors, and pharmaceutically acceptable salts and solvates thereof.

[0117] The compounds of the present invention can be administered therapeutically as pure chemicals, but it is preferred that the NHE1 inhibitors be administered as pharmaceutical compositions or formulations. Therefore, the present invention further provides pharmaceutical compositions or formulations comprising: NHE1 inhibitors, or pharmaceutically acceptable salts thereof, together with one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients and / or preventive ingredients. Suitable carriers are compatible with the other ingredients of the formulation and are harmless to the recipient. The compounds of the present invention can also be carried in a delivery system to provide sustained release of the compound or to enhance the absorption or activity of the compound, such as liposomes or hydrogel systems for injection, microparticles, nanoparticles or micelle systems for oral or parenteral delivery, or graded capsule systems for oral delivery.

[0118] Example

[0119] The following examples describe the use of some compounds according to the present invention. These examples are not limiting and are merely illustrative of the present invention.

[0120] Example 1 - In vitro Model of More Residual Muscle Force (Less Fatigue) by Inhibiting Sodium Overload and Detrimental Cellular Calcium Changes and Skeletal Muscle Calcium Overload

[0121] Model Introduction

[0122] The patent claims that NHE1 inhibitors can improve the physical dysfunction and symptoms of ME / CFS, such as disorders associated with chronic fatigue (DACF). Muscle fatigue and weakness (e.g., loss of grip strength) are hallmarks of ME / CFS patients. The primary target of NHE1 inhibition is skeletal muscle, whose energetic function is severely disturbed in ME / CFS and is presumed to be very important in the symptomatology of ME / CSF. NHE1 activity plays a key role in the emergence of a disorder of elevated intramuscular sodium that ultimately leads to calcium overload. The latter can lead to profound functional energetic disorders or damage. Isolated rat muscle (either soleus or extensor digitorum longus) is a well-characterized and well-validated model for studying the function of ion exchangers. Isolated rat soleus muscle is particularly suitable for in vitro experiments because it is very thin, so that no blood supply is required. It has been demonstrated under different experimental conditions that cellular sodium ion output reduces the decrease in contractile force that occurs after measures such as the administration of carbachol or high ion concentrations (e.g., 12.5 mM KCl) or high extracellular NaCl concentrations that worsen the energy situation or cause cellular sodium overload during electrical stimulation. In this experimental setting, high NaCl concentration buffers can cause sodium influx to interfere with and reduce excitability and interfere with calcium homeostasis, thereby subsequently reducing muscle strength. Inhibition of NHE1 (i.e., exchanging one proton for one sodium ion uptake) reduces sodium entry and overload. The prevention and reduction of the loss of muscle strength in this process indicates that the test compound with the described NHE1 inhibition mode of action effectively inhibits NHE1.

[0123] The decrease in force in the soleus muscle can be seen as muscle fatigue and is associated with the loss of grip muscle strength in ME / CFS patients. The in vitro model is suitable for testing the effects of NHE1 inhibitors, particularly their effects on force production that is disturbed by mechanisms that deteriorate excitability or energy status or calcium homeostasis via the sodium-calcium exchanger. High NaCl (200 mM) in the buffer is used to cause an intracellular sodium load, resulting in a decrease in contractile force. After a period of exposure to high NaCl buffer, In physiological extracellular NaCl Concentration Electrical stimulation was performed showing a decrease in force values.

[0124] The resulting decrease in muscle strength is considered muscle fatigue. In the absence of electrical stimulation to prevent sodium entry through sodium channels, sodium influx during high NaCl exposure is due to NHE1. Inhibition of NHE1 prevents sodium overload and the decrease in muscle strength and fatigue.

[0125] method

[0126] Muscle preparation and culture: 5-week-old female Sprague Dawley rats were used. After isoflurane anesthesia and cervical dislocation, the intact soleus muscle of the animals was dissected. The soleus muscle was vertically mounted between two field stimulation electrodes (thick copper wire) at rest length in an organ bath containing 15 mL of culture medium. The lower end of the muscle was fixed to a clamp fixed to the bottom of the organ bath, and the upper end of the muscle was fixed to an isometric force sensor (force sensor K300 HugoSachs) through a clamp that allows the measurement of isometric contractions, and a tension of 100 mN was applied.

[0127] Force Measurement: The experimental setup for force measurement allows data from 6 muscles to be recorded in parallel simultaneously in separate culture chambers. Force values ​​were recorded and stored using the Notocord HEM data acquisition system. The standard culture medium was Krebs-Ringer (KR) bicarbonate buffer (pH 7.4), which contained (in mM): 120.1 NaCl, 25.1 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.3 CaCl2, and 5 glucose. The culture was carried out at 30°C. The buffer was continuously aerated with a mixture of 95% O2 and 5% CO2. Before stimulation, the muscles were equilibrated in standard culture medium with a mixture of 95% oxygen and 5% carbon dioxide for 30 minutes (min).

[0128] Electrical stimulation protocol: After a 30-min equilibration period, the muscles were field stimulated using a Hugo Sachs stimulator (HSE) at 83 Hz, 2-3 ms pulses, 12 V (biphasic), 0.2-0.5 s training width to deliver optimal force values. After optimizing the stimulation protocol, the muscles were stimulated every 10 minutes for 30 minutes in the above buffer to obtain control force values, and then the contractile force was weakened by exposure to high NaCl in the buffer.

[0129] Study of test compounds: At time 0 (T0), the above buffer was exchanged with a buffer containing NaCl 200 mM. No electrical stimulation was performed in the next 30 minutes. The buffer was then exchanged for the initial buffer with physiological NaCl concentration and electrical stimulation was performed 1 minute later. The high NaCl buffer resulted in a strong loss of force values ​​in the control group.

[0130] 10 minutes before exchanging the initial buffer for a new buffer with 200 mM high NaCl (10 minutes before T0), a volume of 0.1 mL of NHE1 inhibitor was added to the organ bath containing 15 mL of buffer solution and added freshly to the buffer containing physiological NaCl after the exchange. Electrical stimulation was then performed. Electrical stimulation was performed only in the presence of physiological NaCl concentrations; the buffer containing high NaCl was used only to load the cells with sodium. The control group received only buffer instead of NHE1 inhibitor.

[0131] evaluate

[0132] High NaCl 200mM causes a strong loss of force value in the control group. Effective drugs will show a lower loss of contractile force, thus maintaining contractile force. The residual contractile force after 30min in high NaCl buffer is selected as the efficacy parameter and is related to the final value of the force stimulated in the initial buffer in the same organ bath. The percentage of the residual force value between the different groups is compared and used for statistical calculations.

[0133] result

[0134] As an illustrative and non-limiting example, the NHE1 inhibitor compounds according to the invention were tested according to the above protocol and delayed the decrease in soleus muscle force values ​​in rats, thus demonstrating their advantage as therapeutically active substances.

[0135] Under the influence of high extracellular sodium concentrations (200 mM), soleus muscle force values ​​dropped significantly to 10% to 20% of the force values ​​measured under baseline normal sodium concentrations. The NHE1 inhibitors cariporide and ramipril at drug concentrations of 30 to 500 nM reduced the loss of force values, thus showing that muscle strength retained 50-70% of the residual force values ​​compared to their baseline controls.

[0136] in conclusion:

[0137] NHE1 inhibitors reduce muscle fatigue by preserving contractile force under conditions of sodium overload caused by high NaCl buffer. The mechanism is that NHE1 inhibition reduces sodium input and sodium overload, while Na + / K + ATPase still exports some sodium. Based on these effects, NHE1 inhibitors could treat muscle fatigue and strength loss.

[0138] In addition, since NHE1 inhibition reduces intracellular sodium (high intramuscular sodium causes calcium overload via the reverse mode of the sodium calcium exchanger (NCX), causing muscle damage and mitochondrial dysfunction), NHE1 inhibitors can treat and prevent the harmful secondary consequences of mitochondrial dysfunction and energy depletion. This depletion would otherwise lead to physiological stimulation of vasoactive mediators from skeletal muscle, which are usually physiologically meant to increase muscle blood flow. These mediators are then overproduced due to the very poor energy situation and are therefore released into the systemic blood flow (osmosis). Therefore, these mediators with pain, hyperalgesia, spasm-inducing and microvascular leakage-inducing properties can reach every organ to cause the various symptoms of diseases associated with chronic fatigue. These symptoms include pain, edema and spasms, which are prevented by treatment involving NHE1 inhibition in the form of indirect effects.

Claims

1. A substance having NHE1 inhibitory activity (NHE1 inhibitor) for use as an active ingredient in a therapeutic agent having sodium-hydrogen antiporter 1 inhibitory activity for the treatment and prevention of chronic fatigue, exhaustion and / or exertion intolerance, and for the treatment and prevention of diseases associated with chronic fatigue, exhaustion and / or exertion intolerance.

2. The substance according to claim 1, wherein the disease associated with chronic fatigue, exhaustion and / or exertion intolerance is a disease diagnosed as: myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS), systemic exertion intolerance, exertion intolerance, post-vac-syndrome after vaccination against viruses and pathogenic agents, post-infectious fatigue after viral, bacterial, or fungal infection, excluding Covid-19 sequelae, long Covid and post-Covid-19 syndrome.

3. The agent of claim 1, wherein chronic fatigue and exhaustion is a symptom of or is associated with cancer (cancer-related fatigue), fibromyalgia, Ehlers-Danlos syndrome, Marfan syndrome, Gulf War illness, the autoimmune diseases rheumatoid arthritis, ANCA vasculitis and Sjögren's syndrome, and other autoimmune diseases with fatigue and exhaustion as debilitating symptoms.

4. The substance according to one or more of claims 1 to 3, wherein the NHE1 inhibitory substance is in the form of IC 50 <50μM, preferably IC 50 ≤1μM, more preferably IC 50 0.1 to 600 nM, particularly preferably IC 50 The range is about 0.2 to about 100 nM, and more preferably, the range is about 0.2 to about 100 nM. 50 A substance that inhibits the transport activity of NHE1 at about 1 to about 100 nM.

5. The substance according to one or more of claims 1 to 4, wherein the NHE1 inhibitor inhibits sodium overload and detrimental cellular calcium changes and skeletal muscle calcium overload.

6. Substances according to one or more of claims 1 to 5, wherein the NHE1 inhibitor is present in a pharmaceutical preparation, preferably in the form of an oral medication, intravenous, subcutaneous, intramuscular, pharyngeal or nasal administration.

7. The substance according to one or more of claims 1 to 6, wherein the NHE1 inhibitor is a chemical compound, a protein or polypeptide, a nucleic acid molecule, a ribozyme, a DNA enzyme, a protein degrading agent, or a material for gene therapy.

8. The substance according to claim 7, wherein the chemical compound is a small molecule inhibitor, including natural substances and synthetic substances with low molecular weight, such as peptides, peptidomimetics and non-peptide inhibitors, such as classical small molecule chemical compounds, preferably with a molecular weight of less than about 750 g / mol and with an IC 50 For IC 50 <50μM, preferably IC 50 ≤1μM, more preferably IC 50 0.1 to 600 nM, particularly preferably IC 50 The range is about 0.2 to about 100 nM, and more preferably, the range is about 0.2 to about 100 nM. 50 Small molecule inhibitors that inhibit NHE1 activity at about 1 to about 100 nM.

9. The substance according to claim 7, wherein the NHE1 inhibitor is a protein or polypeptide, including an NHE1 binding antibody, a nanobody, or a functionally related protein or protein fragment or fusion protein, including a single-chain variable fragment or a designed ankyrin repeat domain protein or a lipocalin / anticalin.

10. The substance according to claim 7, wherein the NHE1 inhibitor inhibits NHE1 expression and is an antisense nucleic acid molecule such as an antisense oligonucleotide, antisense RNA, antisense mRNA, antisense DNA, or an NHE1 RNAi molecule, siRNA, microRNA or other such molecules, or an NHE1 altering ribozyme, or an NHE1 altering DNA enzyme.

11. The substance according to claim 7, wherein the NHE1 inhibitor is a protein degrader, such as PROTAC, SNIPER, HaloPROTAC, HyT, LYTAC, AUTAC, ATTEC, RIBOTEC, monomer degrader, dual mechanism degrader, SARD, TF-PROTAC, dual PROTAC, SERD, bispecific aptamer chimera, AbTAC, GlueTAC, AUTOTAC, CMA-based degrader, MADTAC, ATAC, molecular glue, biodegrader, mRNA of biodegradable protein, mRNA of biodegradable protein in lipid nanoparticle formulation, all targeting NHE1. 12 . The substance according to claim 7 , wherein the NHE1 inhibitor is a gene therapy for modifying, complementing, replacing, or deleting the NHE1 gene.

13. The substance according to one or more of claims 1 to 12, wherein the NHE1 inhibitor is a member selected from the group consisting of substances comprising the structural units benzoyl-guanidine (based on a benzene ring), pyrazinoyl-guanidine (based on a pyrazine ring), (piperidin-1-yl)pyrimidine, aminophenoxazine, pyrrolidine, and piperidine.

14. The substance according to one or more of claims 1 to 13, wherein the NHE1 inhibitor is a member selected from the group consisting of ramipril, cariporide, eniporide, amiloride, EIPA (5-(N-ethyl-N-isopropyl) amiloride), DMA (5-(N,N-dimethyl) amiloride), MIBA (5-(N-methyl-N-isobutyl) amiloride), HMA (5-(N,N-(hexamethylene) amiloride), 2 -aminophenoxazine-3-one, 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one, zonipoli, 5-(4-fluoro-3-methylphenyl)-2-methoxy-4-[4-(5-methyl-1H-imidazol-4-yl)piperidin-1-yl]pyrimidine, and 3-[(cyclopropylcarbonyl)amino]-N-[2-(dimethylamino)ethyl]-4-[4-(4-methyl-1H-imidazol-5-yl)-1-piperidinyl]benzamide.

15. A pharmaceutical composition, characterized in that It comprises at least one NHE1 inhibitor according to one or more of claims 1 to 14 .

16. The pharmaceutical composition according to claim 15, characterized in that It comprises (i) at least one NHE1 inhibitor according to claim 8, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, polymorph, ester, ether, enantiomer, prodrug, or metabolite thereof, and at least one pharmaceutically acceptable diluent or excipient, or (ii) at least A compound of the formula according to claim 13 or claim 14, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, polymorph, ester, ether, enantiomer, prodrug, or metabolite thereof, and at least one pharmaceutically acceptable diluent or excipient.

17. Use of a compound having sodium-hydrogen antiporter 1 inhibitory activity (NHE1 inhibitor) for preparing a medicament for treating and preventing chronic fatigue, exhaustion and / or exertion intolerance, preferably, the NHE1 inhibitory compound is expressed as IC 50 <50μM, preferably IC 50 ≤1μM, more preferably IC 50 0.1 to 600 nM, particularly preferably IC 50 From about 0.2 to about 100 nM, and more preferably from about IC 50 The transport activity of NHE1 is inhibited at about 1 to about 100 nM.

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