WO2023238126A1

WO2023238126A1 - Composition and method for use thereof for treatment and prevention of autoimmune and neurological disorders

Info

Publication number: WO2023238126A1
Application number: PCT/IL2023/050582
Authority: WO
Inventors: Shmuel Levit; Liudmila KOSHELEVA; Daniil KOSHELEV; Elyahu Meir TEILER
Original assignee: Levicure Ltd
Priority date: 2022-06-07
Filing date: 2023-06-06
Publication date: 2023-12-14

Abstract

The present subject matter provides a composition for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, the composition comprising: at least one first agent configured to cause immunomodulation through inhibition of DPP4 enzyme and activation of glucagon-like peptide- 1 receptor (GLP- 1R) in neurological and immune cells; at least one second agent configured to reduce stomach acidity; and at least one third agent, comprising: Gamma aminobutyric acid GABA, or at least one GABA analogue, or at least one GABA receptor agonist, or any combination thereof. A method for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction is provided as well.

Description

COMPOSITION AND METHOD FOR USE THEREOF FOR TREATMENT AND

PREVENTION OF AUTOIMMUNE AND NEUROEOGICAE DISORDERS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to United States Provisional Patent Application No. 63/349,637, filed June 07, 2022, the entire contents of which is incorporated herein by reference in its entirety.

FIELD

[001] The present subject matter relates to treatment and prevention of autoimmune and neurological disorders. More particularly, the present subject matter relates to treatment and prevention of autoimmune and neurological disorders caused by Glutamate/Gamma aminobutyric acid (GABA) imbalance in cells.

BACKGROUND

[002] Neurological disorders are primarily caused by neurons and neurotransmission in the human brain and central nervous system (CNS). One type of neurological disorders is neurodegenerative disorders, which are incurable debilitating conditions that result in progressive degeneration of neurons that may result eventually in death of the neurons. Many neurological disorders are complex and can harbor a variety of characteristics such as damage to axons, synapses and nerve cell bodies, as well as neuro-inflammation and autoimmunity. Examples of neurological disorders include Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), Stiff-person syndrome (SPS), multiple sclerosis (MS) and the like.

[003] Most of the neurological disorders have been linked to a combination of genetic and environmental factors. While some genetic sequences have been known to increase the chance of certain disorders, in most cases the genetic influences on neurological and, more specifically, neurodegenerative disorders, are not well understood. [004] Neurons are nerve cells that communicate through a combination of electrical and chemical signals. Within a single neuron, charged particles drive electrical signals that allow rapid conduction through the cell. Neurons communicate at synapses where specialized parts of presynaptic and postsynaptic neurons allow for chemical transmission. The presynaptic neuron releases chemicals known as neurotransmitters that are received by the postsynaptic neuron's specialized proteins called neurotransmitter receptors. The neurotransmitter molecules affect the postsynaptic neuronal function through binding to the receptor proteins.

[005] Neuronal excitability is characterized by a balance between excitatory input of Glutamate and counterbalancing inhibitory transmission of GABA. During neuronal signaling a neuron is releasing the neurotransmitter at its synapse and the message is conveyed to the adjacent neuron though activation of specific receptors on its surface. [GABA and Glutamate: Their Transmitter Role in the central nervous system (CNS) and Pancreatic Islets (https://www.intechopen.com/chapters/57103), the entire contents of which is incorporated herein by reference] .

[006] Glutamate and GABA are major neurotransmitters in the brain and CNS. Inhibitory GABA and excitatory Glutamate work together to control many processes, including the brain’s overall level of excitation. A balanced interaction is required to maintain physiological homeostasis, while prolonged imbalance can lead to a variety of diseases.

[007] Glutamatergic/GABAergic imbalance can be found in autism spectrum disorders and anxiety disorders with elevated Glutamatergic neurotransmission, while high levels of GABA produce more relaxation and even sedation. Both Glutamatergic and GABAergic neurons are highly diversified in a variety of electrically excitable cells, thus maintaining appropriate and balanced Glutamate/GABA transmission is essential in controlling the level of excitation in the CNS.

[008] The glutamate/GABA balance can also be affected by autoimmunity and genetic disorders. Inhibitory GABA and excitatory Glutamate imbalance can be found in a variety of neurological disorders like epilepsy, seizures and even autism. For example, Huntington’s disease (HD), Temporal lobe epilepsy (TLE) and Parkinson’s disease (PD) are neurodegenerative disorders that have been proven to involve disruptions in GABAergic signaling [GABA signaling: therapeutic targets for epilepsy, Parkinson's disease and Huntington's disease (https://pubmed.ncbi.nlm.nih.gov/15992178/), the entire contents of which is incorporated herein by reference]. TLE seizures reflect excess excitation, which may result from local inhibitory circuit dysfunction. Parkinson’s disease (PD) destroys input to striatal GABAergic neurons and Huntington’s disease (HD) devastates striatal GABAergic neurons.

[009] Glutamate is the major excitatory neurotransmitter and, as such, it inevitably plays a role in optimizing and synchronizing cellular responses to react adequately to external stimuli. An inadequate signal transmission or abnormal response to it may result in the initiation and spread of seizure activity. Misbalance resulting in excess Glutamate is known to have catastrophic effects on the CNS as it induces excitotoxicity and significant loss of brain function [Glutamate, T-cells and multiple sclerosis

(https://pubmed.ncbi.nlm.nih.gov/28236206/), the entire contents of which is incorporated herein by reference}. Effects of glutamate are mediated by a large family of ionotropic and metabotropic glutamate receptors that are expressed by all cells of the nervous system as well as many non-neural and immune cells in various peripheral organs and tissues. In relation to multiple sclerosis (MS) it was found that Glutamate receptors have been highly expressed in immune cells of MS patients, and significantly over engaged during relapse and when there was established neurological evidence of disease activity [The neurotransmitter glutamate and human T-cells: glutamate receptors and glutamate-induced direct and potent effects on normal human T-cells, cancerous human leukemia and lymphoma T-cells, and autoimmune human T- cells (https://pubmed.ncbi.nlm.nih.gov/24584970/), the entire contents of which is incorporated herein by reference].

[010] In recent years, it has become clear that there is extensive cross-communication between the CNS and the immune system. GABA has several effects on the immune cells such as activation or suppression of cytokine secretion, modification of cell proliferation and even migration of the cells. The immune cells encounter GABA when released by the immune cells themselves, or when the immune cells enter the brain. GABA appears to have a role in autoimmune diseases like MS and rheumatoid arthritis and may modulate the immune response to infections. In the near future, it will be important to work out what specific effects GABA has on the function of the different types of immune cells and determine the underlying mechanisms revealing the role of GABA as an immunomodulator [GABA is an effective immunomodulatory molecule (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680704/), the entire contents of which is incorporated herein by reference]. [Oi l] Several types of Glutamate receptors have been recently discovered on the surface of T-cells that allow Glutamate’s role not only as neurotransmitter, but also as an immunomodulator [Role of glutamate on T-cell mediated immunity (https://www.researchgate.net/publication/6502722_Role_of_glutamate_on_T- cell_mediated_immunity), the entire contents of which is incorporated herein by reference]. More specifically, all types of T-cells including cancer and autoimmune human T-cells express functional ionotropic and metabotropic Glutamate receptors [The neurotransmitter glutamate and human T-cells: glutamate receptors and glutamate-induced direct and potent effects on normal human T-cells, cancerous human leukemia and lymphoma T-cells, and autoimmune human T-cells (https://pubmed.ncbi.nlm.nih.gov/24584970/), the entire contents of which is incorporated herein by reference]. The expressed Glutamate receptor subtypes are different for a variety of T-cells, including different expression of these receptors for resting T-cells in comparison to activated T-cells [The neurotransmitter glutamate and human T-cells: glutamate receptors and glutamate-induced direct and potent effects on normal human T-cells, cancerous human leukemia and lymphoma T-cells, and autoimmune human T-cells (https://pubmed.ncbi.nlm.nih.gov/24584970/), the entire contents of which is incorporated herein by reference] .

[012] Glutamate plays an exceptionally important role in the activation of many key T-cell functions including, but not limited to, adhesion, migration, proliferation, intracellular polarization through Ca²⁺ fluxes and outward K⁺ currents [Glutamate, T-cells and multiple sclerosis (https://pubmed.ncbi.nlm.nih.gov/28236206/), the entire contents of which is incorporated herein by reference]. Once T-cells are activated, Glutamate also protects them from antigen-induced apoptotic cell death, drastically influencing their function and survival [The neurotransmitter glutamate and human T-cells: glutamate receptors and glutamate- induced direct and potent effects on normal human T-cells, cancerous human leukemia and lymphoma-T-cells, and autoimmune human-T-cells

(https://pubmed.ncbi.nlm.nih.gov/24584970/), the entire contents of which is incorporated herein by reference] .

[013] Thus, it is expected that Glutamate plays a key role in pathogenic mechanisms of T-cell diseases. In relation to MS it was found that Glutamate receptors have been highly expressed in T-cells of MS patients, and significantly over engaged during relapse and when there was established neurological evidence of disease activity [The neurotransmitter glutamate and human T-cells: glutamate receptors and glutamate-induced direct and potent effects on normal human T-cells, cancerous human leukemia and lymphoma T-cells, and autoimmune human T- cells (https://pubmed.ncbi.nlm.nih.gov/24584970/), the entire contents of which is incorporated herein by reference]. Interestingly, Glutamate or a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptors increase the proliferation of autoreactive T-cells in response to myelin proteins in MS. It was also found that Glutamate is a highly effective initiator of the T-cell mediated immune response during the contact between T-cells and dendritic cells [Glutamate released by dendritic cells as a novel modulator of T-cell activation (https://pubmed.ncbi.nlm.nih.gov/17082582/), the entire contents of which is incorporated herein by reference]. Glutamate transmission cycle in relation to T-cell response, activation and proliferation also seem to be involved in autoimmune rheumatoid arthritis and Systemic lupus erythematosus (SLE) [The neurotransmitter glutamate and human T-cells: glutamate receptors and glutamate-induced direct and potent effects on normal human T-cells, cancerous human leukemia and lymphoma T-cells, and autoimmune human T-cells (https://pubmed.ncbi.nlm.nih.gov/24584970/), the entire contents of which is incorporated herein by reference] .

[014] GABA is a principal inhibitory neurotransmitter in the CNS, maintaining an inhibitory tone counterbalancing neuronal excitation and playing an important role in muscle tone regulation [Anti-glutamic acid decarboxylase antibody positive neurological syndromes (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107286/), the entire contents of which is incorporated herein by reference]. GABA plays a major role in a variety of body functions, and while its significance in many processes is still unclear, it can be found in varying low concentrations throughout the body and in all organ systems.

[015] GABAergic neurons are responsible for GABA production in the CNS and are part of a large network of inhibitory interneurons responsible for inhibitory signals throughout the CNS. GABAergic neurons in the CNS are primarily located in the hippocampus, the cerebellum, basal ganglia, brainstem nuclei, and spinal gray matter [Neurological Syndromes Associated with Anti-GAD Antibodies

(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279468/), the entire contents of which is incorporated herein by reference]. Because GABA modulates most inhibition that is ongoing in the brain, perturbations in GABAergic inhibition have the potential to result in seizures. Therefore, the most common disorder in which GABA is targeted as a treatment is epilepsy. However, other disorders such as psychiatric disease, spasticity, and Stiff-person syndrome (SPS) all have been related to disorders of GABAergic function in the brain. When levels of GABA are abnormally low, the firing frequency of nerve cells increases, and leads, in its turn, to over-excitation and a variety of conditions including anxiety and seizure disorders [Anti- glutamic acid decarboxylase antibody positive neurological syndromes (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107286/), the entire contents of which is incorporated herein by reference] . There are known neurological and cognitive disorders that are directly associated with decreasing or critically low levels of GABA, including cerebellar ataxia and limbic encephalitis along with epilepsy [Anti-glutamic acid decarboxylase antibody positive neurological syndromes (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107286/), the entire contents of which is incorporated herein by reference], anxiety disorders, schizophrenia, SPS, and premenstrual dysphoric disorder [GABA, gamma-hydroxybutyric acid, and neurological disease (https://pubmed.ncbi.nlm.nih.gov/12891648/), the entire contents of which is incorporated herein by reference].

[016] Due to the inhibitory role of GABA, it is well established that modulation of GABA signaling is the basis of many pharmacologic treatments in neurology, psychiatry, and anesthesia [Physiology, GABA (https://www.ncbi.nlm.nih.gov/books/NBK513311/), the entire contents of which is incorporated herein by reference] . There is extensive research that indicates an important role played by GABA transmission in the mechanism and treatment of epilepsy, including abnormalities of GABAergic function observed in genetic and acquired animal models of epilepsy. Additional research of human epileptic brain tissue and cerebrospinal fluid have also outlined reduced GABA-mediated inhibition and excessive activity of Glutamate [GABAergic mechanisms in epilepsy (https://pubmed.ncbi.nlm.nih.gov/11520315/), the entire contents of which is incorporated herein by reference]. This is consistent with findings that GABA agonists suppress seizures while GABA antagonists produce seizures; medicaments that inhibit GABA synthesis cause seizures; and medicaments that increase synaptic GABA are potent anticonvulsants [Molecular mechanisms of antiseizure drug activity at GABAA receptors (https://www. seizure - joumal.com/article/S1059- 1311(13)00114-3/fulltext), the entire contents of which is incorporated herein by reference].

SUMMARY [017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present subject matter, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[018] According to one aspect of the present subject matter, there is provided a composition for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, the composition comprising: at least one first agent configured to cause immunomodulation through inhibition of DPP4 enzyme and activation of glucagon-like peptide- 1 receptor (GLP-1R) in neurological and immune cells; at least one second agent configured to reduce stomach acidity; and at least one third agent, comprising: Gamma aminobutyric acid (GABA), or at least one GABA analogue, or at least one GABA receptor agonist, or any combination thereof.

[019] According to another aspect of the present subject matter, there is provided a method for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, in a patient, the method comprising: administering to the patient at least one first agent configured to cause immunomodulation through inhibition of DPP4 enzyme and activation of glucagon-like peptide- 1 receptor (GLP-1R) in neurological and immune cells; administering to the patient at least one second agent configured to reduce stomach acidity; and administering to the patient at least one third agent, comprising: Gamma aminobutyric acid (GABA), or at least one GABA analogue, or at least one GABA receptor agonist, or any combination thereof.

[020] The composition of claim 1, wherein the at least one first agent is at least one Dipeptidyl peptidase-4 (DPP-4) inhibitor. [021] According to yet another aspect of the present subject matter, there is provided a composition for use in the treatment and prevention of a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, in a patient, the treatment and prevention comprising: administering to the patient at least one first agent configured to cause immunomodulation through inhibition of DPP4 enzyme and activation of glucagon-like peptide- 1 receptor (GLP-1R) in neurological and immune cells; administering to the patient at least one second agent configured to reduce stomach acidity; and administering to the patient at least one third agent, comprising: Gamma aminobutyric acid (GABA), or at least one GABA analogue, or at least one GABA receptor agonist, or any combination thereof.

[022] According to one embodiment, the at least one DPP-4 inhibitor is configured to be administered orally at a daily dose in a range of substantially 1 to 400 mg.

[023] According to one embodiment, the at least one DPP-4 inhibitor is configured to be administered orally once daily.

[024] According to one embodiment, the at least one DPP-4 inhibitor is in a form of a tablet, or a capsule, or a powder, or granules, or an oral liquid.

[025] According to one embodiment, the at least one DPP-4 inhibitor is selected from the group consisting of: Alogliptin, Linagliptin, Saxagliptin, Sitagliptin, Vildagliptin, or any combination thereof.

[026] According to one embodiment, the at least one DPP-4 inhibitor is Sitagliptin.

[027] According to one embodiment, the Sitagliptin is configured to be administered orally in an amount in a range of substantially 25-400 mg once daily, or in a range of substantially 12.5-200 mg twice daily.

[028] According to one embodiment, the Sitagliptin is configured to be administered orally in an amount in a range of substantially 25-100 mg once daily, or substantially 12.5-50 mg twice daily. [029] According to one embodiment, the at least one DPP-4 inhibitor is Saxagliptin.

[030] According to one embodiment, the Saxagliptin is configured to be administered orally in a range of substantially 1.25-5 mg once daily, or in an amount of substantially 2.5 mg once daily, or in a range of substantially 0.625-2.5 mg twice daily, or substantially 1.25 mg twice daily.

[031] According to one embodiment, the Saxagliptin is configured to be administered orally in a range of substantially 2.5-10 mg once daily, or in an amount of substantially 5 mg once daily, or in a range of substantially 1.25-5 mg twice daily, or in an amount of substantially 2.5 mg twice daily.

[032] According to one embodiment, at least one DPP-4 inhibitor is Linagliptin.

[033] According to one embodiment, the Linagliptin is configured to be administered orally in a range of substantially 2.5-10 mg once daily, or an amount of substantially 5 mg once daily, or in a range of substantially 1.25-5 mg twice daily, or in an amount of substantially 2.5 mg twice daily.

[034] According to one embodiment, the at least one DPP-4 inhibitor is Alogliptin.

[035] According to one embodiment, the Alogliptin is configured to be administered orally in a range of substantially 12.5-50 mg once daily, or in an amount of substantially 25 mg once daily, or in a range of substantially 6.25-25 mg twice daily, or in an amount of or substantially 12.5 mg twice daily.

[036] According to one embodiment, the Alogliptin is configured to be administered orally in a range of substantially 6.25-25 mg once daily, or in an amount of substantially 12.5 mg, or in a range of substantially 3.125-12.5 mg twice daily, or in an amount of substantially 6.25 mg twice daily.

[037] According to one embodiment, the Alogliptin is configured to be administered orally in a range of substantially 3.125-12.5 mg once daily, or in an amount of substantially 6.25 mg once daily, or in a range of substantially 1.56-3.25 mg twice daily, or in an amount of substantially 3.125 mg twice daily. [038] According to one embodiment, the at least one first agent is at least one (GLP-1R) agonist.

[039] According to one embodiment, the GLP-1R agonist is in a form of a pharmaceutical composition for parenteral administration.

[040] According to one embodiment, the at least one GLP-1R agonist is in a form of tablets configured to be orally administered to a patient.

[041] According to one embodiment, a formulation of the at least one GLP-1R agonist comprises an insulinotropic GLP-1 compound, a basal insulin peptide and pharmaceutically acceptable additives.

[042] According to one embodiment, the at least one GLP-1R agonist is a short time-acting Exenatide.

[043] According to one embodiment, the short time-acting Exenatide is configured to be administered in an amount of substantially 5 pg subcutaneously twice daily within substantially 1 hour before meals.

[044] According to one embodiment, a dose of the short time-acting Exenatide is increased to substantially 10 pg twice daily after substantially 1 month of therapy.

[045] According to one embodiment, the at least one GLP-1R agonist is Lixisenatide.

[046] According to one embodiment, the Lixisenatide is configured to be administered initially during 14 days in an amount of substantially 10 pg subcutaneously once daily within substantially 1 hour before a first meal of a day, and on day 15, the amount of Lixisenatide is increased to substantially 20 pg once daily.

[047] According to one embodiment, the at least one GLP-1R agonist is an intermediateacting Liraglutide.

[048] According to one embodiment, the intermediate- acting Liraglutide is configured to be administered initially in an amount of substantially 0.6 mg daily for one week. [049] According to one embodiment, after the administration of the intermediate-acting Liraglutide in an amount of substantially 0.6 mg daily for one week, the intermediate -acting Liraglutide is configured to be administered in an amount of substantially 1.2 mg daily for at least one week.

[050] According to one embodiment, after the administration of the intermediate-acting Liraglutide in an amount of substantially 1.2 mg daily for at least one week, the intermediateacting Liraglutide is configured to be administered in an amount of substantially 1.8 mg daily for at least one week.

[051] According to one embodiment, after the administration of the intermediate-acting Liraglutide in an amount of substantially 1.8 mg daily for at least one week, the intermediateacting Liraglutide is configured to be administered in an amount of substantially 2.4 mg daily for at least one week.

[052] According to one embodiment, after the administration of the intermediate-acting Liraglutide in an amount of substantially 2.4 mg daily for at least one week, the intermediateacting Liraglutide is configured to be administered in an amount of substantially 3.0 mg daily for at least one week.

[053] According to one embodiment, the at least one GLP-1R agonist is a long-acting Exenatide weekly (QW).

[054] According to one embodiment, the Exenatide QW is configured to be administered in an amount of substantially 2 mg subcutaneously once every 7 days.

[055] According to one embodiment, the Exenatide QW is configured to be administered at any time of day, with or without meals.

[056] According to one embodiment, the at least one GLP-1R agonist is Albiglutide.

[057] According to one embodiment, the Albiglutide is configured to be administered in an amount of substantially 30 mg subcutaneously once weekly.

[058] According to one embodiment, the Albiglutide is configured to be administered in an amount of substantially 50 mg subcutaneously once weekly. [059] According to one embodiment, the at least one GLP-1R agonist is Dulaglutide.

[060] According to one embodiment, the Dulaglutide is configured to be administered in an amount of substantially 0.75 mg subcutaneously once weekly.

[061] According to one embodiment, the Dulaglutide is configured to be administered in an amount of substantially 1.5 mg subcutaneously once weekly.

[062] According to one embodiment, the at least one GLP-1R agonist is Semaglutide.

[063] According to one embodiment, the Semaglutide is configured to be firstly administered in an amount of substantially 0.25 mg, by subcutaneous injection, once weekly for 4 weeks.

[064] According to one embodiment, after the administration of the Semaglutide in an amount of substantially 0.25 mg, by subcutaneous injection, once weekly for 4 weeks, the Semaglutide is configured to be administered in an amount of substantially 0.5 mg, by subcutaneous injection, once weekly for at least 4 weeks.

[065] According to one embodiment, after the administration of the Semaglutide in an amount of substantially 0. 5 mg, by subcutaneous injection, once weekly for 4 weeks, the Semaglutide is configured to be administered in an amount of substantially 1.0 mg, by subcutaneous injection, once weekly for at least 4 weeks.

[066] According to one embodiment, the Semaglutide is configured to be administered by subcutaneous injection, once weekly, according to a dose escalation schedule.

[067] According to one embodiment, the Semaglutide is configured to be firstly administered orally in an amount in a range of substantially 0.1-3 mg once daily for 30 days.

[068] According to one embodiment, after the administration of the Semaglutide in an amount in a range of substantially 0.1-3 mg once daily for 30 days, the Semaglutide is configured to be orally administered in an amount in a range of substantially 0.2-7 mg once daily, for 30 days.

[069] According to one embodiment, after the administration of the Semaglutide in an amount in a range of substantially 0.2-7 mg once daily for 30 days, the Semaglutide is configured to be orally administered in an amount in a range of substantially 0.4-14 mg once daily. [070] According to one embodiment, the at least second agent is selected from the group consisting of: at least one proton pump inhibitor (PPI), or at least one H2-receptor antagonist, or at least one Bombesin-like peptide, or any combination thereof.

[071] According to one embodiment, the at least on PPI is in a pharmaceutically acceptable formulation.

[072] According to one embodiment, the at least one PPI is in a form of tablets, or delayed- release tablets, or capsules, or delayed-release capsules, or granules, or delayed-release granules, or injection.

[073] According to one embodiment, the at least one PPI is configured to be administered by intravenous (IV) injection.

[074] According to one embodiment, the at least one PPI is selected from the group consisting of: Omeprazole, Pantoprazole, Lansoprazole, Rabeprazole, Esomeprazole, or any combination thereof.

[075] According to one embodiment, the at least one PPI is Omeprazole.

[076] According to one embodiment, the Omeprazole is configured to be administered to a patient in a form of delayed-release oral tablets, or delayed-release oral capsules, or delayed- release oral granules, which can be swallowed as-is, with liquid, or mixed in a food or liquid to be swallowed.

[077] According to one embodiment, the Omeprazole is configured to be administered in a range of substantially 10 to 120 mg daily.

[078] According to one embodiment, the at least one PPI is Lansoprazole.

[079] According to one embodiment, the Lansoprazole is configured to be administered in a range of substantially 15-60 mg daily.

[080] According to one embodiment, the at least one PPI is Pantoprazole.

[081] According to one embodiment, the Pantoprazole is configured to be administered in a range of substantially 20 to 80 mg daily. [082] According to one embodiment, the at least one PPI is Esomeprazole.

[083] According to one embodiment, the Esomeprazole is configured to be administered in a range of substantially 20 to 240 mg daily.

[084] According to one embodiment, the at least one H2-receptor antagonist is selected from the group consisting of: Cimetidine, Ranitidine, Nizatidine, Famotidine, or any combination thereof.

[085] According to one embodiment, the at least one Bombesin-like peptide is a gastrinreleasing peptide (GRP), or gastrin-releasing peptide receptor agonist, or a combination thereof.

[086] According to one embodiment, the at least one third agent is configured to be administered to a patient in a daily dose of up to substantially 3000 mg.

[087] According to one embodiment, the at least one third agent is configured to be administered as capsules, or tablets, or oral powder, or oral granules.

[088] According to one embodiment, the at least one third agent is configured to allow modification of absorption of GABA by increasing bioavailability or GABA.

[089] According to one embodiment, the at least one third agent is an extended-release formulation of GABA.

[090] According to one embodiment, the at least one third agent is in a form of GABA producing bacteria.

[091] According to one embodiment, an amount of the GABA that is produced by the GABA producing bacteria in up to substantially 3,000 mg daily.

[092] According to one embodiment, the at least one GABA receptor agonist is selected from the group consisting of: Baclofen, Aminophenyl butyric acid, Hopantenic acid, Zolpidem, or any combination thereof.

[093] According to one embodiment, the at least one third agent is at least one anti-epileptic medication. [094] According to one embodiment, the at least one anti-epileptic medication is selected from the group consisting of: Valproic acid, Valproate sodium, Topiramate, Carbamazepine, medical cannabis, or any combination thereof.

[095] According to one embodiment, each component of the composition is in a separate formulation.

[096] According to one embodiment, any combination of two of the components of the composition is in one formulation, and the third component of the composition is in another formulation.

[097] According to one embodiment, all the components of the composition are in one formulation.

[098] According to one embodiment, at least one of the components of the composition is configured to be slowly released.

[099] According to one embodiment, the at least one component is enteric coated.

[100] According to one embodiment, at least one of the components of the composition is in a formulation that is configured to enhance bioavailability of the at least one component of the composition.

[101] According to a further aspect of the present subject matter, there is provided a composition for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, the composition comprising:

DPP-4 antagonist;

PPI; and

GABA.

[102] According to one embodiment, the PPI is configured to be administered in an amount in a range of substantially 10-240 mg daily.

[103] According to one embodiment, the PPI is in a delayed release formulation.

[104] According to one embodiment, the PPI is enteric-coated. [105] According to one embodiment, the GABA is configured to be administered in an amount in a range of substantially 50-3,000 mg daily.

[106] According to one embodiment, the GABA is in an extended release form, or has enhanced bioavailability, or a combination thereof.

[107] According to one embodiment, the GABA is configured to be absorbed to a patient's body from the gastrointestinal tract.

[108] According to an additional aspect of the present subject matter, there is provided a composition for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, the composition comprising:

Sitagliptin;

Omeprazole; and

GABA.

[109] According to one embodiment, the Sitagliptin is configured to be administered in an amount in a range of substantially 25-100 mg daily.

[110] According to one embodiment, the Omeprazole is configured to be administered in an amount in a range of substantially 10-40 mg daily.

[111] According to one embodiment, the Omeprazole is configured to be administered in an amount of up to substantially 120 mg daily.

[112] According to one embodiment, the GABA is configured to be administered in an amount in a range of substantially 50-3,000 mg daily.

[113] According to one embodiment, the composition is configured to be orally administered to a patient.

[114] According to one embodiment, the composition is formulated in tablets, or delayed- release tablets, or capsules, or delayed-release capsules, or a powder, or a delayed -release powder, or a powder for oral suspension, or a delayed-release powder for oral suspension, or granules, or delayed-release granules, or granules for oral suspension, or delayed-release granules for oral suspension, or dispersible tablets, or orally dispersible (orodispersible) tablets, or effervescent tablets.

[115] According to one embodiment, the composition is configured to be administered once daily.

[116] According to one embodiment, the composition is configured to be administered twice daily.

[117] According to one embodiment, the composition is configured to be administered three times daily.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[118] Before explaining at least one embodiment in detail, it is to be understood that the subject matter is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The subject matter is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. In discussion of the various figures described herein below, like numbers refer to like parts. The drawings are generally not to scale.

[119] The present subject matter provides a composition for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction. For the sake of simplicity, the term "composition" will occasionally be used hereinafter instead of the phrase "composition of therapeutic agents for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction". The present subject matter additionally provides a method for treating and preventing a variety of diseases that can be caused by, or linked to, GABAergic transmission dysfunction, with the aforementioned composition.

[120] According to one embodiment, the composition for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction comprises: at least one first agent configured to cause immunomodulation through inhibition of DPP4 enzyme, or activation of glucagon-like peptide- 1 receptor (GLP- 1R) in neurological and immune cells; at least one second agent configured to reduce stomach acidity; and at least one third agent comprising: Gamma aminobutyric acid (GABA), or at least one GABA analogue, or at least one GABA receptor agonist, or any combination thereof.

[121] Referring now to the at least one first agent. Any agent that is configured to cause immunomodulation through inhibition of DPP4 enzyme, or activation of GLP-1R in neurological and immune cells is under the scope of the present subject matter. Some exemplary first agents include at least one Dipeptidyl peptidase-4 (DPP-4) inhibitor, or at least one (GLP-1R) agonist, or any combination of at least one Dipeptidyl peptidase-4 (DPP-4) inhibitor and at least one (GLP-1R) agonist.

[122] GLP-1R is a receptor protein found on neurons of the brain and it has been shown to effectively potentiate GABAergic signaling in neurons [GLP-1 and Exendin-4 Transiently Enhance GABAA Receptor-Mediated Synaptic and Tonic Currents in Rat Hippocampal CA3 Pyramidal Neurons (https://diabetes.diabetesjoumals.org/content/64Zl/79), the entire contents of which is incorporated herein by reference]. GLP-lRs are also found in the hippocampus, the center for memory and learning. Glucagon-like peptide-1 (GLP-1) and GLP-1R agonist (exendin-4) were found to transiently enhance synaptic and tonic currents studied for GABA signaling in hippocampal CA3 pyramidal neurons demonstrating that GLP-1R agonists enhance GABA(A) signaling by pre- and post-synaptic mechanisms [GLP-1 and exendin-4 transiently enhance GABA(A) receptor-mediated synaptic and tonic currents in rat hippocampal CA3 pyramidal neurons (https://pubmed.ncbi.nlm.nih.gov/25114295/), the entire contents of which is incorporated herein by reference] .

[123] When assessing the role of GLP-1 in the CNS research specifically points out the inhibitory effects of GLP-1 R that greatly influence seizure activity through regulation of GABA neuronal receptors [Inhibition of DPP-4 enhances inhibitory synaptic transmission through activating the GLP-1/GLP-1R signaling pathway in a rat model of febrile seizures (https://www.sciencedirect.com/science/article/abs/pii/S00062952183032417via%3Dihub), the entire contents of which is incorporated herein by reference]. Administration of DPP-4 inhibitor will suppress hyperthermia-induced neuronal excitability through activation of the GLP-1R pathway, and will also enhance GABAergic transmission.

[124] Dysfunction of DPP-4 impairs GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) function, breaks the GABA/Glutamate cycle and can become a contributing factor to development of a variety of neurological disorders.

[125] DPP-4 is an enzyme expressed on many cell types, and is associated with immune regulation, signal transmission and apoptosis. DPP-4 is broadly distributed in different organs and tissues such as the lungs, liver, intestinal epithelium, placenta, kidney and neurons. It also appears in soluble form in body fluids including plasma and cerebrospinal fluid. DPP-4 has been shown to rapidly cleave and inactivate GIP and GLP-1. DPP-4 inhibitors are a class of oral antihyperglycemic agents that enhance the body's ability to regulate blood glucose by increasing the active levels of incretins, GLP-1 and GIP. There are numerous DPP-4 inhibitors in development with Sitagliptin (Januvia) as the first approved and most investigated DPP-4 inhibitory agent for the treatment of patients with type 2 diabetes.

[126] DPP-4, also known as CD26, is a lymphocyte cell surface protein which is present and widely expressed in a variety of immune cells including CD8⁺ and CD4⁺ T-cells, B-cells, natural killer (NK) cells, dendritic cells, and macrophages. Its primary function is to regulate the differentiation, maturation, or proliferation of these cells [Sitagliptin, a DPP-4 inhibitor, alters the subsets of circulating CD4⁺ T-cells in patients with type 2 diabetes (https://pubmed.ncbi.nlm.nih.gov/26508675/), the entire contents of which is incorporated herein by reference]. Expansive research has been dedicated to show that DPP-4 plays an important role in T-cell activation [Cut to the chase: a review of CD26/dipeptidyl peptidase-4's (DPP-4) entanglement in the immune system

(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908298/), the entire contents of which is incorporated herein by reference] . Application of DPP-4 inhibitors has been shown to regulate T-cell-dependent immune regulation [Cut to the chase: a review of CD26/dipeptidyl peptidase- 4's (DPP-4) entanglement in the immune system

(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908298/), the entire contents of which is incorporated herein by reference]. Application of the DPP-4 inhibitor Sitagliptin for 12 weeks reduced the number of circulating CD4⁺ T-cells in patients with type 2 diabetes [Sitagliptin, a DPP-4 inhibitor, alters the subsets of circulating CD4⁺ T-cells in patients with type 2 diabetes (https://pubmed.ncbi.nlm.nih. ov/26508675/). the entire contents of which is incorporated herein by reference]. Additionally, to support the key role played by DPP-4, it was shown that inhibiting its enzymatic activity suppresses T-cell proliferation in vitro [CD26 - The emerging role of a co stimulatory molecule in allograft rejection

(https://www.nature.com/articles/s41423-018-0028-9), the entire contents of which is incorporated herein by reference].

[127] DPP-4 is a surface T-cell activation antigen [Cut to the chase: a review of CD26/dipeptidyl peptidase-4's (DPP4) entanglement in the immune system (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908298]. Therefore, it is assumed that inhibition of DPP-4 overexpression in T-cells might also be a contributing factor for promoting GABAergic transmission through GABA(A) channels.

[128] Any type of administration, and administration regime, of the DPP-4 inhibitor to a patient is under the scope of the present subject matter. According to one embodiment, the at least one DPP-4 inhibitor is configured to be administered orally at a daily dose in a range of substantially 1 to 400 mg.

[129] Any type of DPP-4 inhibitor is under the scope of the present subject matter. Some examples of the at least one DPP-4 inhibitor include: Alogliptin, Linagliptin, Saxagliptin, Sitagliptin, Vildagliptin and the like, or any combination thereof. According to one embodiment, the DPP-4 inhibitor is Sitagliptin.

[130] Any type of formulation of the DPP-4 inhibitor is under the scope of the present subject matter. For example, the DPP-4 inhibitor, for example Sitagliptin, can be formulated in tablets.

[131] According to one embodiment, the at least one DPP-4 inhibitor is configured to be orally administered to the patient. According to another embodiment, the at least one DPP-4 inhibitor is in a form of a tablet, or a capsule, or a powder, or granules, or an oral liquid and the like. According to yet another embodiment, the at least one DPP-4 inhibitor is configured to be administered orally once daily.

[132] According to one embodiment, the at least one DPP-4 inhibitor is Sitagliptin. According to another embodiment, Sitagliptin is configured to be administered orally in an amount in a range of substantially 25-400 mg once daily, or in a range of substantially 12.5-200 mg twice daily. According to yet another embodiment, Sitagliptin is configured to be administered orally in an amount in a range of substantially 25-100 mg once daily, or substantially 12.5-50 mg twice daily. According to a further embodiment, embodiment, lower doses of Sitagliptin can be administered in some cases, for example in cases of pediatric use, low body weight of the patient, impaired renal function, and the like. Thus, according to an additional embodiment, Sitagliptin is configured to be administered orally in an amount of substantially 25 mg once daily, or substantially 12.5 mg twice daily. According to yet an additional embodiment, Sitagliptin is configured to be administered orally in an amount of substantially 50 mg Sitagliptin once daily, or substantially 25 mg Sitagliptin twice daily.

[133] According to one embodiment, the at least one DPP-4 inhibitor is Saxagliptin. According to another embodiment, Saxagliptin is configured to be administered orally in a range of substantially 1.25-5 mg once daily, or in an amount of substantially 2.5 mg once daily, or in a range of substantially 0.625-2.5 mg twice daily, or substantially 1.25 mg twice daily. According to yet another embodiment, Saxagliptin is configured to be administered orally in a range of substantially 2.5-10 mg once daily, or in an amount of substantially 5 mg once daily, or in a range of substantially 1.25-5 mg twice daily, or in an amount of substantially 2.5 mg twice daily.

[134] According to one embodiment, the at least one DPP-4 inhibitor is Linagliptin. According to another embodiment, Linagliptin is configured to be administered orally in a range of substantially 2.5-10 mg once daily, or an amount of substantially 5 mg once daily, or in a range of substantially 1.25-5 mg twice daily, or in an amount of substantially 2.5 mg twice daily.

[135] According to one embodiment, the at least one DPP-4 inhibitor is Alogliptin. According to another embodiment, Alogliptin is configured to be administered orally in a range of substantially 12.5-50 mg once daily, or in an amount of substantially 25 mg once daily, or in a range of substantially 6.25-25 mg twice daily, or in an amount of or substantially 12.5 mg twice daily. In some cases there is a need to administer lower doses of Alogliptin, according to the following embodiments. According to yet another embodiment, Alogliptin is configured to be administered orally in a range of substantially 6.25-25 mg once daily, or in an amount of substantially 12.5 mg, or in a range of substantially 3.125-12.5 mg twice daily, or in an amount of substantially 6.25 mg twice daily. According to still another embodiment, Alogliptin is configured to be administered orally in a range of substantially 3.125-12.5 mg once daily, or in an amount of substantially 6.25 mg once daily, or in a range of substantially 1.56-3.25 mg twice daily, or in an amount of substantially 3.125 mg twice daily.

[136] Any type of GLP-1R agonist is under the scope of the present subject matter. In addition, any type of formulation of the GLP-1R agonist, and any type of administration and administration regime of the GLP-1R agonist is under the scope of the present subject matter. Embodiments of some exemplary GLP-1R agonists are described hereinafter.

[137] According to one embodiment, the at least one GLP-1R agonist is in a form of a pharmaceutical composition for parenteral administration. According to another embodiment, the at least one GLP-1R agonist is in a form of tablets configured to be orally administered to a patient.

[138] According to one embodiment, a formulation of the at least one GLP-1R agonist comprises an insulinotropic GLP-1 compound, a basal insulin peptide and pharmaceutically acceptable additives. According to another embodiment, the formulation of the at least one GLP-1R agonist further comprises zinc.

[139] According to one embodiment, the at least one GLP-1R agonist is a short time-acting Exenatide (Byetta). According to another embodiment, the short time-acting Exenatide (Byetta) is configured to be administered in an amount of substantially 5 pg subcutaneously twice daily within substantially 1 hour before meals. According to yet another embodiment, based on clinical response, a dose of the short time-acting Exenatide (Byetta) is increased to substantially 10 pg twice daily after substantially 1 month of therapy.

[140] According to one embodiment, the at least one GLP-1R agonist is Lixisenatide (Adlyxin). According to another embodiment, Lixisenatide (Adlyxin) is configured to be administered initially during 14 days in an amount of substantially 10 pg subcutaneously once daily within substantially 1 hour before a first meal of a day, and on day 15, the amount of Lixisenatide is increased to substantially 20 pg once daily.

[141] According to one embodiment, the at least one GLP-1R agonist is an intermediateacting Liraglutide (Victoza, Saxenda). According to another embodiment, the intermediateacting Liraglutide (Victoza, Saxenda) is configured to be administered initially in an amount of substantially 0.6 mg daily for one week. The substantially 0.6 mg dose is a starting dose '? intended to reduce gastrointestinal symptoms during initial titration, and is not effective for glycemic control in adults. According to yet another embodiment, after one week at substantially 0.6 mg per day, the dose is increased to substantially 1.2 mg daily for at least one week. According to yet another embodiment, if additional glycemic control is required, the dose is increased to substantially 1.8 mg daily after at least one week of treatment with the substantially 1.2 mg daily dose. According to another embodiment, the dose can be increased to substantially 2.4 mg daily after at least one week of treatment with the substantially 1.8 mg daily dose, and to substantially 3.0 mg daily after at least one week on the substantially 2.4 mg dose.

[142] According to one embodiment, the at least one GLP-1R agonist is a long-acting Exenatide weekly (QW) (Bydureon, or Bydureon Bcise). According to another embodiment, the Exenatide QW (Bydureon, or Bydureon Bcise) is configured to be administered in an amount of substantially 2 mg subcutaneously once every 7 days (weekly). According to yet another embodiment, the Exenatide QW (Bydureon, or Bydureon Bcise), is configured to be administered at any time of day, with or without meals.

[143] According to one embodiment, the at least one GLP-1R agonist is Albiglutide (Tanzeum). According to another embodiment, the Albiglutide (Tanzeum) is configured to be administered in an amount of substantially 30 mg subcutaneously once weekly. According to yet another embodiment, the Albiglutide (Tanzeum) is configured to be administered in an amount of substantially 50 mg subcutaneously once weekly.

[144] According to one embodiment, the at least one GLP-1R agonist is Dulaglutide

(Trulicity). According to another embodiment, the Dulaglutide (Trulicity) is configured to be administered in an amount of substantially 0.75 mg subcutaneously once weekly. According to yet another embodiment, the Dulaglutide (Trulicity) is configured to be administered in an amount of substantially 1.5 mg subcutaneously once weekly. [Gastrin-Releasing Peptide and Glucose Metabolism Following Pancreatitis

(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593441/), the entire contents of which is incorporated herein by reference].

[145] According to one embodiment, the at least one GLP-1R agonist is Semaglutide [Ozempic, or Wegovy (Novo Nordisk)]. [146] According to one embodiment, the Semaglutide [Ozempic, or Wegovy (Novo Nordisk)] is configured to be firstly administered in an amount of substantially 0.25 mg, by subcutaneous injection, once weekly for 4 weeks. The substantially 0.25 mg dose is intended for treatment initiation and is not effective for glycemic control. After the 4 weeks on the substantially 0.25 mg dose, the dosage is increased to substantially 0.5 mg once weekly for at least 4 weeks. If additional glycemic control is needed after at least 4 weeks on the substantially 0.5 mg dose, the dosage is increased to substantially 1 mg once weekly. In all the embodiment, the Semaglutide [Ozempic, or Wegovy (Novo Nordisk)] is configured to be administered by subcutaneous injection,

[147] According to one embodiment, the Semaglutide [Ozempic, or Wegovy (Novo Nordisk)] is configured to be administered by subcutaneous injection, once weekly, according to a dose escalation schedule, in order to minimize gastrointestinal adverse reactions. An exemplary dose escalation schedule is provided in the following table 1.

[148] Table 1: An exemplary dose escalation schedule of Wegovy;

[149] According to one embodiment, if a patient does not tolerate a dose of Wegovy during the dose escalation, the start of the dose escalation schedule can be delayed, for example for 4 weeks. According to another embodiment, if a patient does not tolerate the substantially 2.4 mg maintenance dose of Wegovy, the dose of Wegovy can be temporarily decreased to substantially 1.7 mg once weekly, for up to 4 weeks, for example, and then increased back to substantially 2.4 mg once weekly. If the patient still does not tolerate the substantially 2.4 mg maintenance dose of Wegovy, it should be considered to discontinue the administration of Wegovy to the patient. [150] According to one embodiment, the at least one GLP-1R agonist is Semaglutide, also known as Rybelsus (Novo Nordisk). According to another embodiment, the Semaglutide [Rybelsus (Novo Nordisk)] is configured to be firstly administered orally in an amount in a range of substantially 0.1-3 mg once daily for 30 days. The substantially 0.1-3 mg dose of Rybelsus is for treatment initiation and is not effective for glycemic control. Then, the dose of Rybelsus is increased to a range of substantially 0.2-7 mg once daily, for 30 days. If after 30 days on the substantially 0.2-7 mg daily dose there is a need for an additional glycemic control, the dose of Rybelsus can be increased to a range of substantially 0.4-14 mg once daily.

[151] Referring now to the at least one second agent. Any agent that is configured to reduce stomach acidity is under the scope of the present matter. Some exemplary second agents include at least one proton pump inhibitor (PPI), or at least one H2 -receptor antagonist, or at least one Bombesin-like peptide, or any combination thereof.

[152] PPI belongs to a group of agents that block gastric acid secretion, causing the body to increase serum gastrin concentration to compensate for the decreased acidity in the stomach. Other groups of drugs, in addition to PPI, that also decrease the acidity of the stomach include stomach acid reducers, such as antacids and H2 -receptor antagonists (blockers). [Does fasting serum gastrin predict gastric acid suppression in patients on proton-pump inhibitors? (https://pubmed.ncbi.nlm.nih.gov/10695611/), the entire contents of which is incorporated herein by reference] . As serum gastrin production in G-cells is stimulated by GRP, it can be assumed that PPIs contribute to GRP elevation through negative feedback of inhibiting gastric acid secretion. Hence, while some of the effects of PPIs still remain to be discovered, it is important to consider two factors facilitated by PPIs that might play a role in the GABAergic transmission: elevation of GRP and gastrin.

[153] It should be noted that the second agent contributes to gastrin and gastrin-releasing peptide (GRP) elevation through negative feedback of inhibiting gastric acid secretion. It should be further noted that the PPIs and H2 antagonists (blockers), both decrease gastric acid secretion. On the other hand, the antacids do not decrease gastric acid secretion, but rather neutralize gastric acid after it has been secreted.

[154] Any type of PPI is under the scope of the present subject matter. Some exemplary PPIs include Omeprazole, Pantoprazole, Lansoprazole, Rabeprazole, Esomeprazole, or any combination thereof, and the like. [155] According to one embodiment, the at least one PPI is in a pharmaceutically acceptable formulation. According to another embodiment, the at least one PPI is in a form of tablets, or delayed-release tablets, or capsules, or delayed-release capsules, or granules, or delayed- release granules, or injection, and the like. According to yet another embodiment, the at least one PPI is configured to be administered by intravenous (IV) injection.

[156] According to one embodiment, the at least one PPI is configured to be administered to a patient orally at a daily dose suitable for any type of PPI. Following are some types of PPI and the doses for administration.

[157] According to one embodiment, the at least one PPI is Omeprazole. According to another embodiment, the Omeprazole is configured to be administered to a patient in a form of delayed- release oral tablets, or delayed-release oral capsules, or delayed-release oral granules, which can be swallowed as-is, with liquid, or mixed in a food or liquid to be swallowed. According to another embodiment, the Omeprazole is configured to be administered in a range of substantially 10 to 120 mg daily.

[158] According to one embodiment, the at least one PPI is Lansoprazole. According to another embodiment, the Lansoprazole is configured to be administered in a range of substantially 15-60 mg daily. According to one embodiment, the at least one PPI is Pantoprazole. According to another embodiment, the Pantoprazole is configured to be administered in a range of substantially 20 to 80 mg daily. According to one embodiment, the at least one PPI is Esomeprazole. According to another embodiment, the Esomeprazole is configured to be administered in a range of substantially 20 to 240 mg daily.

[159] Any type of H2-receptor antagonist is under the scope of the present subject matter. As mentioned above, the H2 -receptor antagonist is a stomach acid reducer. Any type of stomach acid reducer is under the scope of the present subject matter, for example an antacid. According to one embodiment, the at least one H2-receptor antagonist includes: Cimetidine, Ranitidine, Nizatidine, Famotidine, or any combination thereof.

[160] The Bombesin-like peptides were initially characterized in frog skin; then subsequently Bombesin was found to have many potent effects on the CNS effects mammals, including, but not limited to, effects on temperature regulation, behavior, appetite, heart rate, and gastric acidity regulation. Outside of the CNS, Bombesin is a potent stimulant of gastrin secretion as well as other gastrointestinal hormones, pancreatic exocrine secretion, and a stimulus for gastrointestinal growth. Early studies of Bombesin used radioimmunoassay to establish that Bombesin-like immunoreactivity is widespread in the brain [Cohen, Ronald N MD, REVIEWER ENCYCLOPEDIA OF HORMONES— 3 VOLUME SET, Shock: May 2004 - Volume 21 - Issue 5, the entire contents of which is incorporated herein by reference].

[161] Any type of Bombesin-like peptide is under the scope of the present subject matter.

[162] According to one embodiment, the at least one Bombesin-like peptide is a gastrinreleasing peptide (GRP), or gastrin-releasing peptide receptor agonist, or a combination thereof. GRP is a neuropeptide that has a number of functions primarily including stimulation of the release of gastrin from G-cells of the stomach. A lesser known function of GRP is that it is reported to depolarize GABAergic interneurons in the hippocampus [Gastrin-Releasing Peptide and Glucose Metabolism Following Pancreatitis (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593441/), the entire contents of which is incorporated herein by reference]. GRP binds preferentially to the GRP receptor (GRPR), located predominantly in the CNS [Gastrin-Releasing Peptide and Glucose Metabolism Following Pancreatitis (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593441/), the entire contents of which is incorporated herein by reference] . While many of the mechanisms of GRP are still unclear, there is evidence that activation of the GRP receptor may modulate GABAergic transmission and confer anticonvulsant properties on agonists acting at the GRP receptor in the variety of electrically excitable cells [Effect of gastrin-releasing peptide on rat hippocampal extracellular GABA levels and seizures in the audiogenic seizure-prone DBA/2 mouse (https://pubmed.ncbi.nlm.nih.gov/10719092/), the entire contents of which is incorporated herein by reference]. The GRP receptor is expressed by various cell types, and recent studies have suggested the relationship between GRP and inflammatory diseases. RC- 3095, a selective GRPR antagonist, was found to have anti-inflammatory properties in models of arthritis, gastritis, uveitis and sepsis. It is generally assessed that GRP and its receptor are relevant to the inflammatory response, being a potential therapeutic target several diseases are related to autoimmunity and inflammation [Gastrin-releasing peptide as a molecular target for inflammatory diseases: an update (https://pubmed.ncbi.nlm.nih.gov/23621446/), the entire contents of which is incorporated herein by reference]. [163] Referring now to the GABA, or at least one GABA receptor agonist.

[164] Due to the inhibitory role of GABA it is well established that modulation of GABA signaling is a basis of many pharmacologic treatments in neurology, psychiatry, and anesthesia [Physiology, GABA (https://www.ncbi.nlm.nih.gov/books/NBK513311/), the entire contents of which is under the scope of the present subject matter]. There is extensive research that indicates an important role played by GABA transmission in the mechanism and treatment of epilepsy, including abnormalities of GABAergic function observed in genetic and acquired animal models of epilepsy. Additional research of human epileptic brain tissue and cerebrospinal fluid have also outlined reduced GABA-mediated inhibition and excessive activity of Glutamate [GABAergic mechanisms in epilepsy (https://pubmed.ncbi.nlm.nih.gov/11520315/), the entire contents of which is incorporated herein by reference]. This is consistent with findings that GABA agonists suppress seizures while GABA antagonists produce seizures; medicaments that inhibit GABA synthesis cause seizures; and medicaments that increase synaptic GABA are potent anticonvulsants [Molecular mechanisms of antiseizure drug activity at GABA(A) receptors (https://www.seizure- joumal.com/article/S1059- 1311(13)00114-3/fulltext), the entire contents of which is incorporated herein by reference].

[165] During the transmission GABA binds to two major post-synaptic receptors: GABA(A) and GABA(B). GABA has an inhibitory function regardless of its binding to GABA(A) or GABA(B) receptors. The GABA(A) receptor is an ionotropic receptor, and in the presence of GABA it increases chloride ion conductance into the cell. There is a higher concentration of chloride outside of the cell, therefore the influx of negatively charged chloride ions hyperpolarizes the cell, consequently inhibiting the creation of an action potential. The GABA(B) receptor hyperpolarizes the postsynaptic cell and prevents conduction of an action potential through a metabotropic G-protein coupled receptor.

[166] Aberrations in GABA signaling can be implicated in a large variety of neurologic and psychiatric conditions. When levels of GABA are abnormally low, the firing frequency of nerve cells increases. This leads to over-excitation and a variety of conditions including anxiety and seizure disorders [Anti-glutamic acid decarboxylase antibody positive neurological syndromes (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107286/), the entire contents of which is incorporated herein by reference]. There are known neurological and cognitive problems that are directly associated with decreasing levels of GABA, or with critically low levels of GABA, including cerebellar ataxia and limbic encephalitis along with epilepsy [Anti- glutamic acid decarboxylase antibody positive neurological syndromes (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107286/), the entire contents of which is incorporated herein by reference].

[167] To assess the GABAergic transmission in T-cells it is also necessary to look at the presence of functional GABA(A) receptors. Studies have found that GABA(A) receptors mediate immune inhibition. Pharmacological modulation of GABA(A) receptors can provide new approaches to modulate T-cell responses in inflammation and autoimmune diseases [GABA(A) receptors mediate inhibition of T-cell responses (https://pubmed.ncbi.nlm.nih.gov/10227421/), the entire contents of which is incorporated by reference] .

[168] Similarly to Glutamate, it was also established that GABA also has an immunomodulatory function, in relation to both T-cells infiltrating the brain [GABA, a natural immunomodulator of T-lymphocytes (https://pubmed.ncbi.nlm.nih.gov/18954912/), the entire contents of which is incorporated herein by reference]. GABA has been shown to affect the fate of pathogenic T-lymphocytes entering the brain. It was found that these cells expressed functional GABA(A) channels that could be activated by low (100 nM) concentrations of GABA, which decreased T-cell proliferation. The results are consistent with GABA being immunomodulatory [GABA, a natural immunomodulator of T-lymphocytes (https://pubmed.ncbi.nlm.nih.gov/18954912/), the entire contents of which is incorporated herein by reference] .

[169] Consistent with immunomodulatory role in the brain, pancreatic islets T-cells were also found to express GABA(A) channels in diabetes prone rats and low concentrations of GABA (100 nM) also decreased T-cell proliferation. These findings show that GABA acting through GABA(A) receptor in T-cells can regulate inflammation by inhibition of activated T- lymphocytes [CD8⁺ but not in CD4⁺ T cells in BB rats developing diabetes compared to their congenic littermates (https://pubmed.ncbi.nlm.nih.gov/21112637/), the entire contents of which is incorporated herein by reference] .

[170] According to one embodiment, the at least one third agent, namely, the GABA, or the at least one GABA analogue, or the at least one GABA receptor agonist, or any combination thereof, is configured to be administered to a patient in a daily dose of up to substantially 3000 mg, depending on the body weight. According to one embodiment, the at least one third agent is configured to be administered as capsules, or tablets, or oral powder, or oral granules. According to another embodiment, the third agent is configured to allow modification of absorption of GABA by increasing bioavailability or GABA, for example in order to reduce the dose of GABA that is configured to be administered daily. Another option is to use an extended-release formulation of GABA to reduce the frequency of administration. In another option, both the aforementioned options - increasing the bioavailability or GABA and using an extended-release formulation of GABA, can be used.

[171] Any analogue of GABA is under the scope of the present subject matter, and any source of GABA that can be administered to a patient is under the scope of the present subject matter, for example GABA producing bacteria [EP2828375B 1, the entire contents of which is incorporated herein by reference]. Thus, according to one embodiment, the at least one third agent is in a form of GABA producing bacteria. According to another embodiment, an amount of the GABA that is produced by the GABA producing bacteria in up to substantially 3,000 mg daily.

[172] According to yet another embodiment, the third agent is Aminalone in a form of tablets.

[173] Any type of GABA agonist is under the scope of the present subject matter. Thus, according to one embodiment, some example of a GABA receptor agonist include: Baclofen, Aminophenyl butyric acid (Phenibut), Hopantenoic acid (Pantogam), Zolpidem, or any combination thereof.

[174] According to one embodiment, the at least one third agent is at least one anti-epileptic medication. According to another embodiment, some example of an anti-epileptic medication include: Valproic acid (Depalept), Valproate sodium, Topiramate (Topamax), Carbamazepine, medical cannabis, or any combination thereof.

[175] According to one embodiment, each component of the composition is in a separate formulation, as described above. According to another embodiment, any combination of two of the components of the composition is in one formulation, and the third component of the composition is in another formulation. According to yet another embodiment, all the components of the composition are in one formulation. Any type of formulation of the combined components is under the scope of the present subject matter. Some exemplary types of formulation include an orally administered tablet, or capsule, or a sachet, or any type of container containing powder or granules; an injectable formulation, and the like.

[176] According to one embodiment, at least one of the components of the composition is configured to be slowly released. Any type of mechanism of slow release, or delayed release, of any one of the components of the composition is under the scope of the present subject matter. For example, the at least one component is enteric coated. According to another embodiment, at least one of the components of the composition is in a formulation that is configured to enhance bioavailability of the at least one component of the composition.

[177] According to one embodiment, the composition comprises:

DPP-4 antagonist;

PPI; and GABA.

[178] According to one embodiment, the DPP-4 antagonist is configured to be administered in an amount in a range of substantially 1-400 mg daily.

[179] According to one embodiment, the PPI is configured to be administered in an amount in a range of substantially 10-240 mg daily. According to another embodiment, the PPI is in a delayed release formulation. According to yet another embodiment, the PPI is enteric-coated.

[180] According to one embodiment, the GABA is configured to be administered in an amount in a range of substantially 50-3,000 mg daily. According to another embodiment, the GABA is in an extended release form, or has enhanced bioavailability, or a combination thereof. According to yet another embodiment, the GABA is configured to be absorbed to a patient's body from the gastrointestinal tract.

[181] According to one embodiment, the composition comprises:

Sitagliptin;

Omeprazole; and GABA. [182] According to one embodiment, the Sitagliptin is configured to be administered in an amount in a range of substantially 12.5-400 mg daily.

[183] According to one embodiment, the Omeprazole is configured to be administered in an amount in a range of substantially 10-40 mg daily. According to another embodiment, the Omeprazole is configured to be administered in an amount of up to substantially 120 mg daily.

[184] According to one embodiment, the GABA is configured to be administered in an amount in a range of substantially 50-3,000 mg daily.

[185] According to one embodiment, the composition is configured to be orally administered to a patient. According to another embodiment, the composition is in any known pharmaceutical acceptable oral form. According to yet another embodiment, the composition is formulated in tablets, or delayed-release tablets, or capsules, or delayed-release capsules, or a powder, or a delayed-release powder, or a powder for oral suspension, or a delayed-release powder for oral suspension, or granules, or delayed-release granules, or granules for oral suspension, or delayed-release granules for oral suspension, or dispersible tablets, or orally dispersible (orodispersible) tablets, or effervescent tablets and the like.

[186] According to one embodiment, the composition is configured to be administered once daily. According to another embodiment, the composition is configured to be administered twice daily. According to yet another embodiment, the composition is configured to be administered three times daily.

[187] The present subject matter further provides a method for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, in a patient, the method comprising: administering to the patient at least one first agent configured to cause immunomodulation through inhibition of DPP4 enzyme and activation of glucagon-like peptide- 1 receptor (GLP-1R) in neurological and immune cells; administering to the patient at least one second agent configured to reduce stomach acidity; and administering to the patient at least one third agent, comprising: Gamma aminobutyric acid (GABA), or at least one GABA analogue, or at least one GABA receptor agonist, or any combination thereof. [188] It should be noted that all the embodiment of the composition described above relate also to the method for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, in a patient.

[189] The present subject matter additionally provides, a composition for use in the treatment and prevention of a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, in a patient, the treatment and prevention comprising: administering to the patient at least one first agent configured to cause immunomodulation through inhibition of DPP4 enzyme and activation of glucagon-like peptide- 1 receptor (GLP-1R) in neurological and immune cells; administering to the patient at least one second agent configured to reduce stomach acidity; and administering to the patient at least one third agent, comprising: Gamma aminobutyric acid (GABA), or at least one GABA analogue, or at least one GABA receptor agonist, or any combination thereof.

[190] It should be noted that all the embodiments of the composition and method described above relate also to the composition for use in the treatment and prevention of a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, in a patient.

[191] It should be noted that based on the description of the various embodiments of the composition, the present subject matter includes also methods for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, as well as compositions .

EXAMPLES

Effect of GABA, Sitagliptin, Omeprazole and combinations thereof on glutamate excitotoxicity

Introduction [192] Glutamate excitotoxicity is one of the leading pathogenic mechanisms in neurodegenerative diseases associated with the activation of glutamate receptors, in particular N-methyl-D-aspartate (NMDA) receptors, and the launch of intracellular signaling cascades leading to neuronal death. N-methyl-D-aspartate (NMDA) receptor is a receptor of glutamate, the primary excitatory neurotransmitter in the human brain. The NMDA receptor plays an integral role in synaptic plasticity, which is a neuronal mechanism believed to be the basis of memory formation.

Aim

[193] To study the effect of three drugs: GABA, Sitagliptin, Omeprazole and combinations thereof, on a model of glutamate excitotoxicity.

Information regarding the investigated drugs

GABA

[194] Gamma-aminobutyric acid (GABA) is a non-protein amino acid that is widely distributed in nature. In particular, GABA is present in high concentrations in various areas of the brain. Typically, GABA is produced from 1-glutamic acid by the catalysis of glutamic acid decarboxylase. In the nervous system, newly synthesized GABA is packaged into synaptic vesicles and then released into the synaptic cleft for diffusion to target receptors on the postsynaptic surface.

[195] The damage of nervous tissue triggers an inflammatory response, causing the release of various inflammatory mediators such as reactive oxygen species (ROS), nitric oxide, and cytokines. Numerous studies have been reported regarding the important role of GABA in protecting the nervous system from degeneration caused by toxin or trauma.

[196] In the present study on the glutamate excitotoxicity model, GABA (Sigma- Aldrich (St. Louis, MO, USA) was used in a concentration of 100 pM.

Sitagliptin [197] Sitagliptin is a highly selective inhibitor of the DPP-4 enzyme intended for the treatment of type 2 diabetes mellitus. Sitagliptin differs in chemical structure and pharmacological action from GLP-1 analogues, insulin, sulfonylurea derivatives, biguanides, peroxisome proliferator-activated receptor gamma agonists (PPAR-y), alpha-glucosidase inhibitors, and amylin analogues. By inhibiting DPP-4, Sitagliptin increases the concentration of two hormones of the incretin family: GLP-1 and GIP. Hormones of the incretin family are secreted in the intestine during the day and their concentration increases in response to food intake. Incretins are part of the internal physiological system for the regulation of glucose homeostasis. At normal or elevated blood glucose concentrations, hormones of the incretin family contribute to an increase in insulin synthesis, as well as its secretion by pancreatic beta cells due to intracellular signaling mechanisms associated with cAMP.

[198] GLP-1 also contributes to the suppression of increased secretion of glucagon by pancreatic alpha cells. A decrease in the concentration of glucagon against the background of an increase in the concentration of insulin contributes to a decrease in the production of glucose by the liver, which ultimately leads to a decrease in glycemia. This mechanism of action differs from the mechanism of action of sulfonylurea derivatives, which stimulate the release of insulin even at low blood glucose concentrations, which is fraught with the development of sulfonylated hypoglycemia not only in patients with type 2 diabetes mellitus, but also in healthy individuals.

[199] In the present study on the glutamate excitotoxicity model, Sitagliptin (Carbosynth Limited, Berkshire, UK) was used in a concentration of 100 pM.

Omeprazole

[200] Omeprazole is a weak base, concentrated and converted into an active form in the acidic environment of the secretory tubules of the parietal cells of the gastric mucosa, where it inhibits the enzyme H⁺, K⁺-ATPase (proton pump). Omeprazole has a dose-dependent effect on the final stage of hydrochloric acid synthesis and inhibits both basal and stimulated acid secretion in the stomach, regardless of the nature of the stimulus.

[201] Gastrin, a peptide hormone produced by gastric G-cells, is involved in the development of protodifferentiated islet progenitors in fetal pancreatic ducts and has also been reported to induce neoplasm of P-cells from exocrine pancreatic duct cells in rodents. The literature indicates that the combination of gastrin with GLP-1 analogs stimulates cell neogenesis in animal models of diabetes. Proton pump inhibitors (PPIs), used to treat excess stomach acid, indigestion, gastroesophageal reflux disease, and stomach ulcers, can lead to a persistent increase in serum gastrin levels. Therefore, the use of proton pump inhibitors, which indirectly increase serum gastrin levels, may be useful in optimizing the efficacy of GLP-1 treatment.

[202] In the present study on the glutamate excitotoxicity model, Omeprazole (Premier Pharmacy Labs, Weeki Wachee, FL) was used in a concentration of 100 pM.

Methods

Isolation of cerebellar neurons

[203] Six to eight days old Wistar rat pups were decapitated in accordance with the ethical requirements for working with animals. The cerebellum was extracted and washed 2-3 times in Hanks’ solution (PanEco, Russia) with the addition of 10% HEPES solution (PanEco, Russia), crushed, and transferred to a 0.05% trypsin solution preliminarily heated to 37°C with 0.02% EDTA (PanEco, Russia). After 15 minutes incubation, the cell suspension was washed twice with Hank's solution, the cells were disaggregated until a homogeneous suspension was obtained and centrifuged for 2.5 minutes at 3000 rpm. The cell pellet was resuspended in the appropriate volume of Neurobasal Medium culture medium (Gibco, USA) containing 2% B27 supplement (Gibco, USA), 20 mM KC1 (Sigma-Aldrich, USA) and transferred into the wells of 12-well plates (Corning, USA), pre-coated with poly-DL-lysine (PanEco, Russia) at a concentration of 10 pg/ml. Two ml of cell suspension was added to each well, the seeding density was 2.5xl0⁵ cells/cm². The culture of cerebellar neurons was incubated for 6-7 days. (37°C, 5% CO2, 98% relative humidity) to achieve the desired expression of glutamate receptors. On the second day of cultivation, 10 pM arabinosine monocytoside (Sigma- Aldrich, USA) was added to the medium to suppress the proliferation of mitotically active cells.

Modeling of glutamate excitotoxicity

[204] To simulate glutamate excitotoxicity, the culture of cerebellar neurons was washed with a Dulbecco's phosphate buffered saline (DPBS) solution without Ca²⁺ and Mg²⁺ (PanEco, Russia) and was divided to treatment groups listed in Table 1. The cells were incubated for 1 h in the presence of 200 pM glutamate in a buffer solution comprising: 140 mM NaCl, 5 mM KC1, 2 mM CaCh, 10 mM glycine, 20 mM 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), 5 mM glucose, pH 7.4 (Sigma-Aldrich, USA), and in the presence of the various combinations of drugs as indicated in Table 1 (the concentration of each drug - GABA, Sitagliptin, Omeprazole was 100 M). The cells of the positive control group were not exposed to glutamate and not to any one of the drugs. After the 1 h incubation the cells were washed twice with buffer containing: 20 mM HEPES, 5 mM glucose, pH 7.4 (Sigma-Aldrich, USA); and the original culture medium was added.

Table 1: Treatment groups of isolated cerebellar neurons.

Assessment of neuronal death

[205] The death of cerebellar neurons in culture was determined 24 hours after the addition of glutamate. Viability was assessed using the MTT test. [4,5dimethylthiazol-2-yl]-2,5- diphenyl-tetrazolium bromide (MTT) (PanEco, Russia) was introduced into the culture medium to a final concentration of 0.5 mg/ml and incubated at 37°C for 2 hours followed by dissolution of formazan crystals in dimethyl sulfoxide (DMSO) (PanEco, Russia). The optical density of the eluate was measured at 570 nm and 620 nm, using a BioRad Reader xMark plate spectrophotometer (Bio-Rad Laboratories, USA). Then, for each sample, the absorbance at 620 nm was subtracted from the absorbance at 570 nm. The results were evaluated in relation to the control, which was taken as 100%. Data analysis

[206] For all quantitative data, the group arithmetic mean (M) and standard error of the mean (SEM) or standard deviation (SD) were calculated.

[207] The homogeneity of the samples (the equality of the variances of the three compared groups in each experiment) was checked using the Bartlett test.

[208] If the null hypothesis of equality of group variances was confirmed, and after successful testing of other assumptions, one-way ANOVA analysis of variance was used to determine the significance of differences in group means. At the same time, if the null hypothesis ANOVA about the equality of the general means was rejected, subsequent pairwise comparison of the experimental groups with the control was carried out using the Dunnett test.

[209] In case of rejecting the null hypothesis of equality of group variances, or failing to test other assumptions for applying ANOVA, the nonparametric Kruskal-Wallis test was used to determine the significance of differences in group medians. At the same time, if the null hypothesis about the equality of the general medians was rejected, the subsequent pairwise comparison of the experimental groups with the control group was carried out using the nonparametric Dunn test.

[210] Statistical data processing was carried out using the Statistica 12 program for Windows 10.

Results

[211] The results of the experiment are summarized in Table 2, showing the average absorbance at 570 nm minus average absorbance at 620 (n=8), standard deviation (SD) of absorbance values, percent survival of the cerebellar neurons with reference to the control treatment that was calculated as 100% survival, and SD of the percentages.

Table 2: Distribution of isolated cerebellar neurons into treatment groups.

[212] Using the MTT test, it was shown that 24 hours after incubation of rat cerebellar neurons in the presence of 200 pM glutamate, the number of viable neurons decreased by 50.31+12.88% . Incubation of neurons with the studied compounds separately did not affect the cytotoxic effect of glutamate. The double combinations contributed to non- significant increase in the number of viable cells. However, the use of a combination of all three drugs (GABA + Sitagliptin + Omeprazole) contributed to an increase in the number of viable cells up to 79.15+12.21%, which was statistically significantly different from the viability of the cerebellar neurons treated with Glutamate + Ca²⁺ (P<0.05) (Table 2). The triple therapy group was the only treatment that showed a statistically significant increase in the number of viable cells.

Conclusions

[213] This experiment presents data on the study of the specific pharmacological activity of a triple combination of drugs: GABA+ Sitagliptin + Omeprazole in a model of glutamate excito toxicity. As a result of the analysis of the results obtained in this study, the following conclusions can be drawn:

1. Incubation of neurons with the studied compounds separately did not affect the cytotoxic effect of glutamate.

2. A combination of the two drugs GABA + Sitagliptin contributed to a non-significant increase in the number of viable cells up to 71.49+11.18%. 3. A combination of the two drugs (GABA + Omeprazole) contributed to a non-significant increase in the number of viable cells up to 65.12+11.34%.

4. A combination of the two drugs (Sitagliptin + Omeprazole) contributed to a non-significant increase in the number of viable cells up to 68.42+8.59%.

5. A combination of all three drugs (GABA + Sitagliptin + Omeprazole) contributed to a maximal and significant increase in the number of viable cells up to 79.15+12.21 %.

[214] The triple therapy group was the only treatment that showed a statistically significant increase in the number of viable cells. Thus, it can be concluded that therapy with the composition comprising GABA+ Sitagliptin + Omeprazole has a superior pharmacological effect compared to any of these drugs alone, or compared to any combination of any two of these drugs.

Animal model of multiple sclerosis

[215] Multiple sclerosis is an autoimmune neurodegenerative disease in which leukocytes pass through the blood-brain barrier and attack and destroy the myelin sheaths that encase the axons of nerve cells in the central nervous system. It has been shown that abnormal glutamatedependent synaptic excitation contributes to neuronal damage in multiple sclerosis [Inflammation inhibits GABA transmission in multiple sclerosis (https://pubmed.ncbi.nlm.nih.gov/22419673/), the entire contents of which is incorporated herein by reference]. There is also growing evidence for dysfunctional glutamatergic excitation and GABAergic inhibition in patients with MS [Altered hippocampal GABA and glutamate levels and uncoupling from functional connectivity in multiple sclerosis (https://pubmed.ncbi.nlm.nih.gov/30069963/), the entire contents of which is incorporated herein by reference]. It is hypothesized that strategies that modulate glutamate and GABA neurotransmission may represent new therapeutic treatments for patients with multiple sclerosis.

[216] An animal model of multiple sclerosis is produced by inducing animals, like mice and guinea pigs, with experimental autoimmune encephalomyelitis (EAE) via injections of myelin peptides, such as myelin basic protein (MBP), proteolipid protein (PLP), or myelin oligodendrocyte glycoprotein (MOG), in complete Freund's adjuvant. This animal model is referred to hereinafter as "MS animal model". [217] The composition of the present subject matter, comprising a DPP-4 inhibitor, GABA and PPI I administered via subcutaneous (SC), intraperitoneal (IP) and oral administration routes to the MS animal model to explore the therapeutic potential of the combination therapy in regulation of glutamate/GABAergic neurotransmission and inhibition for autoimmunity. It is expected that the combination therapy in vivo will decrease and delay clinical and neuropathological signs of adoptive transfer EAE. In addition, the combination therapy can assist in modulation of glutamate/GABAergic transmission.

Mice

[218] The experiment begins with mice in the age of 7-14 weeks. The mice are split into the following groups based on treatment and administration method:

1. Placebo

2. DPP-4 inhibitor, GABA and PPI via oral administration

3. DPP-4 inhibitor, GABA and PPI via IP or SC

4. DPP-4 inhibitor

Antigens

[219] Whole myelin basic protein (MBP) is prepared according to the method of Deibler [Deibler, G. E., R. E. Martenson, and M. W. Kies. 1972. Large scale preparation of myelin basic protein from central nervous system tissue of several mammalian species. Prep. Biochem. 2:139, the entire contents of which is incorporated herein by reference] from guinea pig spinal cords [Brocke, S., L. Quigley, H. F. McFarland, and L. Steinman. 1996. Isolation and characterization of autoreactive T-cells in experimental autoimmune encephalomyelitis of the mouse. Methods 9:458, the entire contents of which is incorporated herein by reference]. Peptide 139-151 from proteolipid protein (PLP) (PLP139-151), is prepared by continuous flow solid phase synthesis.

Induction of adoptive transfer EAE

[220] Each recipient mouse is injected I.V. with 3 3xl0⁷ activated MBP - or PLP139-151- specific lymph node cells (LNC). Immunization (400 mg MBP or 200 mg PLP139-151) and the preparation of primary LNC cultures and short term T-cell lines follow in accordance to the method of Brocke, S., et al. [Brocke, S., L. Quigley, H. F. McFarland, and L. Steinman. 1996. Isolation and characterization of autoreactive T-cells in experimental autoimmune encephalomyelitis of the mouse. Methods 9:458, the entire contents of which is incorporated herein by reference]. Mice are examined daily for signs of disease and graded on a scale of increasing severity from 0 to 5 as follows:

0 no signs;

0.5 - partial tail weakness;

1 - limp tail or slight slowing of righting from supine position;

1.5 - limp tail and slight slowing of righting;

2 - partial hind limb weakness or marked slowing of righting;

2.5 - dragging of hind limb(s) without complete paralysis;

3 - complete paralysis of at least one hind limb;

3.5 - hind limb paralysis and slight weakness of forelimbs;

4 - severe forelimb weakness;

5 - moribund or dead.

[221] Treatment effects are assessed using the nonparametric Mann-Whitney rank sum test. All statistical tests are performed with SigmaStat software (Jandel, San Rafael, CA).

Histology

[222] Selected mice are sacrificed with CO2. CNS tissues are fixed in 10% phosphate buffer saline (PBS) buffered formalin. Paraffin sections (4 mm) are stained with hematoxylineosin or Luxol fast blue (American Histolabs, Gaithersburg, MD). At least two coronal sections from three brain levels and at least two longitudinal and coronal sections from cervical, thoracic, and lumbosacral levels of the spinal cord are evaluated in a blinded manner by an experienced neuropathologist.

Inhibitors and treatment

[223] For the experiments described as DPP-4 inhibitor, GABA and PPI via IP or SC administration, the DPP-4 inhibitor, GABA and PPI are dissolved in PBS at 10’² M and adjusted to neutral pH. In treatment experiments, 1 mg of the combination therapy solution is injected from once every other day to three times daily, SC or IP. [224] For the experiments described as DPP-4 inhibitor, GABA and PPI via oral administration, the DPP-4 inhibitor, GABA and PPI are administered to the mice twice a day, orally, in a volume of 1ml in the morning and the evening. A 1% starch solution is used as solvent for the following dosages: GABA - 300 mg/kg, DPP-4 inhibitor - 30mg/kg and PPI - 15mg/kg. The treatment is administered for 21 days before the induction of EAE.

[225] For the experiments described as DPP-4 inhibitor, the DPP-4 inhibitor is dissolved in PBS at 10’² M and adjusted to neutral pH. In treatment experiments, 1 mg of the DPP-4 inhibitor solution is injected from once every other day to three times daily, SC or IP.

[226] For the experiments described as Placebo, the 1% starch solution is administered to the mice either SC, or IP, or orally, twice a day, in a volume of 1ml in the morning and the evening. The treatment is administered for 14 days before the induction of EAE.

Determination of DPP-4 activity

[227] The enzymatic activity of DPP-4 is determined according to the method published by Schon et al. [Schon, E., H. U. Demuth, A. Barth, and S. Ansorge. 1984. Dipeptidyl peptidase IV of human lymphocytes: evidence for specific hydrolysis of glycylproline pnitroanilide in T- lymphocytes. Biochem. J. 223:255 the entire contents of which is incorporated herein by reference] using 1.6 mM Gly-Pro-4-nitroanilide as substrate for DPP-4. All measurements with substrate and PBS controls are performed in duplicates. To measure DPP-4 activity in serum, two to three mice per treatment group are bled several times during the experiment period. Pooled sera are diluted 1:10 before the assay. Brains and spinal cords (caudal from C2) of mice from treatment and Placebo groups are removed after transcardial perfusion with PBS at the end of the experiment period.

Proliferation assays

[228] MBP- or PLPi39-i5i-specific proliferation of primed LNC or short term T-cell lines are measured. To determine the effect of the inhibitors, varying concentrations are added at a fixed antigenic concentration (25 mg/ml MBP or 10 mg/ml peptide). Cytokine measurements

[229] Cytokine secretion is measured by sandwich Enzyme Linked Immuno-Sorbent Assay (ELISA) on culture supernatants.

Results

[230] The combination therapy administration, orally and SC/IP, suppresses the clinical severity of EAE, with reduced or suppressed CNS cell inflammation and reduced myelin loss in treated mice.

[231] It is appreciated that certain features of the subject matter, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the subject matter, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination.

[232] Although the subject matter has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. A composition for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, the composition comprising: at least one first agent configured to cause immunomodulation through inhibition of DPP4 enzyme and activation of glucagon-like peptide- 1 receptor (GLP-1R) in neurological and immune cells; at least one second agent configured to reduce stomach acidity; and at least one third agent, comprising: Gamma aminobutyric acid GABA, or at least one GABA analogue, or at least one GABA receptor agonist, or any combination thereof.

2. The composition of claim 1 , wherein the at least one first agent is at least one Dipeptidyl peptidase-4 (DPP-4) inhibitor.

3. The composition of claim 2, wherein the at least one DPP-4 inhibitor is configured to be administered orally at a daily dose in a range of substantially 1 to 400 mg.

4. The composition of claim 3, wherein the at least one DPP-4 inhibitor is configured to be administered orally once daily.

5. The composition of any one of claims 2-4, wherein the at least one DPP-4 inhibitor is in a form of a tablet, or a capsule, or a powder, or granules, or an oral liquid.

6. The composition of any one of claims 2-5, wherein the at least one DPP-4 inhibitor is selected from the group consisting of: Alogliptin, Linagliptin, Saxagliptin, Sitagliptin, Vildagliptin, or any combination thereof.

7. The composition of any one of claims 2-5, wherein the at least one DPP-4 inhibitor is Sitagliptin.

8. The composition of claim 7, wherein the Sitagliptin is configured to be administered orally in an amount in a range of substantially 25-400 mg once daily, or in a range of substantially 12.5-200 mg twice daily.

9. The composition of claim 7, wherein the Sitagliptin is configured to be administered orally in an amount in a range of substantially 25-100 mg once daily, or substantially 12.5-50 mg twice daily.

10. The composition of any one of claims 2-9, wherein the at least one DPP-4 inhibitor is Saxagliptin.

11. The composition of claim 10, wherein the Saxagliptin is configured to be administered orally in a range of substantially 1.25-5 mg once daily, or in an amount of substantially 2.5 mg once daily, or in a range of substantially 0.625-2.5 mg twice daily, or substantially 1.25 mg twice daily.

12. The composition of claim 10, wherein the Saxagliptin is configured to be administered orally in a range of substantially 2.5-10 mg once daily, or in an amount of substantially 5 mg once daily, or in a range of substantially 1.25-5 mg twice daily, or in an amount of substantially 2.5 mg twice daily.

13. The composition of any one of claims 2-12, wherein at least one DPP-4 inhibitor is Linagliptin.

14. The composition of claim 13, wherein the Linagliptin is configured to be administered orally in a range of substantially 2.5-10 mg once daily, or an amount of substantially 5 mg once daily, or in a range of substantially 1.25-5 mg twice daily, or in an amount of substantially 2.5 mg twice daily.

15. The composition of any one of claims 2-14, wherein the at least one DPP-4 inhibitor is Alogliptin.

16. The composition of claim 15, wherein the Alogliptin is configured to be administered orally in a range of substantially 12.5-50 mg once daily, or in an amount of substantially 25 mg once daily, or in a range of substantially 6.25-25 mg twice daily, or in an amount of or substantially 12.5 mg twice daily.

17. The composition of claim 15, wherein the Alogliptin is configured to be administered orally in a range of substantially 6.25-25 mg once daily, or in an amount of substantially 12.5 mg, or in a range of substantially 3.125-12.5 mg twice daily, or in an amount of substantially 6.25 mg twice daily.

18. The composition of claim 15, wherein the Alogliptin is configured to be administered orally in a range of substantially 3.125-12.5 mg once daily, or in an amount of substantially 6.25 mg once daily, or in a range of substantially 1.56-3.25 mg twice daily, or in an amount of substantially 3.125 mg twice daily.

19. The composition of any one claims 1-18, wherein the at least one first agent is at least one (GLP-1R) agonist.

20. The composition of claim 19, wherein the GLP-1R agonist is in a form of a pharmaceutical composition for parenteral administration.

21. The composition of any one of claims 19-20, wherein the at least one GLP-1R agonist is in a form of tablets configured to be orally administered to a patient.

22. The composition of any one of claims 19-21, wherein a formulation of the at least one GLP-1R agonist comprises an insulinotropic GLP-1 compound, a basal insulin peptide and pharmaceutically acceptable additives.

23. The composition of any one of claims 19-22, wherein the at least one GLP-1R agonist is a short time-acting Exenatide.

24. The composition of claim 23, wherein the short time-acting Exenatide is configured to be administered in an amount of substantially 5 pg subcutaneously twice daily within substantially 1 hour before meals.

25. The composition of claim 24, wherein a dose of the short time-acting Exenatide is increased to substantially 10 pg twice daily after substantially 1 month of therapy.

41

26. The composition of any one of claims 19-25, wherein the at least one GLP-1R agonist is Lixisenatide.

27. The composition of claim 26, wherein the Lixisenatide is configured to be administered initially during 14 days in an amount of substantially 10 pg subcutaneously once daily within substantially 1 hour before a first meal of a day, and on day 15, the amount of Lixisenatide is increased to substantially 20 pg once daily.

28. The composition of any one of claims 19-27, wherein the at least one GLP-1R agonist is an intermediate- acting Liraglutide.

29. The composition of claim 28, wherein the intermediate-acting Liraglutide is configured to be administered initially in an amount of substantially 0.6 mg daily for one week.

30. The composition of claim 29, wherein after the administration of the intermediateacting Liraglutide in an amount of substantially 0.6 mg daily for one week, the intermediateacting Liraglutide is configured to be administered in an amount of substantially 1.2 mg daily for at least one week.

31. The composition of claim 30, wherein after the administration of the intermediateacting Liraglutide in an amount of substantially 1.2 mg daily for at least one week, the intermediate- acting Liraglutide is configured to be administered in an amount of substantially 1.8 mg daily for at least one week.

32. The composition of claim 31, wherein after the administration of the intermediateacting Liraglutide in an amount of substantially 1.8 mg daily for at least one week, the intermediate- acting Liraglutide is configured to be administered in an amount of substantially 2.4 mg daily for at least one week.

33. The composition of claim 32, wherein after the administration of the intermediateacting Liraglutide in an amount of substantially 2.4 mg daily for at least one week, the intermediate- acting Liraglutide is configured to be administered in an amount of substantially 3.0 mg daily for at least one week.

34. The composition of any one of claims 19-33, wherein the at least one GLP-1R agonist is a long-acting Exenatide weekly (QW).

35. The composition of Claim 34, wherein the Exenatide QW is configured to be administered in an amount of substantially 2 mg subcutaneously once every 7 days.

36. The composition of any one of claims 34-35, wherein the Exenatide QW is configured to be administered at any time of day, with or without meals.

37. The composition of any one of claims 19-36, wherein the at least one GLP-1R agonist is Albiglutide.

38. The composition of claim 37, wherein the Albiglutide is configured to be administered in an amount of substantially 30 mg subcutaneously once weekly.

39. The composition of claim 37, wherein the Albiglutide is configured to be administered in an amount of substantially 50 mg subcutaneously once weekly.

40. The composition of any one of claims 19-39, wherein the at least one GLP-1R agonist is Dulaglutide.

41. The composition of claim 40, wherein the Dulaglutide is configured to be administered in an amount of substantially 0.75 mg subcutaneously once weekly.

42. The composition of claim 40, wherein the Dulaglutide is configured to be administered in an amount of substantially 1.5 mg subcutaneously once weekly.

43. The composition of any one of claims 19-42, wherein the at least one GLP-1R agonist is Semaglutide.

44. The composition of claim 43, wherein the Semaglutide is configured to be firstly administered in an amount of substantially 0.25 mg, by subcutaneous injection, once weekly for 4 weeks.

45. The composition of claim 44, wherein after the administration of the Semaglutide in an amount of substantially 0.25 mg, by subcutaneous injection, once weekly for 4 weeks, the Semaglutide is configured to be administered in an amount of substantially 0.5 mg, by subcutaneous injection, once weekly for at least 4 weeks.

46. The composition of claim 45, wherein after the administration of the Semaglutide in an amount of substantially 0. 5 mg, by subcutaneous injection, once weekly for 4 weeks, the Semaglutide is configured to be administered in an amount of substantially 1.0 mg, by subcutaneous injection, once weekly for at least 4 weeks.

47. The composition of claim 43, wherein the Semaglutide is configured to be administered by subcutaneous injection, once weekly, according to a dose escalation schedule.

48. The composition of claim 43, wherein the Semaglutide is configured to be firstly administered orally in an amount in a range of substantially 0.1-3 mg once daily for 30 days.

49. The composition of claim 48, wherein after the administration of the Semaglutide in an amount in a range of substantially 0.1-3 mg once daily for 30 days, the Semaglutide is configured to be orally administered in an amount in a range of substantially 0.2-7 mg once daily, for 30 days.

50. The composition of claim 49, wherein after the administration of the Semaglutide in an amount in a range of substantially 0.2-7 mg once daily for 30 days, the Semaglutide is configured to be orally administered in an amount in a range of substantially 0.4-14 mg once daily.

51. The composition of any one claims 1-50, wherein the at least second agent is selected from the group consisting of: at least one proton pump inhibitor (PPI), or at least one H2- receptor antagonist, or at least one Bombesin-like peptide, or any combination thereof.

52. The composition of claim 51, wherein the at least on PPI is in a pharmaceutically acceptable formulation.

53. The composition of claim 51, wherein the at least one PPI is in a form of tablets, or delayed-release tablets, or capsules, or delayed-release capsules, or granules, or delayed- release granules, or injection.

54. The composition of claim 51, wherein the at least one PPI is configured to be administered by intravenous (IV) injection.

55. The composition of claim any one of claims 51-54, wherein the at least one PPI is selected from the group consisting of: Omeprazole, Pantoprazole, Lansoprazole, Rabeprazole, Esomeprazole, or any combination thereof.

56. The composition of claim 51-54, wherein the at least one PPI is Omeprazole.

57. The composition of claim 56, wherein the Omeprazole is configured to be administered to a patient in a form of delayed-release oral tablets, or delayed-release oral capsules, or delayed-release oral granules, which can be swallowed as-is, with liquid, or mixed in a food or liquid to be swallowed.

58. The composition of any one of claims 56-57, wherein the Omeprazole is configured to be administered in a range of substantially 10 to 120 mg daily.

59. The composition of ant one of claims 51-58, wherein the at least one PPI is Lansoprazole.

60. The composition of claim 59, wherein the Lansoprazole is configured to be administered in a range of substantially 15-60 mg daily.

61. The composition of any one of claims 51-60, wherein the at least one PPI is Pantoprazole.

62. The composition of claim 61 , wherein the Pantoprazole is configured to be administered in a range of substantially 20 to 80 mg daily.

63. The composition of any one of claim 51-62, wherein the at least one PPI is Esomeprazole.

64. The composition of claim 63, wherein the Esomeprazole is configured to be administered in a range of substantially 20 to 240 mg daily.

65. The composition of any one of claims 51-64, wherein the at least one H2-receptor antagonist is selected from the group consisting of: Cimetidine, Ranitidine, Nizatidine, Famotidine, or any combination thereof.

66. The composition of any one of claims 51-65, wherein the at least one Bombesin-like peptide is a gastrin -releasing peptide (GRP), or gastrin-releasing peptide receptor agonist, or a combination thereof.

67. The composition of any one of claims 1-66, wherein the at least one third agent is configured to be administered to a patient in a daily dose of up to substantially 3000 mg.

68. The composition of any one of claims 1-67, wherein the at least one third agent is configured to be administered as capsules, or tablets, or oral powder, or oral granules.

69. The composition of any one of claims 1-68, wherein the at least one third agent is configured to allow modification of absorption of GABA by increasing bioavailability or GABA.

70. The composition of any one of claims 1-68, wherein the at least one third agent is an extended-release formulation of GABA.

71. The composition of any one of claims 1-67, wherein the at least one third agent is in a form of GABA producing bacteria.

72. The composition of claim 70, wherein an amount of the GABA that is produced by the GABA producing bacteria in up to substantially 3,000 mg daily.

73. The composition of any one of claims 1-72, wherein the at least one GABA receptor agonist is selected from the group consisting of: Baclofen, Aminophenyl butyric acid, Hopantenic acid, Zolpidem, or any combination thereof.

74. The composition of any one of claims 1-73, wherein the at least one third agent is at least one anti-epileptic medication.

75. The composition of claim 74, wherein the at least one anti-epileptic medication is selected from the group consisting of: Valproic acid, Valproate sodium, Topiramate, Carbamazepine, medical cannabis, or any combination thereof.

76. The composition of any one of claims 1-75, wherein each component of the composition is in a separate formulation.

77. The composition of any one of claims 1-75, wherein any combination of two of the components of the composition is in one formulation, and the third component of the composition is in another formulation.

78. The composition of any one of claims 1-75, wherein all the components of the composition are in one formulation.

79. The composition of any one of claims 1-78, wherein at least one of the components of the composition is configured to be slowly released.

80. The composition of claim 79, wherein the at least one component is enteric coated.

81. The composition of any one of claims 1-80, wherein at least one of the components of the composition is in a formulation that is configured to enhance bioavailability of the at least one component of the composition.

82. A composition for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, the composition comprising:

DPP-4 antagonist;

PPI; and GABA.

83. The composition of claim 82, wherein the DPP-4 antagonist is configured to be administered in an amount in a range of substantially 1-400 mg daily.

84. The composition of any one of claims 82-83, wherein the PPI is configured to be administered in an amount in a range of substantially 10-240 mg daily.

85. The composition of any one of claims 82-84, wherein the PPI is in a delayed release formulation.

86. The composition of any one of claims 82-85, wherein the PPI is enteric-coated.

87. The composition of any one of claims 82-86, wherein the GABA is configured to be administered in an amount in a range of substantially 50-3,000 mg daily.

88. The composition of any one of claims 82-87, wherein the GABA is in an extended release form, or has enhanced bioavailability, or a combination thereof.

89. The composition of any one of claims 82-88, wherein the GABA is configured to be absorbed to a patient's body from the gastrointestinal tract.

90. A composition for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, the composition comprising:

Sitagliptin;

Omeprazole; and GABA.

91. The composition of claim 90, wherein the Sitagliptin is configured to be administered in an amount in a range of substantially 25-100 mg daily.

92. The composition of any one of claims 90-91, wherein the Omeprazole is configured to be administered in an amount in a range of substantially 10-40 mg daily.

93. The composition of any one of claims 90-92, wherein the Omeprazole is configured to be administered in an amount of up to substantially 120 mg daily.

94. The composition of any one of claims 90-93, wherein the GABA is configured to be administered in an amount in a range of substantially 50-3,000 mg daily.

95. The composition of any one of claims 90-94, wherein the composition is configured to be orally administered to a patient.

96. The composition of claim 95, wherein the composition is formulated in tablets, or delayed-release tablets, or capsules, or delayed-release capsules, or a powder, or a delayed- release powder, or a powder for oral suspension, or a delayed-release powder for oral suspension, or granules, or delayed-release granules, or granules for oral suspension, or delayed-release granules for oral suspension, or dispersible tablets, or orally dispersible (orodispersible) tablets, or effervescent tablets.

97. The composition of any one of claims 90-96, wherein the composition is configured to be administered once daily.

98. The composition of any one of claims 90-96, wherein the composition is configured to be administered twice daily.

99. The composition of any one of claims 90-96, wherein the composition is configured to be administered three times daily.

100. A method for treating and preventing a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, in a patient, the method comprising: administering to the patient at least one first agent configured to cause immunomodulation through inhibition of DPP4 enzyme and activation of glucagon-like peptide- 1 receptor (GLP-1R) in neurological and immune cells; administering to the patient at least one second agent configured to reduce stomach acidity; and administering to the patient at least one third agent, comprising: Gamma aminobutyric acid (GABA), or at least one GABA analogue, or at least one GABA receptor agonist, or any combination thereof.

101. A composition for use in the treatment and prevention of a variety of diseases that are caused by, or linked to, GABAergic transmission dysfunction, in a patient, the treatment and prevention comprising: administering to the patient at least one first agent configured to cause immunomodulation through inhibition of DPP4 enzyme and activation of glucagon-like peptide- 1 receptor (GLP-1R) in neurological and immune cells; administering to the patient at least one second agent configured to reduce stomach acidity; and administering to the patient at least one third agent, comprising: Gamma aminobutyric acid GABA, or at least one GABA analogue, or at least one GABA receptor agonist, or any combination thereof.